Figure 3

Ras overexpression increases association of RbAp46 with HDAC1 at the SP1 site of RECK promoter in 7–4 cells. (A) The 7–4 cells were transiently transfected with plasmid DNA pcDNA-RbAp46 (0.2 μg) or RbAp46-specific siRNA (55.6 nM) in the presence of IPTG for 48 hr. After treatment, co-immunoprecipitation was conducted using anti-RbAp46, anti-HDAC1 antibodies, or anti-Sp1 antibody. (B) The cells were co-transfected with 0.2 μg of plasmid DNA of pGL3-RECK and pGL3-Sp1 mutant in the presence or absence of pcDNA-RbAp46 plasmid. RECK promoter activity was measured at 48 hr post-transfection. **: statistical significance at p < 0.001 by Student’s t test analysis. (C) The 7–4 cells were transfected with pcDNA-RbAp46 (0.2 μg) or RbAp46-specific siRNA (55.6 nM) and incubated with IPTG for 48 hr. DNA affinity precipitation assay was performed using streptavidin-coated beads to bind the biotinylated DNA probe, which was used to interact with nuclear extract proteins. (D) The 7–4 cells were incubated with IPTG for 24 hr. Chromatin was extracted, and then immunoprecipitated by Sp1, RbAp46, or HDAC1 antibody. After elution, DNA samples were analyzed using PCR for Sp1 binding sequence in the RECK promoter region. NC: negative control.