Fig. 4: Effect of RNF4-mediated SUMOylation on PDHA1 ubiquitination and degradation.
From: RNF4 mediated degradation of PDHA1 promotes colorectal cancer metabolism and metastasis
A Co-transfection of FLAG-PDHA1 and EGFP-SUMO (SUMO1, SUMO2, or SUMO3) in SW620 cells, with or without treatment of 20 μM MG132 for 6 h. Ni-NTA analyzed SUMOylation of PDHA1; B Co-transfection of FLAG-PDHA1-His, HA-Ub, and T7-SUMO3 in SW620 cells, followed by treatment with CHX (20 μg/ml), MG132 (20 μM), or CHX + MG132 for 6 h. SUMOylation and ubiquitination of PDHA1 were examined by Ni2+ pull-down assay; C Co-transfection of SW620 cells with FLAG-PDHA1-His (WT, 2 KR (UMO-deleted double mutation), K77 (SUMO-deleted single mutation), K186 (another SUMO-deleted single mutation)), HA-Ub, and T7-SUMO3, followed by treatment with 20 μM MG132 for 6 h and Ni2+ pull-down experiments to detect the SUMOylation and ubiquitination of PDHA1; D Performance of Western blot analysis to evaluate the protein expression of PDHA1, SAE1, SAE2, Ubc9, SUMO-1, and SUMO-2 in different SW620 cell groups; E Transfection of SW620 cells with Flag-PDHA1 WT, Flag-PDHA1 2 KR, Flag-PDHA1 K77, and Flag-PDHA1 K186 for 48 h. The protein expression of PDHA1 was measured by Western blot after different incubation durations with 20 μg/ml CHX. * indicates difference compared to the Flag-PDHA1 WT group, P < 0.05. Cell experiments were repeated three times.
