Fig. 3: TRIM8 promotes K63-linked ubiquitination of PGK1 and its stability.

A Mass spectrometric analysis of TRIM8-binding proteins commonly related to metabolism in SGC7901 cells. B Endogenous co-immunoprecipitation of TRIM8 and PGK1 in SGC7901 cells and BGC823 cells. C SGC7901 cells were transfected with indicated plasmids for ubiquitination assay. D In screening for potential lysine ubiquitination types, the ubiquitination of HA-PGK1 in response to TRIM8 overexpression was examined in GC cells transfected with the wild-type (WT) and mutated His-Ub plasmids. K63R, Ub only Lys63 residue was mutated. K48R, Ub only Lys48 residue was mutated. E An immunoprecipitation assay was used to examine PGK1 ubiquitination in HEK293T cells that were transfected with HA-PGK1, His-Ub, Flag-TRIM8, and/or a Flag-TRIM8 mutant (C15A, C18A). F Representative WB analyses of PGK1 expression in shTRIM8 SGC7901 cells that were treated with the protein synthesis inhibitor cycloheximide (CHX; 50 µg/ml) for 8 h before extraction. G Real-time qPCR of PGK1 mRNA levels after silencing of TRIM8 in SGC7901 and BGC823 cell lines.