Fig. 4: TRIM65 promoted K48-linked ubiquitination of BTG3 and its degradation.

A Overexpression of TRIM65 suppressed the protein levels of BTG3 in a dose-dependent manner. B MG132 can restore the protein decline of BTG3 induced by TRIM65. C Overexpression of TRIM65 but not TRIM65 mutant accelerated the degradation of BTG3 protein. HEK293T cells expressing EV or TRIM65 or TRIM65 mutant were treated with cycloheximide (CHX) for indicated times. Data was shown as mean ± SEM (n = 3). **p < 0.01, ***p < 0.001. D HEK293T cells were co-transfected with BTG3 plus HA-Ub mutants as indicated (e.g. K6O means K6 only) and/or GFP-TRIM65. E HEK293T cells were co-transfected with BTG3 plus HA-ubiquitin (HA-Ub) and/or FLAG-TRIM65. F BTG3 and FLAG-TRIM65 were co-transfected with HA-Ub or HA-Ub K48R into HEK293T cells. G HEK293T cells were co-transfected with BTG3 and EV, FLAG-TRIM65 (WT) or FLAG-TRIM65 mutant (Mutant). H HEK293T cells were co-transfected with HA-Ub and GFP-TRIM65 plus BTG3 (WT), BTG3 K41R, or BTG3-K192R. Cell lysates were harvested and immunoprecipitated with anti-BTG3 (anti-Myc) antibody, and were also detected using the indicated antibodies by western blotting (D–H). I Ectopic transfection of TRIM65 can only modulate the protein levels of BTG3 WT, but not BTG3 K41R. J Upregulation or knockdown of TRIM65 cannot affect the ubiquitination levels of BTG3 K41R.