Fig. 2
From: Multiple molecular interactions redundantly contribute to RB-mediated cell cycle control
Multiple point mutations are needed to overcome RB mediated cell cycle arrest. a GST-tagged RB large pocket proteins corresponding to the RBG, RBS, RBL, and RBGSL mutant versions of pRB were produced and purified. These GST-fusions were incubated with C33A extracts transfected with the indicated expression constructs. Bound proteins were isolated by precipitation and identified by western blotting. b Constructs containing full-length RB harboring the indicated mutations under the control of a CMV promoter were transfected into SAOS2 cells along with a CD20 reporter. Cells were then stained with propidium iodide and the percentage of cells in G1 were determined by DNA content of CD20 positive cells. Bars indicate the mean of three separate experiments, and error bars indicate one standard deviation from the mean. Letters indicate groups that are significantly different from one another (ANOVA, Tukey test, p < 0.05). c Full length CMV-RB constructs were transfected into C33A cells and extracts were incubated with recombinant HPV-E7. Bound proteins were isolated by precipitation and western blotted to detect pRB. d Full length RBWT and RBGSC were transfected into C33A cells prior to cycloheximide treatment (CHX). Extracts were prepared over a 15 h time course and stability was monitored by Western blotting. e Constructs containing full-length RB harboring the various mutations, or combinations of mutations, under the control of a CMV promoter were transfected into SAOS2 cells along with a CD20 reporter. Cells were then stained with propidium iodide and the percentage of cells in G1 were determined by DNA content of CD20 positive cells. Bars indicate the mean of three separate experiments, and error bars indicate one standard deviation from the mean. f Transfections and cell cycle analysis were performed as in b and e, except the increase in G1 cells is shown as Change in % G1 (relative to β-Gal control). Letters indicate groups that are significantly different from one another (ANOVA, Tukey test, p < 0.05)