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. 1998 Aug 1;12(15):2278–2292. doi: 10.1101/gad.12.15.2278

Figure 2.

Figure 2

Figure 2

Figure 2

Figure 2

 PSM-RB inhibits cyclin A expression in Rat-1 cells. (A) Rat-1 cells were cotransfected with a puromycin-resistance plasmid (0.5 μg) and vector (lane 3), WT-LP (lane 4), or PSM.7-LP (lane 5) expression plasmids (5.0 μg). Transfected cells were selected with puromycin for 72 hr and then harvested. As controls, quiescent (lane 1) or asynchronously growing (lane 2) Rat-1 cells were also harvested. Total protein (15 μg) was resolved by SDS-PAGE, and the endogenous cyclin D1, cyclin E, cyclin A, and cyclin B proteins were detected by immunoblotting with the respective antibodies. (B) Rat-1 cells were cotransfected with a puromycinresistance plasmid and the indicated expression plasmids, selected, and harvested as in A. Total protein (15 μg) was resolved by SDS-PAGE, and the endogenous cdk2, cdc2, p21cip1, and p27kip1 proteins were detected by immunoblotting with the respective antibodies. (C) Rat-1 cells were cotransfected with a puromycin-resistance plasmid and the indicated expression plasmids, selected, and harvested as in A. Twenty micrograms of total protein was utilized in in vitro kinase reactions with histone H1 as a substrate. Cdk/cyclin complexes were recovered by immunoprecipitation with the indicated antibodies against cyclin E, cyclin A, cdk2, or cdc2 and protein A–Sepharose. A nonspecific rabbit anti-mouse antibody was utilized as a negative control. Incorporation of 32P into histone H1 was visualized by autoradiography. (D) Rat-1 cells were cotransfected with a puromycin-resistance plasmid and the indicated expression plasmids, selected, and harvested as in A. Quiescent and asynchronous growing Rat-1 cells were used as controls. Total RNA was prepared. Quantitative RT-PCR was performed with 1–16 ng of total RNA as template. The level of cyclin A PCR product was normalized to an internal control PCR with primers that amplify the sequence of GAPDH (see Materials and Methods). Relative cyclin A mRNA level from five independent PCR reactions is shown, with the level found in asynchronous growing cells set to 100%.