Name: Human Reactive Cell Death and Autophagy Antibody Sampler Kit
Price: 735.0 USD
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Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 1 — Simple Western™ analysis of lysates (1 mg/mL) from Jurkat cells treated with Etoposide (25 μM, 5 hours) using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb #5625. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 2 — Simple Western™ analysis of lysates (0.1 mg/mL) from HT-29 cells treated with ZVAD (20uM, 7.5 hours) + hTNF-alpha (20ng/mL, 7 hours) + SM-164 (100nM, 7 hours) using Phospho-RIP (Ser166) (D1L3S) Rabbit mAb #65746. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 3 — Flow cytometric analysis of HCT-116 cells, untreated (blue) or treated with Chloroquine #14774 (50 μM, 18 hr; green) using LC3B (E5Q2K) Mouse mAb Antibody (solid lines) or concentration-matched Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 (dashed lines). Anti-mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408 was used as a secondary antibody.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 4 — Simple Western™ analysis of lysates (0.1 mg/mL) from Jurkat cells treated with Cytochrome C using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb #9664. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 5 — Western blot analysis of extracts from THP-1 cells, differentiated with TPA #4174 (50 ng/ml, overnight) and then treated with LPS #14011 (5 μg/ml, indicated times), using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb (upper), total Gasdermin D (L60) Antibody #93709 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 6 — Western blot analysis of extracts from HeLa cells, untreated or treated with Staurosporine #9953 (1 μM, 3 hr), Jurkat cells, untreated or etoposide-treated (25 μM, overnight), and THP-1 cells, untreated or cycloheximide-treated (CHX, 10 μg/ml, overnight) followed by treatment with TNF-α #8902 (20 ng/ml, 4 hr), using Cleaved PARP (Asp214) (D64E10) XP^® Rabbit mAb (upper), or total PARP Antibody #9542 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 7 — Western blot analysis of HT-29 cells, untreated (-) or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP (Ser166) (D1L3S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 8 — After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO^® is added and emits light during enzyme catalyzed decomposition.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 9 — Western blot analysis of recombinant Human Interleukin-1β (hIL-1β) #8900 using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 10 — Western blot analysis of extracts from HCT 116 and HCT 116 LC3B knockout cells, untreated (-) or treated with Chloroquine #14774 (50 μM, 18 hr) using LC3B (E5Q2K) Mouse mAb #83506 (upper) or GAPDH (D16H11) XP^® Rabbit mAb #5174 (lower). The absence of signal in the HCT 116 knockout cells confirms the specificity of the antibody for LC3B.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 11 — Confocal immunofluorescent analysis of fixed frozen mouse cerebellum, labeled with LC3B (E5Q2K) Mouse mAb #83506 (left, green) and co-labeled with GFAP (E4L7M) XP^® Rabbit mAb #80788 (right, red) and DAPI #4083 (right, blue).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 12 — Confocal immunofluorescent analysis of fixed frozen mouse pons, labeled with LC3B (E5Q2K) Mouse mAb #83506 (left, green) and co-labeled with GFAP (E4L7M) XP^® Rabbit mAb #80788 (right, red) and DAPI #4083 (right, blue).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 13 — Western blot analysis of extracts from HeLa cells (lane 1) or SQSTM1 knock-out cells (lane 2) using SQSTM1/p62 (D5L7G) Mouse mAb #88588 (upper), and β-actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the SQSTM1 knock-out HeLa cells confirms specificity of the antibody for SQSTM1.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 14 — Western blot analysis of HT-29 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb (upper), or MLKL (D2I6N) Rabbit mAb #14993 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 15 — Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb. To confirm phospho-specificity, membranes were either untreated (left) or treated with Calf Intestinal Phosphatase (CIP; right).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 16 — Western blot analysis of extracts from C6 (rat), NIH/3T3 (mouse), and Jurkat (human) cells, untreated or treated with staurosporine #9953 (1uM, 3hrs) or etoposide #2200 (25uM, 5hrs) as indicated, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 17 — Immunoprecipitation of Cleaved Gasdermin D (Asp725) from THP-1 cells differentiated with TPA #4174 (50 ng/ml, overnight) and then treated with LPS #14011 (5 μg/ml, 6 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP^® Isotype Control #3900, and lane 3 is Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb. Western blot was performed using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 18 — Immunohistochemical analysis of paraffin-embedded human tonsil using Cleaved PARP (Asp214) (D64E10) XP^® Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 19 — Western blot analysis of extracts from cells or media collected from THP-1 cells, differentiated with TPA #4147 (80 nM, overnight) and subsequently treated with (+) or without (-) Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr), using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 20 — Western blot analysis of extracts from HeLa, C2C12, and KNRK cells, untreated (-) or treated with Chloroquine (50 μM, overnight; +) #14774 using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 21 — Western blot analysis of extracts from various cell lines using SQSTM1/p62 (D5L7G) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 22 — Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 23 — Immunoprecipitation of extracts from Jurkat cells, untreated or etoposide-treated (25uM, 5hrs), using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb. Western blot was performed using the same antibody.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 24 — Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 25 — Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Staurosporine #9953 (right), using Cleaved PARP (Asp214) (D64E10) XP^® Rabbit mAb (green). Actin filament were labeled with DY-554 phalloidin. Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 26 — Western blot analysis of extracts from HeLa cells or HeLa cells with a knockout of LC3B (HeLa/LC3B KO) using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 27 — Western blot analysis of extract from HCT 116 cells, untreated (-) or starved with Earles Basic Salt Solution (EBSS; 4 hr; +) using SQSTM1/p62 (D5L7G) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 28 — Western blot analysis of HT-29 cells or HT-29 RIPK1 KO cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle) or β-Actin (D6A8) Rabbit mAb #8457 (lower). HT-29 RIPK1 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston, MA.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 29 — Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb in the presence of non-cleaved Gasdermin D peptide (left) or Asp275 cleavage-specific Gasdermin D peptide (right).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 30 — Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with Etoposide #2200 (25 uM, 18 hr; green) using Cleaved PARP (Asp214) (D64E10) XP^® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP^® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412 was used as a secondary antibody.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 31 — Confocal immunofluorescent analysis of THP-1 cells, differentiated with TPA #4174 (80 nM, 24 hr) and subsequently treated with (right) or without (left) Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr), using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb (green). Actin filaments were labeled with DyLight^™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 32 — Western blot analysis of extracts from A549 cells, untreated (-) or starved with Earle's Balanced Salt Solution (EBSS) (indicated times) using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 33 — Western blot anlaysis of extracts from HeLa cells, untreated (-) or treated with Chloroquine #14774 (50 μM, overnight; +) using SQSTM1/p62 (D5L7G) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 34 — Confocal immunofluorescent analysis of HT-29 cells, untreated (left), pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 6 hr; center), or pre-treated with Z-VAD followed by treatment with SM-164 and hTNF-α and post-processed with λ-phosphatase (right), using Phospho-RIPK3 (Ser227) (D6W2T) Rabbit mAb (green). Actin filaments were labeled with DyLight^™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 35 — Immunohistochemical analysis of paraffin-embedded mouse embryo, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb in the presence of control peptide (left) or Cleaved Caspase-3 (Asp175) Blocking Peptide (#1050) (right).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 36 — Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 37 — Western blot analysis of extracts from MCF7 and Raji cells, untreated (-) or treated with Torin 1 (250 nM, 5 hr; +) #14379, using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 38 — Western blot analysis of extracts from HeLa cells transfected with 100 nM SignalSilence^® Control siRNA (Unconjugated) #6568 (-), SignalSilence^® SQSTM1/p62 siRNA I #6394 (+), or SignalSilence^® SQSTM1/p62 siRNA II #6399 (+), using SQSTM1/p62 (D5L7G) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 39 — Immunohistochemical analysis using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb on SignalSlide^® Cleaved Caspase-3 IHC Controls #8104 (paraffin-embedded Jurkat cells, untreated (left) or etoposide-treated (right)).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 40 — Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's Lymphoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 41 — Immunoprecipitation of LC3B from HeLa cells treated with Chloroquine (50 μM, overnight) # 14774. Lane 1 is 10% input, lane 2 is Mouse (G3A1) mAb IgG1 Isotype Control, and lane 3 is LC3B (E5Q2K) Mouse mAb. Western blot was performed using LC3B (E5Q2K) Mouse mAb. Anti-mouse IgG, HRP-linked Antibody #7076 was used as a secondary antibody.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 42 — Immunoprecipitation of SQSTM1/p62 protein from PANC-1 cell extracts. Lane 1 is 10% input, lane 2 is Mouse (G3A1) mAb IgG1 Isotype Control #5415, and lane 3 is SQSTM1/p62 (D5L7G) Mouse mAb. Western blot was performed using SQSTM1/p62 (D5L7G) Mouse mAb. A light chain-specific secondary antibody was used to avoid reactivity with heavy chain IgG.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 43 — Immunohistochemical staining of paraffin-embedded mouse embryo, showing cytoplasmic localization in apoptotic cells, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 44 — Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 45 — Immunohistochemical analysis of paraffin-embedded human colon carcinoma using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 46 — Immunohistochemical analysis of paraffin-embedded human colon carcinoma using SQSTM1/p62 (D5L7G) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 47 — Immunohistochemical analysis of paraffin-embedded human spleen (left, positive) and skeletal muscle (right, negative) using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 48 — Immunohistochemical analysis of paraffin-embedded human esophageal carcinoma using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 49 — Immunohistochemical analysis of paraffin-embedded human lung carcinoma using SQSTM1/p62 (D5L7G) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 50 — Immunohistochemical analysis of paraffin-embedded THP-1 cell pellets, differentiated with TPA #4174 (left) and then treated with LPS #14011 (right), using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 51 — Immunohistochemical analysis of paraffin-embedded human lung carcinoma using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 52 — Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using SQSTM1/p62 (D5L7G) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 53 — Immunohistochemical analysis of paraffin-embedded human ulcerative colitis using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 54 — Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's lymphoma using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 55 — Immunohistochemical analysis of paraffin-embedded HDLM-2 (left) and Daudi (right) cell pellets using SQSTM1/p62 (D5L7G) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 56 — Confocal immunofluorescent images of HT-29 cells, untreated (left) or Staurosporine #9953 treated (right) labeled with Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor^® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 57 — Immunohistochemical analysis of paraffin-embedded HCT116 cell pellets, untreated (left-top) or treated with Chloroquine #14774 (right-top), and HCT116 LC3B knockout cell pellets, untreated (left-bottom) or treated with Chloroquine #14774 (right-bottom), using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 58 — Immunohistochemical analysis of paraffin-embedded human inflamed gall bladder using SQSTM1/p62 (D5L7G) Mouse mAb in the presence of control peptide (left) or antigen-specific peptide (right).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 59 — Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with etoposide #2200 (green), using Cleaved Caspase-3(Asp175) (5A1E) Rabbit mAb compared to a nonspecific negative control antibody (red).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 60 — Immunohistochemical analysis of paraffin-embedded normal human brain using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 61 — Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with chloroquine (50 μM overnight; right), using SQSTM1/p62 (D5L7G) Mouse mAb (green). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 62 — Immunohistochemical analysis of paraffin-embedded KARPAS 299 cell pellet (left, high-expressing) or VCaP cell pellet (right, low-expressing) using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 63 — Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with Chloroquine #14774 (right), using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 64 — Immunohistochemical analysis of paraffin-embedded normal human spleen using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 65 — Confocal immunofluorescent analysis of HCT 116 cells either untreated (left) or treated with Chloroquine #14774 (50 µM, overnight) (center) or LC3B HCT 116 knockout cells treated with Chloroquine #14774 (50 µM, overnight) (right) using LC3B (E5Q2K) Mouse mAb (green). Actin filaments were labeled with β-Actin (13E5) Rabbit mAb #4970 (red) and nuclei were labeled with DAPI #4083 (blue).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 66 — Flow cytometric analysis of HCT-116 cells, wild-type (green, high expression) or LC3B knockdown (blue, negative expression), using LC3B (E5Q2K) Mouse mAb or a concentration-matched Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 (dashed lines). Anti-mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408 was used as a secondary antibody.

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To Purchase # 42867

Cat. #	Size	Qty.	Price
42867T	1 Kit (9 x 20 microliters)

Product Includes
Related Products

Product Includes	Quantity	Applications	Reactivity	MW(kDa)	Isotype
Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb 9664	20 µl	WB IP IHC IF F	H M R Mk	17, 19	Rabbit IgG
Cleaved PARP (Asp214) (D64E10) XP^® Rabbit mAb 5625	20 µl	WB IP IHC IF F	H Mk	89	Rabbit IgG
Phospho-RIP (Ser166) (D1L3S) Rabbit mAb 65746	20 µl	WB	H	78-82	Rabbit IgG
Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb 93654	20 µl	WB IF	H	46-62	Rabbit IgG
Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb 91689	20 µl	WB	H	54	Rabbit IgG
Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb 36425	20 µl	WB IP IHC	H	30	Rabbit IgG
Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb 83186	20 µl	WB IF	H	17	Rabbit IgG
LC3B (E5Q2K) Mouse mAb 83506	20 µl	WB IP IHC IF F	H M R	14, 16	Mouse IgG2b
SQSTM1/p62 (D5L7G) Mouse mAb 88588	20 µl	WB IP IHC IF	H	62	Mouse IgG1
Anti-rabbit IgG, HRP-linked Antibody 7074	100 µl	WB			Goat

Kit Usage Information

Protocols

5625: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Paraffin), Immunofluorescence, Flow
7074: Western Blotting
9664: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Paraffin), Immunofluorescence, Flow
36425: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Paraffin)
65746: Western Blotting
83186: Western Blotting, Immunofluorescence
83506: Western Blotting, Immunoprecipitation (Magnetic), Immunohistochemistry (Paraffin), IF-F Citrate Retrieval (Mouse), Immunofluorescence*, Flow Cytometry, Methanol Fixation for Rabbit
88588: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Paraffin), Immunofluorescence
91689: Western Blotting
93654: Western Blotting, Immunofluorescence

Product Description

The Human Reactive Cell Death and Autophagy Antibody Sampler Kit provides an economical means of detecting common readouts in apoptosis, necroptosis, pyroptosis, and autophagy. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the Human Reactive Cell Death and Autophagy Antibody Sampler Kit detects endogenous levels of its target protein. Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb detects endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. This antibody does not recognize full-length caspase-3 or other cleaved caspases. Non-specific labeling may be observed by immunofluorescence in specific sub-types of healthy cells in fixed-frozen tissues (e.g., pancreatic alpha-cells). Cytoplasmic background may be observed in human and monkey samples. Cleaved PARP (Asp214) (D64E10) XP^® Rabbit mAb detects endogenous levels of the large fragment (89 kDa) of human PARP1 protein produced by caspase cleavage. The antibody does not recognize full-length PARP1 or other PARP isoforms. Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb recognizes endogenous levels of the amino-terminal fragment of Gasdermin D only when cleaved at Asp275. Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb recognizes endogenous levels of mature IL-1β protein only when cleaved at Asp116. Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb detected a band at 30 kDa that appears to be a cleavage product of RIP3. Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb may also bind to MLKL when dually phosphorylated at Thr357 and Ser358. LC3B (E5Q2K) Mouse mAb detects both type I and type II forms of LC3B. Cross reactivity was not detected with other family members.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Asp175 of human caspase-3, Asp214 of human PARP, Asp275 of human Gasdermin D, Asp116 of human IL-1β, Pro220 of human SQSTM1/p62, residues near the amino terminus of human LC3B, and synthetic phosphopeptides corresponding to Ser166 of human RIP, Ser227 of human RIP3, and Ser358 of human MLKL.

Background

Regulated cell death has been classified based on distinct morphological and biochemical pathways (1). Type I cell death, or apoptosis, is characterized by cytoplasmic shrinkage, chromatin condensation, nuclear fragmentation, plasma membrane blebbing, and phagocytic update of dead cells. Apoptosis can occur through extrinsic pathways involving extracellular factors, including the activation of death receptors, or through intrinsic pathways involving intracellular perturbations, including mitochondrial outer membrane permeabilization (2). Both of these apoptotic pathways lead to activation of caspases, a family of cysteine acid proteases that are synthesized as inactive zymogens containing pro-domains, followed by large (p20) and small (p10) subunits which are proteolytically activated in a cascade-like fashion. Caspase-3 is a key downstream protease activated by both extrinsic and intrinsic apoptotic pathways and cleaves a large number of proteins involved in the disassembly of the cell, including poly(ADP-ribose) polymerase (PARP), a protein involved in the DNA damage response.

Type II cell death, or autophagy, manifests with extensive cytoplasmic vacuolization, and like apoptosis, can include phagocytic update. Autophagy is a catabolic process for the degradation of cellular components including protein aggregates, damaged organelles, and pathogens (3). The process involves the engulfment of these components into a double membrane structure, the autophagosome, which fuses to the lysosome for degradation. Autophagy requires, and can be monitored by, the conversion of LC3 family members, such as LC3B, from a type I form to a lipidated type II form that is incorporated into the autophagosome membrane and binds to a variety of cargo receptors. Cargo receptors such as SQSTM1/p62 bind LC3 along with ubiquitinated proteins that are targeted for degradation. SQSTM1/p62 is also degraded during this process, and thus its expression is frequently used to monitor this process.

Type III cell death, or necrosis, manifests with plasma membrane permeability with cellular swelling and fragmentation, and lacks a clear phagocytic response which then leads to an inflammatory signaling with the release of damage-associated molecular patterns (DAMPs). Necrosis can be triggered by multiple regulated pathways including necroptosis and pyroptosis. Necroptosis is regulated by the kinase activities of RIP and RIP3 and the pore forming ability of MLKL (4). Necroptosis requires the activation of RIP3 which then phosphorylates MLKL at Ser358 (Ser345 in mouse). Phosphorylation of MLKL leads to generation of a pore complex involved in cell swelling and the secretion of DAMPs. RIP3 activation is triggered through several RIP homotypic interaction motif (RHIM) domain interactions including RIP, TRIF, and ZBP1 and results in the phosphorylation of RIP3 at Ser227 (Thr231/Ser232 in mouse). Canonical necroptosis signaling is mediated by RIP, and this can be inhibited by necrostatins, small molecules that directly inhibit RIP kinase activity. Activation of RIP can be monitored through autophosphorylation sites including Ser166. Pyroptosis is generally induced in cells of the innate immune system, and is characterized by cleavage of Gasdermin D (5). The amino-terminal fragment of Gasdermin D produced following cleavage by inflammatory caspases (Caspase-1, -4, -5), oligomerizes to form a pore. Canonical cleavage of Gasdermin D occurs through a two-step process. The first step involves transcriptional regulation of targets such as NLRP3 and the pro-forms of IL-1β and IL-18. In the second execution step, Caspase-1 is activated through formation of inflammasome complexes. Activated Caspase-1 cleaves Gasdermin D as well as IL-1β and IL-18 to their mature forms, and these active cytokines are secreted through pores formed by Gasdermin D.

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UniProt ID: Q13546 , P01584 , Q13501 , Q9Y572 , P57764 , Q8NB16 , P42574 , Q9GZQ8 , P09874

Entrez-Gene Id: 8737 , 3553 , 8878 , 11035 , 79792 , 197259 , 836 , 81631 , 142

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Human Reactive Cell Death and Autophagy Antibody Sampler Kit #42867