Raw (264.7) cell extracts (1mg protein) were immunoprecipitated with increasing amounts of sheep anti-IRAK2 antibody (lanes 1 - 3) or pre-immune serum (lanes 4 - 6) for 2 hours along with 10 μl of protein G-Sepharose. Immunoprecipitated proteins were denatured in SDS and subjected to SDS-PAGE on 8% gels. Lanes 7- 12 indicate the amount of mouse IRAK2 protein remaining in the supernatant after immunoprecipitation, the amounts loaded being equivalent to 25 μg of cell extract protein. Proteins were transferred to PVDF membrane and immunoblotting was carried out overnight using rabbit polyclonal antibody against IRAK2 (Abcam) at 1:5000.