Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Methylation of H4R3 by PRMT1 and PRMT5.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Effects of acetylation on Arg-3 methylation catalyzed by PRMT5. Reaction buffer contained 50 mm Hepes, pH 8.0, 10 mm NaCl, and 1 mm DTT. The concentrations of PRMT5, [14C]AdoMet, and H4 peptide were 0.1, 30, and 200 μm respectively. The reaction time was 1 h.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Kinetic parameters of PRMT5 catalysis The catalytic activity of PRMT5 on each peptide was tested with the radioactive filter binding assay. Varied concentrations of peptide (0–10 μ m ) and 30 μ m of [ 14 C]AdoMet were incubated at 30 °C for 5 min in the reaction buffer (50 m m HEPES (pH 8.0), 50 m m NaCl, 1 m m EDTA and 0.5 m m DTT) prior to the addition of PRMT5. The methylated products were loaded onto P81 filter paper and quantified by liquid scintillation. Calculated methylation rate was plotted as a function of peptide concentration, and the data were fitted with Michaelis-Menten equation.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Activity Assay, Filter-binding Assay, Incubation, Methylation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Acetylation on H4 protein inhibits its methylation by PRMT1, although it promotes its methylation by PRMT5. A, p300 acetylation inhibits H4 methylation by PRMT1. H4 protein was incubated with acetyl-CoA in the absence or presence of p300 before submission to PRMT1 methylation with [14C]AdoMet. Methylated H4 bands were separated by 15% SDS-PAGE and visualized by storage phosphor scan, which is the same method as for B–D. B, p300 acetylation promotes H4 methylation by PRMT5. H4 protein was incubated with acetyl-CoA in the absence or presence of p300 before submission to PRMT5 methylation. C, MOF acetylation inhibits H4 methylation by PRMT1. H4 protein was incubated with acetyl-CoA in the absence or presence of MOF before submission to PRMT1 methylation. D, MOF acetylation promotes H4 methylation by PRMT5. H4 protein was incubated with acetyl-CoA in the absence or presence of MOF before submission to PRMT5 methylation.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Methylation, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Summary of the effects of lysine acetylation on Arg-3 methylation in H4. Acetylation of the N-terminal H4 tail reciprocally affects PRMT1-mediated Arg-3 methylation and PRMT5-mediated Arg-3 methylation. A solid line means that the effect of acetylation is appreciably strong, and a dotted line means that the effect of acetylation is relatively weak.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Methylation