(A) Co-immunoprecipitation of endogenous p190 with ECT2 variants. HeLa cells were transfected with wild type FLAG-ECT2, FLAG-ECT2N, or FLAG-ECT2C and synchronized at mitosis (M) or left as a cycling (C) population. One mg protein lysate was immunoprecipitated with mouse anti-p190 antibody (UBI). Immunoprecipitates were immunoblotted with mouse anti-p190 antibody (Becton-Dickinson) or anti-FLAG antibody. FLAG-ECT2, FLAG-ECT2N, and FLAG-ECT2C co-immunoprecipitated with p190 from both cycling and mitotic cell extracts, with preferential association observed between p190 and ECT2C.
(B) Western blotting of endogenous p190 or FLAG-tagged, wild type ECT2, ECT2N, and ECT2C overexpressing cells. Thirty µg whole cell lysates from cells in Panel A were immunoblotted with anti-FLAG antibody to detect levels of ECT2 or with anti-p190 (Becton Dickinson) to detect p190.