FIG. 5.
Conditional activation of FKHR-L1 causes growth arrest and increased p130 protein levels in human colon carcinoma cells. (A) DL23 and DLD-1 cells were left untreated (0 h) or treated with 500 nM 4OHT for 8, 12, 16, or 24 h. Total cellular lysates were electrophoresed and blotted for the presence of p27kip1, pRb, HA-FKHR-L1.A3-ER,or actin. Cell lysates were analyzed for cdk2 kinase activity by cdk2 immunoprecipitation (IP) and in vitro kinase reaction using histone H1 as a substrate. The cdk2 kinase reaction products were blotted, autoradiographed, and probed for the presence of p27kip1. WB, Western blot. (B) BrdU incorporation of DL23 and DLD-1 cells left untreated (Con) or treated with 500 nM 4OHT for 16 h. (C) Total cellular lysates from DLD-1 and DL23 cells treated without (0 h) or with 500 nM 4OHT for 4, 8, 12, 16, or 24 h were analyzed by Western blotting for the presence of the p130 retinoblastoma-like protein. Anti-HA immunoprecipitations of A14 cells transiently transfected with empty vector or pCMV-HA-p130 served as controls. (D) DL23 and DLD-1 cells, left untreated (0 h) or treated with 4OHT for 24 or 48 h, were lysed, and cdk2 was immunoprecipitated. The cdk2 immunoprecipitates were subsequently analyzed for the presence of p130 by Western blotting. WCL, whole-cell lysate; IP-Ab, immunoprecipitating antibody.