Fig. 5

NCEH1 upregulated the expression of Cav-1 through the deubiquitination pathway in ECs. (A) Effects of NCEH1 shRNA on the protein expression of Cav-1. (B) Effects of NCEH1 overexpression on the protein expression of Cav-1. (C, E) Effects of NCEH1 shRNA on the Cav-1/eNOS complex. (D, F) Effects of NCEH1 overexpression on the Cav-1/eNOS complex. (G, I) MG-132 reversed the inhibitory effects of NCEH1 overexpression on the protein expression of Cav-1. (H, J) Chloroquine had no effect on the inhibitory effects of NCEH1 overexpression on the protein expression of Cav-1. (K, L) NCEH1 overexpression accelerated the degradation of Cav-1. (M) Effects of NCEH1 downregulation or overexpression on the ubiquitination levels of Cav-1 after transfection of NCEH1 shRNA or NCEH1 overexpression plasmid for 48 h. n = 4–6. *P < 0.05 versus Con shRNA, Vector or CHX. †P < 0.05 versus NCEH1 shRNA, NG or NCEH1 OE. Differences between groups were assessed with ANOVA followed by Bonferroni post-hoc test (A-J). The P-value was calculated by unpaired two-tailed Student’s t-test (L). For immunoblotting assay, the ratio of the grayscale values of the target protein and β-actin in each group was normalized by the average value of the control group. NCEH1, neutral cholesterol ester hydrolase 1; NG, normal glucose; HG, high glucose; OE, overexpression; NO, nitric oxide; Cav-1, caveolin-1; eNOS, endothelial nitric oxide synthase; CHX, cycloheximide; IP, co-immunoprecipitation