Revision 6

#2258Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) orders@cellsignal.com

Support: 877-678-TECH (8324)

Web: info@cellsignal.com cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IHC-P, IF-F, IF-IC, FC-FP, C&R

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

42

Source/Isotype:

Rabbit IgG

UniProt ID:

#P17947

Entrez-Gene Id:

6688

Product Information

Product Usage Information

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:100
Immunofluorescence (Frozen) 1:200 - 1:400
Immunofluorescence (Immunocytochemistry) 1:200 - 1:400
Flow Cytometry (Fixed/Permeabilized) 1:200
CUT&RUN 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #45150.

Specificity / Sensitivity

This antibody detects endogenous levels of total PU.1 protein. The antibody does not cross react with other Ets family members.

Species Reactivity:

Human, Mouse

Species predicted to react based on 100% sequence homology

Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence of human PU.1 protein.

Background

PU.1 is a member of the Ets family of transcription factors and activates target genes through the purine-rich PU-box (1). PU.1 plays a pivotal role in the differentiation of myeloid cells and lymphocytes and is expressed in several hematopoietic cells including B lymphocytes, macrophages, neutrophils, mast cells, early erythroid cells, and megakaryocytes (1,2). The concentration of PU.1 is critical for both the determination of hematopoietic cell lineage and the regulation of differentiation versus stem cell proliferation (3,4). In addition, PU.1 activity is influenced by phosphorylation and interactions with other hematopoietic transcription factors. Phosphorylation of PU.1 at Ser146 by CK2 promotes binding to IRF4 and synergistic activation through the immunoglobulin κ 3' enhancer (5). Treatment of pro-B cells with IL-3 leads to phosphorylation of PU.1 at Ser140, resulting in increased PU.1 activity and activation of the anti-apoptotic gene MCL-1 (6). GATA1 binding blocks PU.1 activity during erythroid cell development (7). Overexpression of PU.1 resulting from proviral insertion during Friend virus infection can induce erythroleukemia, while reduced expression has been associated with acute myeloid leukemia (8).

  1. Lloberas, J. et al. (1999) Immunol Today 20, 184-9.
  2. Klemsz, M.J. et al. (1990) Cell 61, 113-24.
  3. Dahl, R. and Simon, M.C. (2003) Blood Cells Mol Dis 31, 229-33.
  4. DeKoter, R.P. and Singh, H. (2000) Science 288, 1439-41.
  5. Pongubala, J.M. et al. (1993) Science 259, 1622-5.
  6. Wang, J.M. et al. (2003) Mol Cell Biol 23, 1896-909.
  7. Zhang, P. et al. (1999) Proc Natl Acad Sci U S A 96, 8705-10.
  8. Moreau-Gachelin, F. et al. (1988) Nature 331, 277-80.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-F: Immunofluorescence (Frozen) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 6
#2258

PU.1 (9G7) Rabbit mAb

Western Blotting Image 1: PU.1 (9G7) Rabbit mAb Expand Image
Western blot analysis of extracts from RAW, THP1 and p388D1 cells using PU.1 (9G7) Rabbit mAb.
No image available
Immunohistochemistry Image 1: PU.1 (9G7) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded 4T1 syngeneic mouse tumor using PU.1 (9G7) Rabbit mAb #2258.
Immunohistochemistry Image 2: PU.1 (9G7) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded Non-Hodgkin's lymphoma using PU.1 (9G7) Rabbit mAb in the presence of control peptide (left) or PU.1 Blocking Peptide #1036 (right).
Immunofluorescence Image 1: PU.1 (9G7) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of normal mouse brain using PU.1 (9G7) Rabbit mAb (yellow) and GFAP (GA5) Mouse mAb (red). After blocking free secondary antibody binding sites with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, the tissue was then labeled using ASC (D2W8U) Rabbit mAb (Mouse Specific) (Alexa 488 Conjugate) (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Immunofluorescence Image 1: PU.1 (9G7) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of Raw cells using PU.1 (9G7) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Flow Cytometry Image 1: PU.1 (9G7) Rabbit mAb Expand Image
Flow cytometric analysis of MCF7 cells (blue) and THP1 cells (green) using PU.1 (9G7) Rabbit mAb (solid lines) or a 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.
CUT and RUN Image 1: PU.1 (9G7) Rabbit mAb Expand Image
CUT&RUN was performed with U-937 cells and PU.1 (9G7) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across TOMM40L gene.
CUT and RUN Image 2: PU.1 (9G7) Rabbit mAb Expand Image
CUT&RUN was performed with U-937 cells and PU.1 (9G7) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 1 (upper), including TOMM40L gene (lower).
CUT and RUN Image 3: PU.1 (9G7) Rabbit mAb Expand Image
CUT&RUN was performed with U-937 cells and either PU.1 (9G7) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human ANKRD44 intron 21 primers, human CTSS exon 1 primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.