purified recombinant prmt1  (ProSpec)

Journal: PLoS ONE

Article Title: Methylation of the Tumor Suppressor Protein, BRCA1, Influences Its Transcriptional Cofactor Function

doi: 10.1371/journal.pone.0011379

Figure Lengend Snippet: ( a ) GST-BRCA1 constructs (0.5 µg) and core histones (1 µg) were incubated with purified recombinant PRMT1 enzyme (0.2 µg) in the presence of 0.55 µCi S Adenosyl- L-[ methyl -3H] methionine. GST was used as negative control and all GST-BRCA1 construct methylation levels normalized to background GST methylation levels. An average result of two independent experiments run in triplicates is shown. ( b ) The amino acid sequence of BRCA1 504–802 with arginine (R) residues within the 540–696 minimal region highlighted in blue and bolded, and the predicted methylated arginine residue highlighted in red, underlined and bolded. ( c ) Multiple sequence alignment generated by Clustal W of four non-human primates and two rodent BRCA1 full length sequences. Score represents pairwise alignment against full length human BRCA1. Amino acid residues are color-coded according to physical properties: basic (magenta), small hydrophobic (red) and hydroxyl + amine + basic (green). Consensus symbols for degree of conservation observed is represented by “*” (residues in column are identical in all seven sequences), “:” (conserved substitutions observed), and “.” (semi-conserved substitutions observed). ( d ) Two milligram of HeLa whole cell protein extract was incubated with 0.5 µg GST-BRCA1 constructs, beads were washed twice with TNE 150 + 0.1% NP-40 and once with TNE 50 + 0.1% NP-40, separated on a 4–20% gel by SDS-PAGE, and probed with an antibody against PRMT1. Input represents 1/10 of immunoprecipitated material. ( e ) Two milligram of HeLa whole cell protein extract were immunoprecipitated with anti-BRCA1 and anti-IgG antibodies, beads were washed twice with TNE 150 + 0.1% NP-40 and once with TNE 50 + 0.1% NP-40, separated on a 4–20% by SDS-PAGE, and probed with an anti-PRMT1 antibody. Input represents 1/10 of immunoprecipitated material. Results are representative of two independent experiments.

Article Snippet: Five hundred nanograms of GST-BRCA1 1–500, 452–1079, 504–802, 697–1276, 1021–1552, 1501–1861 and core histones were incubated with 0.2 μg of recombinant purified PRMT1 (Active Motif, Carlsbad, CA, USA) in the presence of 0.55 μCi S-Adenosyl-L-[ methyl - 3 H] methionine (GE Healthcare, Piscataway, NJ, USA) and reaction buffer (50 mM Tris-HCl pH 8.0, 20 mM KCl, 10 mM MgCl 2 , 250 mM sucrose, 10 μM β-mercaptoethanol) overnight at 37°C in a final reaction volume of 20 μl.

Techniques: Construct, Incubation, Purification, Recombinant, Negative Control, Methylation, Sequencing, Residue, Generated, SDS Page, Immunoprecipitation

Journal: PLoS ONE

Article Title: Methylation of the Tumor Suppressor Protein, BRCA1, Influences Its Transcriptional Cofactor Function

doi: 10.1371/journal.pone.0011379

Figure Lengend Snippet: ( a ) HeLa cells were transfected with different concentrations of PRMT1 siRNA (10, 25, 50 nM) following manufacturer's instructions. Results are representative of two independent experiments. ( b ) HeLa cells transfected with 50 nM Luc or PRMT1 siRNA were collected for ChIP analysis. Anti-BRCA1 (10 µg), anti-IgG (10 µg), and anti-histone H3-phosphorylated at S10 (H3-pS10, 5 µg) antibodies were used for ChIP analysis. PCR products were run on a 2% agarose gel and visualized with ethidium bromide staining. Results are representative of two independent experiments.

Article Snippet: Five hundred nanograms of GST-BRCA1 1–500, 452–1079, 504–802, 697–1276, 1021–1552, 1501–1861 and core histones were incubated with 0.2 μg of recombinant purified PRMT1 (Active Motif, Carlsbad, CA, USA) in the presence of 0.55 μCi S-Adenosyl-L-[ methyl - 3 H] methionine (GE Healthcare, Piscataway, NJ, USA) and reaction buffer (50 mM Tris-HCl pH 8.0, 20 mM KCl, 10 mM MgCl 2 , 250 mM sucrose, 10 μM β-mercaptoethanol) overnight at 37°C in a final reaction volume of 20 μl.

Techniques: Transfection, Agarose Gel Electrophoresis, Staining