Skip to main content
. 2007 Apr 23;27(13):4938–4952. doi: 10.1128/MCB.01880-06

FIG. 6.

FIG. 6.

MBD3 overexpression in HEK 293 cells results in decreased methylation of the rRNA promoter. HEK 293 cells were transfected with empty vector pEF6 (control) or MBD3-pEF6 for 72 h. (A) Western blot analysis of the endogenous MBD3 (34 kDa) and exogenous MBD3 with an X-press epitope (40 kDa) visualized with an anti-MBD3 antibody (upper panel). The membrane was reprobed for β-actin as a control for loading (lower panel). (B) Bisulfite analysis of the rRNA promoter from control or MBD3 transfectants. Each line represents an independent clone. Filled circles, methylated CG dinucleotides; empty circles, unmethylated CG dinucleotides. (C) Quantification of the individual methylated CGs in the rRNA promoter from control or MBD3 transfectants. (D) Chromatin immunoprecipitation assays of the association between MBD3 or Pol I binding to the rRNA promoter. Results are from quantitative real-time PCR of the rRNA promoter amplified from MBD3- or RPA-116-immunoprecipitated HEK 293 extracts, normalized to the nonimmunoprecipitated extracts (input), from three independent experiments. One-tenth of the input sample was used for PCR amplification. Student's t test was used for analysis of MBD3 versus control (*, P < 0.05). (E) Quantitative RT-PCR of pre-rRNA sequences normalized to GAPDH mRNA levels from three independent experiments.