Fig. 3: USP36 stabilizes and deubiquitylates RBM28 at K162 residue.
A HCT 116 and RKO cells with knockdown of USP36 were treated with or without the proteasome inhibitor MG132 (10 µM, 6 h), and then proteins were analyzed. B Stability analysis of RBM28 protein in HCT 116-shUSP36 #1/3 cells, LoVo-USP36WT, LoVo-USP36C131A cells and treated with 10 µg/mL cycloheximide (CHX) for indicated times. The lower panels are quantification of RBM28 protein levels. C HCT 116 and RKO cells depleted with USP36 were treated with 10 μM of MG132 for 6 h and then harvested. RBM28 was immunoprecipitated with anti‐RBM28 and immunoblotted with anti‐HA. D LoVo cells transfected with the indicated plasmids were treated with 10 μM of MG132 for 6 h and subjected to the sequential IP and immunoblotting analysis. E Immunoblotting was used to detect the ubiquitination of RBM28 in HEK293T cells co‐transfected with Myc‐RBM28, HA‐Ubiquitin, and Flag‐USP36 (wild‐type or C131A). Cells were treated with 10 μM MG132 for 6 h before harvesting. F USP36 removed the ubiquitin chain of RBM28 in a time- and dose-dependent manner. G Immunoblotting to detect the ubiquitination of RBM28 mutants in HEK293T cells co‐transfected with Myc‐RBM28 mutants, Flag-USP36, and HA‐Ub. Cells were treated with 10 μM MG132 for 6 h before harvesting. H HEK293T cells were transfected with Myc-RBM28-WT or Myc-RBM28-K162R plasmids and treated with 10 µg/mL CHX for the indicated time and then were analyzed by western blot. Quantification of the expression levels of WT and K162R RBM28 was shown in the lower panel. All data are presented as the means ± SD of three independent experiments. ***p < 0.001.
