Fig. 4. Effect of hCAF1 and BTG1 on PRMT1 enzymatic activity in vitro. GST-PRMT1 (1 µg) was incubated with 1 µg of (A) GST-Sam68P3, (B) GST-hnRNPA1 or (C) recombinant histone H4 in the presence of [³H]AdoMet without or with increasing amounts of GST-hCAF1, GST-BTG1 or GST for 90 minutes at 37°C. The reaction mixtures were resolved on SDS-PAGE and visualized by autoradiography. (D) GST-PRMT1 (1 µg) was incubated with 1 µg of recombinant histone H4 in the presence of cold AdoMet without or with increasing amounts of GST-hCAF1, GST-BTG1 or GST for 90 minutes at 37°C. The reaction mixtures were resolved on SDS-PAGE and revealed by western blot using the anti-dimethyl-Histone H4 (Arg3) antibody. Results are representative of several individual experiments. (E) GST-PRMT1 (1 µg) was incubated with [³H]AdoMet, 20 µg of hypomethylated extracts prepared from MCF-7 cells without or with increasing amounts of GST-hCAF1 or GST as described above. The reaction mixtures were resolved on SDS-PAGE and visualized by autoradiography. A longer exposition of the gel is shown in the right-hand panel.