FIGURE 4.

Pin1 enhances LSF transactivation potential by facilitating dephosphorylation of Ser-291 and Ser-309. A, NIH 3T3 cells were infected with either a retrovirus overexpressing FLAG-Pin1 (black bars) or the parental retrovirus as control (gray bars). These cells were cotransfected with either pCMV-QZ (−), pCMV-LSF (WT), or pCMV driving expression of the indicated alanine substitution mutants of LSF, the reporter plasmid pGL3B-WT4E1b and phRL-TK. Expression from the reporter construct in the presence of each mutant is normalized to the degree of activation by wild-type LSF in the absence of exogenous Pin1 expression. The results are a compilation of three independent experiments. Error bars represent S.E. Numbers above the bars represent the fold increase in transactivation by Pin1. Asterisks represent p < 0.05, as determined by a pair-wise t test. B, extracts prepared in passive lysis buffer from a representative experiment described in A were analyzed by immunoblotting with anti-LSF-314, anti-S291p, and anti-S309p, as indicated, following SDS-PAGE through a 7.5% gel.