RAW cell lysates (0.5 mg of protein) were incubated with 2 μg of antibody S522C 2nd bleed (Lanes 1-4) or pre-immune serum (Lane 5). The incubation time was either 1h (Lane 1), 2h (Lane 2), 4h (Lane 3) or 16h (Lane 4, 5). The solutions were then incubated for 1 h with 15 μl of protein G Sepharose, centrifuged and the supernatants collected. The samples were denatured in SDS, subjected to 10% SDS-PAGE and immunoblotted with anti-IRAK4 antibody (Upstate, #07-418, 1:1000 dilution). Lanes 1-5 show IRAK4 that was immunoprecipitated and lanes 6-10 the amount of IRAK4 remaining in the supernatant after immunoprecipitation. Immunoprecipitation was almost complete after 16 h. The amounts loaded on the gel for the supernatants are equivalent to 20 μg of extract protein. IRAK4 can be depleted from cell extracts using the second or third bleed antibodies. The third bleed (S522C, 3rd bleed) works just as well as the second bleed.