Figure 3.

pRB up-regulates the DNA binding and transrepression capabilities of p120^E4F. (A) p120^E4F is a site-dependent transcriptional repressor. NIH 3T3 cells are transiently cotransfected with an E4F-responsive reporter gene E4F-TK-Luc and increasing amounts of the pcDNA–p120^E4F plasmid. TK-Luc (without E4F site) was used as a control. Luciferase activity is shown as fold inhibition of the activity obtained with E4F-TK-Luc or TK-Luc constructs transfected alone. (B) pRB up-regulates the transrepression capabilities of p120^E4F. The maximum amount of p120^E4F plasmid that was unable to inhibit the reporter activity as determined in A is cotransfected with either E4F-TK-Luc or TK-Luc and with (dotted bars) or without (stripped bars) a phosphorylation-defective constitutively active pRB (p34RB). As a control, E4s-TK-Luc and TK-Luc reporters were also transfected alone (filled bars) or with p34RB (open bars). (C) pRB up-regulates the DNA binding activity of p120^E4F in vitro. EMSAs are performed with a probe containing an E4F DNA binding site. Purified GST–p120^E4F is added alone (lane 1) or together with increasing amounts of baculovirus-expressed purified pRB protein (lanes 2–4) or BSA (lane 5).