FIG. 2.
Interactions between cyclin A-Cdk2, Skp2, and p21Cip1/WAF1. (A) HtTA1 cells were transfected with either pUHD-P1 vector alone (lanes 1, 3, and 5) or FLAG-p21 in pUHD-P1 (lanes 2, 4, and 6). Cell extracts were prepared, and 60 μg was immunoprecipitated (IP) with either NRS (lanes 3 and 4) or anti-Cdk2 serum (lanes 5 and 6). The immunoprecipitates and total cell extracts (10 μg) (lanes 1 and 2) were subjected to SDS-PAGE on a 17.5% gel, transferred onto a membrane, and immunoblotted with antibodies against Skp2, cyclin A, Cdk2, or FLAG tag to detect FLAG-p21, as indicated at the left. Positions of Skp2, cyclin A, Cdk2, FLAG-p21 (F-p21), and IgG chains are indicated on the right. The asterisk indicates the position of an extra cyclin A band (which can also be immunoprecipitated with Cdk2) seen after transfection with the p21-expressing construct. The positions of molecular size standards (in kilodaltons) are shown on the left. (B) HeLa cell extracts (120 μg) were immunoprecipitated with antiserum against Skp2 (lanes 2 to 4). Buffer (lane 2) or purified recombinant p21-H6 protein expressed in bacteria (1 μg [lane 3] or 5 μg [lane 4]) was incubated with the Skp2 immunoprecipitates at 30°C for 30 min. After washing, the immunoprecipitates were dissolved in SDS sample buffer and subjected to SDS-PAGE on a 17.5% gel. Total cell lysate (10 μg) was loaded in lane 1. The proteins were transferred onto a membrane and immunoblotted with a monoclonal antibody against PSTAIRE to detect Cdk2 (anti-PSTAIRE monoclonal antibody was used instead of anti-Cdk2 polyclonal antibody because it gave a cleaner background of the IgG bands) (top), anti-cyclin A monoclonal antibody E72 (middle), and anti-Skp2 antiserum (bottom).