Fig. 7

CircRNF13 promotes ubiquitination of GLUT1 via SUMO2. A The interaction between GLUT1, SUMO2, and UBC9 was examined using immunoprecipitation and an anti-Flag (GLUT1) antibody in HNE2 and CNE2 cells transfected with Flag-GLUT1 vector, followed by western blotting using the anti-SUMO2 and anti-UBC9 antibodies. B The interaction between SUMO2, GLUT1, and UBC9 proteins was examined using immunoprecipitation with an anti-HA (SUMO2) antibody in HNE2 and CNE2 cells transfected with HA-SUMO2 vector, followed by western blotting using anti-GLUT1 and anti-UBC9 antibodies. C Immunofluorescence experiments performed using anti-Flag (GLUT1) and anti-SUMO2 antibodies in HNE2 and CNE2 cells showed that GLUT1 and SUMO2 were co-localized. DAPI-stained nucleus: blue; anti-Flag (GLUT1): red; anti-SUMO2: green; co-localization of GLUT1 and SUMO2: yellow; the merged image represents the overlap of DAPI, GLUT1, and SUMO2 (scale bar: 20 μm). D Immunoprecipitation experiments using an anti-Flag-GLUT1 antibody, followed by western blotting using an anti-SUMO2 antibody, were performed, to identify the SUMOylation level of the GLUT1 protein in HNE2 and CNE2 cells transfected with sicircRNF13 or circRNF13 overexpression vector. E The ubiquitination level of GLUT1 protein was detected in HNE2 and CNE2 cells using pull-down with an anti-Flag-GLUT1 antibody, followed by western blotting with an anti-ubiquitin antibody, to determine whether SUMO2 affects GLUT1 ubiquitination. F A pull-down experiment using an anti-Flag-GLUT1 antibody, followed by western blot using an anti-ubiquitin antibody, were performed to identify the ubiquitination level of GLUT1 protein in HNE2 and CNE2 cells co-transfected with sicircRNF13 and SUMO2 overexpression vector