Cell Signaling Technology

Product Pathways - NF-kB Signaling

PhosphoPlus® IκBα (Ser32/36) Antibody Kit #9240

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 100 µl W H M R Mk (B) (Dg) (Pg) (GP) 40 Mouse IgG1
IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 100 µl W IP IHC-P IF-IC F H M R Mk B Pg GP 39 Mouse IgG1
NF-κB Control Cell Extracts #9243 200 µl
Anti-mouse IgG, HRP-linked Antibody #7076 100 µl Horse
Anti-biotin, HRP-linked Antibody #7075 100 µl Goat
20X LumiGLO® Reagent and 20X Peroxide #7003 5 ml each
Biotinylated Protein Ladder Detection Pack #7727 100 µl

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  B=Bovine  Dg=Dog  Pg=Pig  GP=Guinea Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 detects endogenous levels of total IκBα protein. Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 detects endogenous levels of IκBα only when phosphorylated at Ser32/36. Neither antibody cross-reacts with other IκB family members at physiological levels.

Western Blotting

Western Blotting

Western blot analysis of extracts from THP-1 cells, differentiated with TPA (#9905, 80 nM for 24h) and treated with 1 μg/ml LPS for the indicated times, using Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 (upper) and IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa, NIH/3T3 and PC12 cells, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814.

Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated orTNF-α-treated (#2169, 20 ng/ml) for 5 minutes, using Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 (upper) or IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 (lower).


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human leiomyoma, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human renal adenocarcinoma, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast tumor, using Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246.


IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left), or TNF-α-treated (right) using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

Description

The PhosphoPlus® IκBα (Ser32/36) Antibody Kit provides reagents and protocols for the rapid analysis of IκBα phosphorylation at Ser32/36. The kit contains a total IκBα antibody, a phospho-specific antibody, and positive/negative control whole cell lysates, along with secondary antibodies and reagents for Western blotting.

Source / Purification

IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 is produced by immunizing mice with a GST-IκBα fusion protein corresponding the amino-terminus of human IκBα. Phospho-IκBα (Ser32/36) (5A5) Mouse mAb #9246 is produced by immunizing mice with a synthetic phosphopeptide corresponding to residues surrounding Ser32/36 of human IκBα. Total cell extracts from HeLa cells prepared without treatment serve as a negative control. Supplied in SDS Sample Buffer. Total cell extracts from HeLa cells prepared with TNF-α treatment (#2169, 20 ng/ml for 5 minutes) serve as a positive control. Supplied in SDS Sample Buffer.

Background

The NF-κB/Rel transcription factors are present in the cytosol in an inactive state complexed with the inhibitory IκB proteins (1-3). Activation occurs via phosphorylation of IκBα at Ser32 and Ser36 followed by proteasome-mediated degradation that results in the release and nuclear translocation of active NF-κB (3-7). IκBα phosphorylation and resulting Rel-dependent transcription are activated by a highly diverse group of extracellular signals including inflammatory cytokines, growth factors, and chemokines. Kinases that phosphorylate IκB at these activating sites have been identified (8).

  1. Baeuerle, P.A. and Baltimore, D. (1988) Science 242, 540-6.
  2. Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70.
  3. Finco, T.S. et al. (1994) Proc Natl Acad Sci USA 91, 11884-8.
  4. Brown, K. et al. (1995) Science 267, 1485-8.
  5. Brockman, J.A. et al. (1995) Mol Cell Biol 15, 2809-18.
  6. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
  7. Chen, Z.J. et al. (1996) Cell 84, 853-62.
  8. Karin, M. and Ben-Neriah, Y. (2000) Annu Rev Immunol 18, 621-63.

Application References

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

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