Effects of GATA-2 on the Ub/proteasome-dependent degradation of p21^WAF1 and p27^Kip1 in FDC-P1/GATA-2/ER and Ba/F3/GATA-2/ER.

(A) To examine the half-lives of p21^WAF1 and p27^Kip1 proteins, cells (1 × 10⁷ cells for each sample) were radiolabeled with [³⁵S]methionine for 30 minutes. Then, the labeled cells were washed, resuspended in DMEM containing 2 mM unlabeled methionine, and cultured with or without 2 μM estradiol for the times indicated. p21^WAF1 and p27^Kip1 were immunoprecipitated from total cellular lysates and subjected to SDS-PAGE. The gels were dried and subjected to the autoradiography. (B) The cells were cultured in the presence or absence of 1 μM estradiol and/or 0.1 μM lactacystin for 24 hours. The protein expression levels of p21^WAF1 and p27^Kip1 were examined by Western blot analysis on total cellular lysates. (C) The cells were cultured with 1 μM estradiol for the times indicated and subjected to Northern blot analysis on the expressions of Skp1, Skp2, and Cul1. The results show representative data from 3 independent experiments.