Fig. 1: PRMT1 interacts with and methylates USP11.
a Flag-tagged USP11 co-immunoprecipitates endogenous PRMT1 in MCF7 cells. Representative image from three independent repeats. b Recombinant USP11 is methylated by recombinant PRMT1 as determined by in vitro methylation assay. Methylation of histone H4 serves as a positive control. Representative image from three independent repeats. c Recombinant PRMT1 methylates endogenous USP11 isolated from 293T cells as determined by in vitro methylation assay. Incubation with 20 µM AdOx for 24 h generates hypomethylated substrate appropriate for enzymatic modification (compare lanes 4 and 5). Representative image from three independent repeats. d USP11 is methylated in vivo. 293T cells were transfected with the indicated plasmids and de novo protein synthesis inhibited by CAM/CHX treatment. Following [3H]-methyl-methionine labelling, Flag-USP11 was immunoprecipitated and incorporated methyl groups detected by SDS-PAGE and autoradiography. Flag-GFP serves as a control for protein synthesis inhibition. The graph represents quantification of methyl incorporation adjusted for the amount of protein immunoprecipitation (mean ± SD; n = 3 biologically independent experiments). e In vivo endogenous USP11 methylation is PRMT1-dependent. MCF7 cells stably expressing shPRMT1 were treated with CAM/CHX, labelled with [3H]-methyl-methionine, followed by endogenous USP11 immunoprecipitation. The incorporation of methyl groups was determined by SDS-PAGE and autoradiography. Asterisk represents non-specific protein immunoprecipitating with IgG control. Graph represents quantification of methyl-incorporation adjusted for USP11 immunoprecipitation and normalised to MCF7-shCTRL (mean ± SD; n = 3 biological independent experiments; **p = 0.00108 (Student’s t test, two-sided, equal variance). Uncropped blots and processed graphical data are provided as a Source Data file.
