p85 becomes ubiquitinated upon Epo stimulation. (A) γ2A/HA-EpoR/JAK2 cells transiently expressing p85 and Flag-tagged Ub were stimulated with Epo for 15 minutes. Immunoprecipitated p85 under nondenaturing conditions was blotted with the indicated antibodies. (B) Ubiquitinated p85 was also detected by immunoprecipitation using FK2 antibody–conjugated beads under denaturing conditions and immunoblotting for p85. (C) Epo induces endogenous p85 ubiquitination in primary Ter119⁻ erythroid progenitor cells. FK2, anti-ubiquitinated proteins antibody; IP, immunoprecipitation; IB, immunoblot.