Flow cytometric analysis of untreated Raw264.7 cells, IRAK1 shRNA (blue) or +GFP shRNA (green), using IRAK1 (D51G7) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor® 647 conjugate) #4414 was used as a secondary antibody.
Western blot analysis of extracts from Jurkat, Raji and K562 cell lines, using IRAK2 Antibody #4367.
Western blot analysis of extracts from HL-60, Raji and K562 cell lines, using IRAK-M Antibody.
Western blot analysis of extracts from THP-1 (human), RAW 264.7 (mouse), and H-4-II-E (rat) cell lines, using IRAK4 Antibody.
Western blot analysis of extracts from RD, 293 and A20 cells using IRAK1 (D51G7) Rabbit mAb.
Western blot analysis of extracts from COS-7 cells, either mock transfected or transfected with human IRAK1, using IRAK1 (D51G7) Rabbit mAb.
Western blot analysis of extracts from THP-1, mouse embryonic fibroblast (MEF), RAW264.7 and NIH/3T3 cells using IRAK1 (D51G7) Rabbit mAb.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® IRAK1 siRNA I (+) or SignalSilence® IRAK1 siRNA II #6228 (+), using IRAK1 (D51G7) Rabbit mAb #4504 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The IRAK1 (D51G7) Rabbit mAb confirms silencing of IRAK1 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
|IRAK1 (D51G7) Rabbit mAb 4504||20 µl||
||H M Mk||78-105||Rabbit IgG|
|IRAK2 Antibody 4367||20 µl||
||H M R Mk||62||Rabbit|
|IRAK-M Antibody 4369||20 µl||
|IRAK4 Antibody 4363||20 µl||
||H M R Mk||55||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The IRAK Isoform Antibody Sampler Kit provides an economical means to examine total protein levels of the four Interleukin-1 Receptor Associated Kinase family members: IRAK1, IRAK2, IRAK3/IRAK-M, and IRAK4.
Each antibody in the IRAK Isoform Antibody Sampler Kit detects endogenous levels of its indicated target. Cross-reactivity has not been detected with other family members at endogenous levels.
Antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy-terminus of murine IRAK1, human IRAK2, human IRAK-M, and surrounding Lys41 of human IRAK4.
Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is a serine/threonine-specific kinase that can be coprecipitated in an IL-1-inducible manner with the IL-1 receptor (1). The mammalian family of IRAK molecules contains four members (IRAK1, IRAK2, IRAK3/IRAK-M, and IRAK4). The binding of IL-1 to IL-1 receptor type I (IL-1RI) initiates the formation of a complex that includes IL-1RI, AcP, MyD88, and IRAKs (2). IRAK undergoes autophosphorylation shortly after IL-1 stimulation. The subsequent events involve IRAK dissociation from the IL-1RI complex, its ubiquitination, and its association with two membrane-bound proteins: TAB2 and TRAF6. The resulting IRAK-TRAF6-TAB2 complex is then released into the cytoplasm where it activates protein kinase cascades, including TAK1, IKKs, and the stress-activated kinases (3).
Upon IL-1R/TLR (Toll-Like Receptor) ligation, IRAK1 and IRAK4 are rapidly recruited to the receptor by the adaptor MyD88 (4). IRAK1 is phosphorylated by IRAK4 at Thr209 and Thr387 (5), followed by sequential autohyperphosphorylation in various domains. Unlike IRAK1 and IRAK4, IRAK2 and IRAK-M do not have significant kinase activity although they can still activate NF-κB when overexpressed (6,7). Antisense oligonucleotide depletion of IRAK2 can inhibit IL-1 mediated NF-κB activation (8). Expression of IRAK-M is more restricted compared to other family members with highest levels of expression occurring in monocytes/macrophages (6). Studies from IRAK-M knockout mice suggest that it may play a role as a negative regulator of TLR signaling and innate immune responses by preventing the dissociation of IRAK1 and IRAK4 from MyD88 and the subsequent formation of its complex with TRAF6 (9).
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