Fig. 6

NCEH1 required ZNRF1 to induce the ubiquitination-dependent degradation of Cav-1 in ECs. (A) The protein expression of ZNRF1 in NG- or HG-exposed ECs. (B) The interaction of ZNRF1 with NCEH1 in NG- or HG-exposed ECs. (C) The interaction of ZNRF1 with Cav-1 in NG- or HG-exposed ECs. (D) Effects of NCEH1 shRNA on the protein expression of ZNRF1 in NG- or HG-exposed ECs. (E) Effects of NCEH1 overexpression on the protein expression of ZNRF1 in NG- or HG-exposed ECs. (F) Effects of NCEH1 shRNA on the interaction of ZNRF1 with Cav-1 in NG- or HG-exposed ECs. (G) Effects of NCEH1 overexpression on the interaction of ZNRF1 with Cav-1 in NG- or HG-exposed ECs. (H) Effects of ZNRF1 overexpression on the protein expression of Cav-1 in NCEH1-deficient ECs. (I) Effects of ZNRF1 overexpression on the Cav-1/eNOS complex in NCEH1-deficient ECs. (J) Primary ECs were transfected with ZNRF1 overexpression plasmid (1 µg) for 6 h, and then transfected with NCEH1 shRNA (100 nM) for additional 48 h, we then examined the effects of ZNRF1 overexpression on ubiquitination of Cav-1 in NCEH1-deficient ECs. (K, L) Effects of ZNRF1 overexpression on NO production in NCEH1-deficient ECs. Scale Bar, 100 μm. n = 4–6. *P < 0.05 versus Con shRNA or Vector. †P < 0.05 versus NCEH1 shRNA. The P-value was calculated by unpaired two-tailed Student’s t-test (A-C). Differences between groups were assessed with ANOVA followed by Bonferroni post-hoc test (D-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. Veh, Vehicle. 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; IP, immunoprecipitation