Neither chARF nor p16-ACT prevents Cyclin D1 from binding CDK4.

<p>U2OS cells were mock transfected (control) or transfected with p16, p16-ACT, p14ARF or chARF expression constructs and CDK4 was immunoprecipitated under non-denaturing conditions. Immunoprecipitations were analyzed via immunoblot for the presence of CDK4, Cyclin D1, p16, p16-ACT, p14ARF, chARF and p21. The PVDF membrane was cut horizontally at the 25 kDa molecular weight marker and the top half was probed with antibodies against CDK4, Cyclin D1, and β-actin while the bottom half was probed with antibodies for p16, p14ARF, and p21. chARF (19kDa) was detected with antibodies to the amino-terminus (N-term) of p14ARF or the carboxyl terminus (C-term) of p16. p16-ACT (15 kDa) was detected with an antibody to the amino terminus (N-term) of p16. β-actin was measured in the lysate as a loading control. Immunoprecipitations are shown on the right half of the blot (IP:CDK4) while input lysates are shown on the left (Lysate). Immunoprecipitation using normal rabbit IgG was performed on chARF transfected U2OS cells (IgG, far right) to ensure target proteins were not immunoprecipitated nonspecifically. A comparable amount of CDK4 was immunoprecipitated in each CDK4 IP, indicating that differing amounts of co-immunoprecipitated Cyclin D1 was a result of the transfected proteins. chARF was detected in the CDK4 IP using a C-term p16 antibody while p16-ACT was not detected in the CDK4 IP using the N-term p16 antibody. Only wild-type p16 prevented Cyclin D1 from binding CDK4 compared to control. In p14ARF and chARF transfected cells, induction of p21 stabilized the Cyclin D1/CDK4 complex, resulting in greater amounts of co-immunoprecipitated Cyclin D1 compared to control.