Cell Signaling Technology

Product Pathways - Lymphocyte Signaling

PU.1 Antibody #2266

Applications Reactivity Sensitivity MW (kDa) Source
W IP IHC-P IF-IC F ChIP H M (Mk) (Pg) Endogenous 42 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  Mk=Monkey  Pg=Pig
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

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

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino acids at the amino-terminus of human PU.1. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from RAW, p388D1, WEHI-3 and THP1 cells using #2266.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing nuclear localization in lymphocytes, using PU.1 Antibody.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma (infiltrating cells), using PU.1 Antibody in the presence of control peptide (left) or PU.1 Blocking Peptide #1036 (right).


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human tonsil, showing nuclear localization, using PU.1 Antibody.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of RAW cells, using PU.1 antibody (blue) compared to a nonspecific negative control antibody (red).

IF-IC

IF-IC

DAPI staining (left) and immunofluorescent staining (right) of paraformaldehyde-fixed RAW cells using #2266.


Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 U-937 cells and either 20 μl of PU.1 Antibody or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human CD11b promoter primers, human CD18 intron 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.

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, 184-189.
  2. Klemsz, M.J. et al. (1990) Cell 61, 113-124.
  3. Dahl, R. and Simon, M.C. (2003) Blood Cells Mol. Dis. 31, 229-233.
  4. DeKoter, R.P. and Singh, H. (2000) Science 288, 1439-1441.
  5. Pongubala, J.M. et al. (1993) Science 259, 1622-1625.
  6. Wang, J.M. et al. (2003) Mol. Cell Biol. 23, 1896-1909.
  7. Zhang, P. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 8705-8710.
  8. Moreau-Gachelin, F. et al. (1998) Nature 331, 277-280.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

Products