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. 2010 Sep 24;285(48):37733–37740. doi: 10.1074/jbc.M110.137612

FIGURE 6.

FIGURE 6.

RB methylation at Lys-860 increases its interaction with the MBT domains of L3MBTL1 in vivo. A, monomethyl Lys-860 RB can be localized to chromatin. 293T cells were fractionated by detergent lysis to generate a cytoplasmic S2 fraction, a nuclear S3 fraction, and a chromatin (Chr) fraction. Fractions were then analyzed by immunoblot with antibodies against RB and RBK860me1. Tubulin and H3K4tri-me were used to assess the purity of the fractionation. B, overexpression of SMYD2 increases binding of RB to L3MBTL1. 293T cells were transfected with the indicated expression plasmids. Whole cell extracts were immunoprecipitated with FLAG resin and analyzed by immunoblot with antibodies against RB, RBK860me1, and L3MBTL1. Inputs for FLAG-L3MBTL1, MYC-SMYD2, and RB are shown. C, D355N mutation in the methyl-binding pocket of L3MBTL1 reduces the affinity of L3MBTL1 for RB. 293T cells were transfected with the indicated expression plasmids. IP was performed as in B and analyzed by immunoblot with antibodies against RB, RBK860me1, and L3MBTL1. Inputs for FLAG-L3MBTL1, FLAG-L3MBTL1(D355N), RB, and MYC-SMYD2 are shown. D, RB(K860R) mutation reduces the affinity of RB for L3MBTL1. 293T cells were transfected with the indicated expression plasmids. Immunoprecipitation was performed as in B and C and analyzed by immunoblot with antibodies against HA, RBK860me1, and L3MBTL1. Inputs for FLAG-L3MBTL1, HA-RB, HA-860, and MYC-SMYD2 are shown. Tubulin serves as a loading control.