Fig. 4.

RNF4 is methylated at R164 by PRMT5. A HA-tagged RNF4 was immunoprecipitated with the HA antibody from transfected HEK293T cells and methylation of RNF4 was detected using anti-SDMe-Arginine antibodies. B Mass spectrometric (MS) analysis of RNF4 methylation sites. HEK293T cells were transfected with HA-RNF4. HA-RNF4 was immunopurified with anti-HA affinity beads followed by MS. C Immunoblotting of the methylation of RNF4 R164. HEK293T cells were transfected with the indicated plasmids for 48 h. D Amino acid sequences from the indicated species were aligned using the ESPript 3.0 website. Conserved sequences are highlighted. E Assay for in vitro methylation of RNF4 by PRMT5 using purified proteins. Left panel, Coomassie blue staining of GST-fused RNF4 WT or the RNF4 R164K mutant purified from E. coli. Middle panel, schematic representation of the in vitro methylation assay. Right panel, purified Myc-PRMT5 proteins were incubated with purified GST-RNF4 WT or GST-RNF4 R164K mutant proteins. The reaction mixtures were separated by SDS-PAGE and immunoblotted using the indicated antibodies. F Diagram of the in vitro ubiquitination assay used for G. G HEK293T cells were transfected with Flag-PML-RARα and His-SUMO2. Flag-tagged PML-RARα was purified from transfected HEK293T cells by immunoprecipitation using anti-Flag antibodies, after which His-SUMO2-Flag-PML-RARα was purified by Ni²⁺ pulldown and incubated with purified GST-RNF4 WT or GST-RNF4 R164K mutant proteins at 37 °C for 4 h. The effect of R164 methylation on its E3 enzyme activity was analyzed by measuring the ubiquitination level of PML-RARα