BMDM extracts (1mg protein) were immunoprecipitated with 5ug of either sheep anti-NEK7 antibody S669a (Lane 1), S671a (Lane 2) or anti-rabbit NEK7 antibody (Abcam) (Lane 4) or sheep IgG (Lane 3) or rabbit IgG (Lane 5) overnight at 4C followed by addition of 10 ml of protein G-Sepharose for 30 min . Immunoprecipitated proteins were denatured in SDS and subjected to SDS-PAGE on 10% gels. Lanes 7-11 indicate the amount of NEK7 protein remaining in the supernatant after immunoprecipitation, the amounts loaded being equivalent to 10 mg of cell extract protein. Proteins were transferred to PVDF membrane and immunoblotting was carried out for 16h at 4C using anti-rabbit NEK7 antibody (Abcam).