FIG 3.

PRMT4 methylates pRb R787 in vivo. (A and B) ELISA analysis (A) and dot blot analysis (B) of an affinity-purified polyclonal antibody raised against a 15-mer pRb R787-Me2 peptide on a nonmethylated peptide (non-Me) versus a pRb arginine-dimethylated peptide (Me). (C) In vitro methylation reaction mixtures consisting of Flag-tagged full-length pRb, PRMT4 (WT) or PRMT4 (EQ), and cold SAM were assembled. pRb methylation was detected using an anti-pRb R787-Me2 polyclonal antibody. Input pRB is shown by anti-Flag immunoblotting (IB). (D) MCF-7 cells were transfected with shRNA vectors targeting either GFP, as a negative control, or PRMT4, to achieve endogenous PRMT4 knockdown. Cell lysates were immunoprecipitated with an anti-pRb R787-Me2 polyclonal antibody and were immunoblotted with an anti-pRb antibody. As a control, 10% input is included.