Fig. 5: Verification of the protein interaction between RNF4 and PDHA1.
From: RNF4 mediated degradation of PDHA1 promotes colorectal cancer metabolism and metastasis
A Co-immunoprecipitation (co-IP) assay measuring the protein interaction between RNF4 and PDHA1 in SW620 cells after 24 h of transfection or co-transfection with Myc-RNF4 and Flag-PDHA1; B PLA assay measuring the proximity of RNF4 and PDHA1 in SW620 cells treated with 20 μM MG132 for 4 h (scale bar = 25 μm). Red fluorescence indicates the proximity of the two proteins. * indicates P < 0.05; C Western blot analysis of RNF4 protein expression in SW620 cells after knockdown of RNF4. * indicates difference compared to the sh-NC group, P < 0.05; D PLA assay measuring the proximity of RNF4 and PDHA1 in SW620 cells after knockdown of RNF4 (scale bar = 25 μm). Red fluorescence indicates the proximity of the two proteins. * indicates P < 0.05; E, F Co-IP assay measuring the protein interaction between RNF4 and FLAG-PDHA1 (WT, 2 KR, K77, K186) or Myc-RNF4 (WT or mSIM) in SW620 cells after co-transfection; G Ni2+ pull-down assay measuring the SUMOylation and ubiquitination of PDHA1 in SW620 cells co-transfected with FLAG-PDHA1-His and HA-Ub or T7-SUMO3 and Myc-RNF4 (WT or mSIM). Cell experiments were repeated three times.
