Ab62419

Proteins from samples were isolated using RIPA buffer (Vazyme, Nanjing, China) and were segregated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then proteins were transferred onto the polyvinylidene difluoride membranes (Vazyme). The membranes were blocked with 5% skimmed milk (Vazyme). Thereafter, the membranes were incubated with the primary antibodies: anti-Irak2 (1:2000, ab62419, Abcam, Cambridge, UK) or glyceraldehyde 3-phosphate dehydrogenase (1:2500, ab9485, Abcam) overnight. After being rewashed, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 2 h. The membranes were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad) after being treated with ECL kit (Vazyme).

The antibodies recognising IRAK1 (Cell Signalling Technology #4504S) and IRAK2 (Abcam #ab62419) were purchased from the sources indicated in parentheses. The antibody recognising IRAK4 phosphorylated at both Thr345 and Ser346 was a gift from Dr Vikram Rao (Pfizer, Boston, MA, U.S.A.). This antibody was used in this study because it is more sensitive than any commercially available IRAK4 antibody that we have tested.