Journal: Molecular and Cellular Biology

Article Title: MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties

doi: 10.1128/mcb.26.3.843-851.2006

Figure Lengend Snippet: FIG. 3. PRMT5 interacts with and methylates the N-terminal RG-rich repeat of MBD2. (A) Purified TAP-MBD2 and TAP-MBD3 complexes were analyzed by Western blotting using a PRMT5 antibody. (B) Sequence of MBD2 with the RG repeats being underlined. (C) In vitro methylation of purified MBD2 complex upon incubation with S-[14C]adenosylmethionine. Methylated protein is indicated with an arrow. Free label is indicated with an asterisk. MBD2 complex was purified using IgG beads. The left panel depicts a Western blot analysis of the purified MBD2 complex or a 293 control purification using anti-ProtA-horseradish peroxidase antibody. The arrow shows TAP-tagged MBD2. (D) (Left panels) In vitro methylation of recombinant GST-RG(n) and GST-MBD2 in the presence of S-[14C]adenosylmethionine and a purified PRMT5/MEP50 fraction (middle panel) or the purified MBD2 complex (top panel). GST-PAH2 was used as a negative control. (Right panels) In vitro methylation of recombinant GST-RG(n), GST-MBD2 lacking the RG stretch, and GST-MBD3 in the presence of S-[14C]adenosylmethionine and a purified PRMT5/MEP50 fraction (middle panel) or the purified MBD2 complex (top panel). Free label is indicated with an asterisk. Loading controls for GST-PAH2, GST-RG(n), GST-MBD2, GST MBD2 lacking the RG stretch, and GST-MBD3 are shown in the bottom panels. (E) Silver-stained gel of purified N-terminally truncated MBD2 complex from HEK 293 cells. The table shows the FT-MS/MS analysis with all identified proteins and their respective peptide numbers and percent sequence coverage.

Article Snippet: Four micrograms of the following antibodies was used for immunoprecipitations: PRMT5 12-303 (Upstate Biotechnology), MBD2 IMG-147 (Imgenex), MBD3 (IBL, Japan), MTA2 PC656 (Oncogene), and anti-dimethyl-histone H4 (Arg3) (07-213) (Upstate Biotechnology).

Techniques: Western Blot, Sequencing, In Vitro, Methylation, Incubation, Control, Recombinant, Negative Control, Staining, Tandem Mass Spectroscopy

Journal: Molecular and Cellular Biology

Article Title: MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties

doi: 10.1128/mcb.26.3.843-851.2006

Figure Lengend Snippet: FIG. 4. MBD2 recruits PRMT5 to chromatin. (A) Schematic representation of the primer sets used in the ChIP experiments. Exons are indicated with black rectangles. CpG islands are indicated in gray. Primer pairs are indicated with arrows. (B) ChIP analysis of MBD2, MBD3, PRMT5, and MTA2 in MCF7 cells. Relative occupancy over a control BMX region is shown. Values are the means with standard deviations of the results from ChIP experiments from three independent chromatin isolations. (C) ChIP analysis of MCF7 cells treated with 5-azacytidine. Immunoprecipitations were performed with antibodies against MBD2, MBD3, MTA2, PRMT5, and anti-dimethyl-histone H4 (Arg3). Relative occupancy over a control BMX region is shown. Values are the means with standard deviations of the results from ChIP experiments from three independent chromatin isolations.

Article Snippet: Four micrograms of the following antibodies was used for immunoprecipitations: PRMT5 12-303 (Upstate Biotechnology), MBD2 IMG-147 (Imgenex), MBD3 (IBL, Japan), MTA2 PC656 (Oncogene), and anti-dimethyl-histone H4 (Arg3) (07-213) (Upstate Biotechnology).

Techniques: Control