Fig. 3.

USP37 interacted with and enhanced NRF2 deubiquitination. a Co-transfection of plasmids expressing USP37-Flag and empty vector or NRF2-HA into 293 T cells. Treating the cells with MG132 (10 µM) for 6 h prior to harvesting. Cell lysate immunoprecipitation using the indicated antibodies. Analyzing the total cell lysates (TCL) and the immunoprecipitates by immunoblotting using anti-HA or Flag antibodies. b Consistent with (a), but in this case, the co-transfection of the plasmid expressing NRF2-HA was with an empty vector or USP37-Flag into 293 T cells. Treating the cells with MG132 (10 µM) for 6 h prior to harvesting. Immunoprecipitating the cell lysates employing the indicated antibodies while immunoblotting TCL and immunoprecipitates using anti-HA or Flag antibodies. c Similar to (A), but in this case, co-transfecting the plasmid expressing NRF2-HA into the 293 T cells was conducted with an empty vector, USP37-Flag, or the enzymatic domain (aa 341–979). The cell treatment with MG132 (10 µM) for 6 h. Harvesting the cells and subjected them to immunoprecipitation with the indicated antibodies. Analyzing the TCL and immunoprecipitates by immunoblotting using anti-HA or Flag antibodies. d Co-transfecting empty vector, plasmids expressing USP37-MYC, or USP37 enzymatic mutant C350S-MYC into 293 T cells with plasmids expressing NRF2-HA and Flag-ubiquitin (Flag-Ub). Treating the cells with MG132 (10 µM) for 6 h prior to harvesting. Cell lysate immunoprecipitation using anti-HA antibody. Immunoblotting the TCL and immunoprecipitates utilizing antibodies against Flag (Flag-Ub), HA (NRF2-HA), and Myc (USP37-Myc)