Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

RPA32 (4E4) Rat mAb #2208

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC H M R Mk Hm Endogenous 32 Rat IgG1

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Hm=Hamster
Species cross-reactivity is determined by Western blot.

Protocols

Specificity / Sensitivity

RPA32 (4E4) Rat mAb detects endogenous levels of total RPA32 protein. The antibody recognizes the carboxy-terminal seqquence of RPA32.

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant full-length human MBP-RPA32 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines, using RPA32 (4E4) Rat mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using RPA32 (4E4) Rat mAb (green) showing translocation to distinct nuclear foci after UV damage. Actin filaments have been labeled with Alexa Fluor® 555 phalloidin. Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

Background

RPA70 (HSSB, REPA1, RF-A, RP-A, p70) is a component of a heterotrimeric complex, composed of 70, 32/30 and 14 kDa subunits, collectively known as RPA. RPA is a single stranded DNA binding protein, whose DNA binding activity is believed to reside entirely in the 70 kDa subunit. The complex is required for almost all aspects of cellular DNA metabolism such as DNA replication (1-3), recombination, cell cycle and DNA damage checkpoints, and all major types of DNA repair including nucleotide excision, base excision, mismatch and double-strand break repairs (4-7). In response to genotoxic stress in eukaryotic cells, RPA has been shown to associate with the Rad9/Rad1/Hus1 (9-1-1) checkpoint complex (8). RPA is hyperphosphorylated upon DNA damage or replication stress by checkpoint kinases including ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) (9-11). Hyperphosphorylation may alter RPA-DNA and RPA-protein interactions. In addition to the checkpoint partners, RPA interacts with a wide variety of protein partners, including proteins required for normal replication such as RCF, PCNA and Pol α, and also proteins involved in SV40 replication, such as DNA polymerase I and SV40 large T antigen (10,12).

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  7. Sancar, A. et al. (2004) Annu. Rev. Biochem. 73, 39-85.
  8. Guo, S. et al. (2006) J Biol Chem 281, 21607-16.
  9. Wu, X. et al. (2005) Oncogene 24, 4728-4735.
  10. Binz, S.K. et al. DNA Repair (Amst) 3, 1015-1024.
  11. Nuss, J.E. et al. (2005) Biochemistry 44, 8428-8437.
  12. Yuzhakov, A. et al. (1999) EMBO J. 18, 6189-6199.

Application References

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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