A. Lysates of HeLa cells stably overexpressing B56γ3HA or vector transfected with expression vector of Flag-p27 were analyzed by SDS-PAGE and immunoblotting with antibodies specific for phospho-p27 (Thr187), phospho-p27 (Thr157), phospho-p27 (Thr198), phospho-p27 (Ser10), total p27, HA, B56γ, and β-actin. B. Lysates of HeLa cells with vector only or B56γ3 knockdown transfected with expression vector of Flag-p27 were analyzed as described in (A). C. Lysates of HeLa with vector only, B56γ3 overexpression, or B56γ3 knockdown and D. lysates of HCT116 cells with vector only or B56γ3 overexpression were analyzed for levels of phospho-p27 (Thr157), p27, HA, B56γ3 and β-actin as described above. The amounts of protein applied to the gel were adjusted so that the lysates from different cells yielded similar total p27 levels detected on the blot. The relative expression level of each phospho-p27 were quantified by densitometry and normalized with total Flag-p27 or p27. The data shown are expressed as -fold expression level over that of vector control, which was set as 1. Data shown are from one representative experiment of at least two experiments with similar results.