Fig. 6

FBXO45 interacts with GPX4 to promote GPX4 ubiquitination. RAECs were cultured in different glucose concentration conditions (Control: 5.5 mM glucose + 16.5 mM mannitol, HG: 22 mM glucose) with or without siScr or siFbxo45 treatment. A The mRNA level of Fbxo45 and Gpx4 in siScr and siFbxo45 RAECs with or without HG stimulation. B Representative images of immunofluorescence staining of GPX4 in siScr and siFbxo45 RAECs with or without HG stimulation. Scale bars, 50 μm. C The protein expression of FBXO45 and GPX4 in siScr and siFbxo45 RAECs with or without HG stimulation. D Co-IP assay of GPX4 and FBXO45 with indicated FBXO45 antibodies in RAECs protein lysate. E Co-IP assay of the GPX4 protein by an FBXO45 antibody in HEK293T cells transfected with pcDNA3.1-HA-GPX4 and pcDNA3.1-Flag-FBXO45. PcDNA3.1-vector was used as a negative control. F Degradation of the GPX4 protein was measured after the treatment of 200 µg/mL CHX at the indicated time points in HEK293T cells, which transfected with Flag-FBXO45 expression plasmids and siFbxo45. G Analysis of GPX4 ubiquitination was performed by Co-IP using an anti-GPX4 antibody, followed by immunoblot with anti-Ub antibody and anti-GPX4 antibody in HEK293T cells transfected with the indicated constructs with or without MG132 (10 µM for 10 h). H Analysis of GPX4 ubiquitination was performed by Co-IP using an anti-GPX4 antibody, followed by immunoblot with anti-Ub antibody and anti-GPX4 antibody in HEK293T cells transfected with the indicated constructs with or without siFbxo45 at the presence of MG132 (10 µM for 10 h). Statistical significances were calculated using (A, C, F) two-way ANOVA, Tukey’s multiple comparisons tests. Data are expressed as the mean ± SEM, statistical analysis revealed a significant difference with **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001, ns = not significant