Fig. 3: TRIM52 is degraded by the ubiquitin-proteasome system.

a RKO cells expressing Ollas-tagged EGFP-TRIM52 were treated for 4 or 8 h. with the proteasome inhibitor epoxomicin (EPO), the lysosome inhibitor bafilomycin A, or the neddylation inhibitor MLN-4924. WCEs were analysed by WB. b Schematic representation of TRIM52 domain organization. c AlphaFold2 model for TRIM52 structure prediction. d RKO cells expressing Ollas-tagged EGFP-TRIM52 or mCherry-cMYC fusion proteins were treated for 5 h. with the proteasomal 20S catalytic core particle inhibitor EPO, or 19S regulatory particle-specific inhibitor capzimin. Ollas-EGFP-TRIM52 and mCherry-cMYC protein levels were determined by measuring EGFP or mCherry fluorescence using flow cytometry, and their mean fluorescence intensity (MFI) plotted. e RKO cells expressing MYC-tagged mCherry, or MYC-tagged mCherry-TRIM52 were treated with epoxomicin for 5 h., lysed under denaturing conditions, RFP-trap IPs performed, and analysed by WB for endogenous ubiquitin. f HEK-293T cells transfected with 3xHA-tagged TRIM52 or TRIM52-KtoR in which all lysines were mutated to arginines were treated with epoxomicin for 5 h., lysed under denaturing conditions, TRIM52 was immunoprecipitated, and its ubiquitination analysed by WB. g HEK-293T cells expressing MYC-tagged mCherry or mCherry-TRIM52, and His-tagged ubiquitin were treated with epoxomicin for 5 h., and dual-affinity purified by RFP-trap and NiNTA. Eluates were analysed by WB. h Ubiquitination sites identified by nLC-MS/MS plotted on schematic TRIM52 domain representation. i HEK-293T cells were transfected with 3xHA-tagged TRIM52 WT and TRIM52 lysine-to-arginine mutants, treated with EPO for 5 h., and lysates analysed by WB. Source data are provided as a Source Data file.