Fig. 4

PRMT-1 binds to and methylates PFV Gag in a manner that depends on R540. a Following lysis, cells expressing PFV Gag WT or R450A mutant and GFP-PRMT1 were incubated with protein A beads coated with either an anti-GFP (cat.11 814 460 001, Roche, 1:100) or an anti-ADMA (ab5394 (7E6), Abcam,1:100) antibody. Input and immunoprecipitated proteins were separated by SDS-PAGE and visualized by Western blotting with anti-GFP (cat.11 814 460 001, Roche, 1:1000) or rabbit polyclonal anti-PFV antibodies. b Lysates from cells expressing HA-tagged GRs or GRs-R540A were immunoprecipitated with an antibody directed against PRMT1 (Cat A300-722A, Bethyl Laboratories (Euromedex), 1:100), the ADMA modification (ab5394 (7E6), Abcam, 1:100), or the HA epitope (H11, Covance, 1:100). Input and bound proteins were analyzed as in A. c HeLa cells were transfected with siRNA targeting PRMT1 or scrambled control (scr) and, two days later, with GFP-GRs expression plasmid. After 24 h, cells were processed as indicated in Fig. 1b. Images are representative of two independent experiments. The numbers indicate the percentage of GFP-positive cells with significant nucleolar accumulation of 100 counted cells. Scale bar represents 10 µm