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. 2021 Jul 9;7(28):eabg4574. doi: 10.1126/sciadv.abg4574

Fig. 6. TRIM63 E3 ligase and UCHL1 antagonistically regulates PKM ubiquitination and protein stability.

Fig. 6

(A) Left: Immunoblot analysis of PKM ubiquitination in HEK293 cells coexpressing TRIM63. The cells were cotransfected with the plasmids carrying Myc-tagged PKM, HA-tagged ubiquitin, and Flag-tagged TRIM63. Right: Relative quantification of the anti-HA immunoblot band intensity of the immunoprecipitated proteins by Myc antibody. n = 3. (B) Left: Immunoblot analysis of PKM ubiquitination in HEK293 cells coexpressing both TRIM63 and UCHL1 WT or C90S. The cells were cotransfected with the plasmids carrying Myc-tagged PKM, HA-tagged ubiquitin, Flag-tagged TRIM63, and Flag-tagged UCHL1 WT or C90S. Right: Relative quantification of the anti-HA immunoblot band intensity of the immunoprecipitated proteins by Myc antibody. n = 2. (C) Left: Immunoblot analysis of endogenous PKM and AMPK phosphorylation in UCHL1 WT and KO HEK293 cells with TRIM63 siRNA expression. Right: The band intensity of PKM and AMPK phosphorylation. n = 3. (D) Left: Immunoblot analysis of mitochondrial outer (MFN1 and TOM20) and inner (TIM23 and COXIV) membrane proteins upon 20 μM CCCP and TRIM63 siRNA treatment. Right: Relative quantification of the immunoblot band intensity of indicated mitochondrial proteins. n = 3.