Effect of Rap on the expression of cdks inhibitors (A), the formation of the G1 cyclin/cdks-p27^Kip1 complex (B) and [³⁵S] methionine-labeled p27^Kip1 (C and D) in ED-40515(−) cells. Exponentially growing ED-40515(−) cells incubated with or without Rap were harvested 0, 24, 28, or 48 hours after the initiation of the culture. (A) Upper: the expression of p21^Waf1 and p27^Kip1 at mRNA level. A total of 10 μg of total RNA was fractionated by electrophoresis and subjected to Northern blotting with ³²P labeled p21^Waf1or p27^Kip1 probes. The same filter membrane was used for p21^Waf1 and p27^Kip1 mRNA hybridization. Bottom: the expression of p21^Waf1 and p27^Kip1 at protein level. The cell lysates containing 40 μg of the protein were subjected to immunoblotting with anti-p27^Kip1 antibody and anti-p21^Waf1 antibody. (B) Upper: immunoblotting of cyclin A and cyclin E. The cell lysate containing 40 μg of protein from indicated samples was separated with 10% gel, followed by immunoblotting with anticyclin A or E antibody. Bottom: the immunoprecipitation (IP)-immunoblotting of p27^Kip1. The cell lysates prepared from 5 × 10⁶ cells of the indicated samples were immunoprecipitated with anti-cyclin E antibody, anti-cdk4 antibody, or anti-cyclin D₃ antibody followed by SDS-PAGE separation with 12.5% gel and immunoblotting with anti-p27^Kip1 antibody. (C) The de novo synthesis rate of p27^Kip1 evaluated by [³⁵S]methionine metabolic labeling. ED-40515(−) cells cultured in RPMI 1640 plus 10% FCS were incubated with or without 10 nmol/L Rap for 24 or 48 hours. The cells were harvested at the indicated points and labeled with 150 μCi/mL of [³⁵S]methionine for 45 minutes. The cells were harvested again for lysis in hypotonic buffer. The cell lysates were immunoprecipitated with anti-p27^Kip1 antibody. The radioactive signals associated with the p27^Kip1 bands, which were measured by a phosphorimager, were plotted as the relative signal intensity. (D) The pulse chase study of [³⁵S]methionine-labeled p27^Kip1. ED-40515g(−) cells cultured with or without 10 nmol/L Rap for 28 hours were harvested and labeled with [³⁵S]methionine by the same method as in (C). The cells were placed in full medium to chase the metabolic labeling with or without 10 nmol/L Rap and harvested at the indicated points (0, 3, 6, and 9 hours after [³⁵S]methionine labeling) for lysis in hypotonic buffer. The radioactive signals associated with the p27^Kip1 bands, which were measured by a phosphorimager, were plotted as the percent of the signal at the start of the chase of Rap-treated cells.