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

Figure 1.

Figure 1

Figure 1

Figure 1

Figure 1

 PSM-RB arrests cell growth without inhibiting the phosphorylation of p107/p130. (A) Rat-1 cells were cotransfected with a GFP expression plasmid (1.0 μg) and the indicated plasmids (5.0 μg). BrdU was added 24 hr post-transfection for a total labeling period of 20 hr. Cells were stained with anti-BrdU antibody, and the percentage of GFP-positive cells exhibiting BrdU incorporation was determined by indirect immunofluorescence. The average and deviation values shown are from two independent experiments with at least 200 GFP-positive cells counted per experiment. (B) Rat-1 cells were cotransfected with a puromycin-resistance plasmid (0.5 μg) and the indicated expression plasmids (5.0 μg). Transfected cells were selected with puromycin for 7 days and then stained with crystal violet. Pictures were taken at 20× magnification with a phase-contrast microscope. (C) Rat-1 cells were cotransfected with a puromycin-resistance plasmid (0.5 μg) and vector (lane 1), WT-LP (lane 2), or PSM.7-LP (lane 3) expression plasmids (5.0 μg). Alternatively, PSM.7-LP (2.5 μg) and either vector (lane 4) or p16ink4a (lane 5) expression plasmids (2.5 μg) were cotransfected with a puromycin-resistance plasmid (0.5 μg). Transfected cells were selected with puromycin for 72 hr and then lysed. Total protein (15 μg) was resolved by SDS-PAGE and the transfected RB protein was detected by immunoblotting with anti-RB antibody. (ppLP) Hyperphosphorylated large-pocket fragment of RB; (pLP) hypophosphorylated large-pocket fragment. (D) 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) 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 p107 or p130 proteins were detected by immunoblotting with the respective antibodies as indicated. (E) Rat-1 cells were cotransfected with a puromycin-resistance plasmid (0.5 μg) and vector (lanes 1,4), PSM.7-LP (lanes 2,5), or p16ink4a (lanes 3,6) plasmids (5.0 μg). Transfected cells were selected with puromycin for 72 hr and then harvested. Total protein (15 μg) was resolved by SDS-PAGE, and the endogenous p107 proteins were detected by immunoblotting.