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. 2023 Oct 11;10(33):2303561. doi: 10.1002/advs.202303561

Figure 4.

Figure 4

Methylation of Rbfox2 at R341 and R441 by PRMT5 is a prerequisite for its ubiquitination by FBXO7. A) GSC1023 and GSC0910 cell lysates were immunoprecipitated using an antibody against Rbfox2 or PRMT5, and then were subjected to immunoblotting analysis using an anti‐PRMT5 or Rbfox2 antibody, respectively. B) 293T cells were transfected with HA‐PRMT5 and Flag‐tagged wild‐type or different truncation mutants of Rbfox2. Cell lysates were immunoprecipitated with an anti‐Flag antibody, and then analyzed by immunoblotting. C) GSC1023 cell extracts were immunoprecipitated using an anti‐Rbfox2 antibody (up panel) or anti‐dimethyl‐arginine antibody, symmetric (anti‐SYM10), and the resultant precipitates were analyzed by immunoblotting using the anti‐SYM10 or Rbfox2 antibody, respectively. D) 293T cells were transfected with Flag‐Rbfox2 and HA‐PRMT5 plasmids. Cell lysates were immunoprecipitated with an anti‐Flag antibody and then were subjected to immunoblotting using an anti‐SYM10 antibody. E) GSC1023 cells expressing control shRNA or PRMT5 shRNAs were treated with MG132 for 6 h before cell harvest. Cell lysates were immunoprecipitated using an anti‐Rbfox2 antibody, and then analyzed by immunoblotting using the indicated antibodies. F) 293T cells were transfected with HA‐PRMT5 and Flag‐Rbfox2‐WT, Flag‐Rbfox2‐R341A, Flag‐Rbfox2‐R441A, or Flag‐Rbfox2‐R341A/R441A plasmids. Cell lysates were incubated with an anti‐Flag antibody and then were analyzed by immunoblotting. G) GST‐Rbfox2‐WT or Rbfox2‐R341A/R441A proteins were incubated with purified PRMT5 protein in the reaction buffer, and in vitro methylation of Rbfox2 was detected using an anti‐SYM10 antibody. H) 293T cells were transfected with Myc‐FBXO7, Flag‐Rbfox2, and HA‐PRMT5 plasmids. Cell lysates were immunoprecipitated using an anti‐Flag antibody and then analyzed by immunoblotting using the indicated antibodies. I) GSC1023 cells were transfected with PRMT5 siRNAs and then were treated with MG132 for 6 h before harvest. The cell lysates were immunoprecipitated using an anti‐FBXO7 antibody, and the resultant precipitates were analyzed by immunoblotting. J) 293T cells were transfected with Myc‐FBXO7 and Flag‐Rbfox2‐WT or Flag‐Rbfox2‐R341A/R441A. Cell lysates were immunoprecipitated using an anti‐Myc antibody and then analyzed by immunoblotting. K) 293T cells were transfected with Myc‐FBXO7, HA‐PRMT5, Flag‐Rbfox2, and HA‐Ubi‐K63. Cell lysates were immunoprecipitated using an anti‐Flag antibody and then analyzed by immunoblotting using the indicated antibodies. L) GSC1023 cells were transfected with PRMT5 siRNAs and HA‐Ubi‐K63, and then treated with MG132 for 6 h. Cell lysates were immunoprecipitated using an anti‐Rbfox2 antibody, and then analyzed by immunoblotting. M) 293T cells were transfected with Myc‐FBXO7, HA‐PRMT5, HA‐Ubi‐K63, and Flag‐Rbfox2‐wt or Flag‐Rbfox2‐R341A/R441A. Cell lysates were immunoprecipitated using an anti‐Flag antibody and then analyzed by immunoblotting.