FIGURE 3.

Methylation of human RB by SMYD2 at lysine 860 in vivo. A, RBK860me1 antibody recognizes monomethyl Lys-860 RB. The specificity of polyclonal antibodies recognizing monomethylated Lys-860 in the RB protein (RBK860me1) was assessed by dot blot analysis with biotinylated peptides. A peptide from histone H4 (amino acids 1–20) serves as a negative control. Streptavidin was used as a positive control and a loading control. B, overexpressed SMYD2 monomethylates overexpressed RB at lysine 860 in 293T cells. Whole cell extracts from 293T cells transfected with the indicated plasmids were immunoprecipitated with HA resin and analyzed by immunoblot with RB, RBK860me1, and E2F antibodies. Controls include lysine to arginine substitutions in RB at Lys-860 and Lys-870, -873, -874 as well as a mutant form of SMYD2 (Y290F) with decreased methyltransferase activity. Inputs for HA-RB and MYC-SMYD2 are shown. C, overexpressed SMYD2 monomethylates endogenous RB at lysine 860. Whole cell extracts of 293T cells were immunoprecipitated with antibodies against RB or control IgG antibodies followed by immunoblot analysis with RB and RBK860me1 antibodies. Inputs for RB and MYC-SMYD2 are shown. D, methylation of endogenous RB at K860 in U2OS cells is decreased upon knockdown of SMYD2. shRNA molecules against SMYD2 (shSMYD2) were stably expressed from a lentiviral vector. Cells infected with an empty lentivirus serve as a control. Whole cell extracts were immunoprecipitated with antibodies against RBK860me1 or control IgG antibodies followed by immunoblot analysis with RB. Inputs for RB and actin are shown. The efficiency of the knockdown was measured by quantitative RT-PCR. One representative quantification of the knockdown is indicated.