Figure 3

Transcriptional repression by CtBP2 and mutants and competition between HDAC and ZEB for RR-CtBP2 binding. A, Repression of E-cad promoter by CtBP2 and mutants. CtBP1/2 double knock-out cell line MEF90 cells were co-transfected with pE-cad-Luc, phRL-tk (for internal control), and various CtBP2 constructs. Dual luciferase assay was performed as described [11]. Lysates were examined for CtBP expression by western blot with the CtBP2 antibody (lower panel). B, Interaction of wt CtBP2 and RR-CtBP2 with endogenous ZEB and HDAC2 in the presence of GFP-ZEB101. HeLa cells were co-transfected with CtBP2 and GFP-ZEB101. Cell lysates were immunoprecipitated with the Flag antibody beads and precipitated proteins examined by western blots as indicated. Both CtBP proteins and GFP fusion proteins carry an HA tag and are recognized by the HA antibody. C, Competition between GFP-ZEB101 and HDAC2 for binding to RR-CtBP2. HeLa cells were co-transfected with CtBP2 and increasing amounts of GFP-ZEB101. Co-IP and western blot analyses were performed as in B.