Fig 6.

Location of, and complex formation between, cyclin D1 (D1) and CDK4. A): Western blot assay detects protein levels of CDK4, total D1, Thr286 phosphorylated D1, CDK2 and cyclin E in the nuclear or cytoplasmic fraction of MCF7 or MB231 cells 48 hours after treatment with 4 or 8 µM NPCD, with the non-treated (0) cells as control. The Rb protein ⁸⁶,⁸⁷ and β-actin are included as the loading control for the nuclear and the cytoplasmic proteins, respectively. B): Co-immunoprecipitation assay detects the D1-CDK4 complex. Total cell lysates were prepared from MCF7, MB231 and MCF15 cells 48 or 72 hours after treatment with 4 or 8 µM NPCD. The protein lysates were first immunoprecipitated with D1 antibody, and the D1 immunoprecipitates were then fractioned in polyacrilamide gel and subjected to a western blot assay for detection of D1 or CDK4 as indicated. The CDK4 detected in the D1 immunoprecipitates is considered to be associated with D1.