FIG 5.

pRb arginine methylation decreases E2F-1 binding in vivo and in vitro. (A) Immunoblot (IB) analysis was performed on total-cell lysates from isogenic U2OS cells expressing Flag-pRb (WT) or Flag-pRb (R3K) by using the indicated pRb phosphorylation-specific antibodies. (B) U2OS control cells or isogenic cell lines expressing either Flag-pRb (WT) or Flag-pRb (R3F) were lysed in phospholysis buffer; whole-cell lysates were immunoprecipitated (IP) with anti-Flag–M2 agarose beads overnight; and immunoprecipitates were probed for interactions with an anti-E2F-1 antibody. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) The U2OS cell line stably expressing pRb (WT) was transfected with an shControl or shPRMT4 vector. Forty-eight hours later, whole-cell lysates (WCL) were immunoprecipitated with anti-Flag–M2 agarose beads overnight, and immunoprecipitates were probed for interactions with an anti-E2F-1 antibody. (D) (a) CBB staining of baculovirus-derived Flag-tagged E2F-1/DP1 used for in vitro GST pulldown assays. (b) GST pulldowns were performed by incubation of baculovirus-derived Flag-tagged E2F-1/DP1 coiled-coil and marked box domains (labeled CM) with the immobilized wild-type GST-pRb (700–850) deletion protein or the GST-pRb (700–850) deletion protein harboring either single or combinatorial phenylalanine substitutions. Proteins were probed for interactions by immunoblot analysis using an anti-Flag antibody.