HEK293 cell lysates (0.5 mg of protein) were immunoprecipitated overnight with 2 μg of antibody (S517C 1st, 2nd or 3rd bleed) or pre-immune serum. The immunoprecipitates were incubated for 1 h with 15 μl of protein G sepharose. The immunoprecipitates were denatured in SDS and subjected to 8 % SDS-PAGE. Lanes 1-4 indicate the amount of IRAK1 that has been immunoprecipitated with the respective bleed of the antibody, whereas the lanes 5-8 indicate the amount of IRAK1 protein remaining in the supernatant after the immunoprecipitation. Amounts loaded for the supernatants are equivalent to 20 μg of extract protein. After SDS-PAGE, the proteins were transferred to PVDF membrane and immunoblotting was carried out using the rabbit polyclonal antibody against IRAK1 (Santa Cruz, SC-7883) overnight at an antibody concentration of 1:1000.