Figure 5.

Release of MBD2 protein from the 14-3-3σ promoter after reactivation in non-14-3-3σ-expressing prostate cancer cells.

1. ChIP assay of MBD2. The formaldehyde crosslinked chromatin was immunoprecipitated with antibodies specific for MBD2. Purified DNA was amplified for the 14-3-3σ promoter as described in Fig. 4. MBD2 dissociate from the 14-3-3σ promoter-associated CpG island following treatment of LNCaP and Tramp-C1 cells with 5-aza and TSA, as described in Materials and Methods. Controls show input genomic DNA before the addition of antibody and eluants from no antibody immunoprecipitations.
2. Quantification of the MBD2 ChIP fractions on the 14-3-3σ promoter as described in Fig. 4. Each bar represents triplicate analyses of mean ± SD where significant difference from cells treated with vehicle alone (control) is represented by an asterisk * (P <0.05).
3. MBD2 protein expression in total cell lysates from 5-aza and TSA-treated and untreated PC3, DU145, LNCaP and Tramp-C1 cells. Equal amounts (100 μg) of protein from control or treated cells were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblot analysis with antibodies specific for MBD2 and GAPDH. GAPDH was utilized as a loading control.
4. Bar diagram showing densitometry quantified data of MBD2/GAPDH protein expression ratios from three independent experiments. Each bar represents triplicate analyses of mean ± SD.