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. 2017 Feb 28;108(2):208–215. doi: 10.1111/cas.13133

Figure 2.

Figure 2

NEDD8‐activating enzyme activity essential for vascular endothelial (VE)‐cadherin protein production. (a) HUVECs were transfected with MLN4924 (80 nM) or DMSO for 72 h. The proteins were extracted and separated using 10% SDS–polyacrylamide gels. VE‐cadherin, p120 catenin, and β‐catenin proteins were then detected by Western blotting. β‐Actin was used as a loading control. (b) HUVECs cultured on gelatin‐coated cover slips were treated with MLN4924 (100 nM) or DMSO. The cells were fixed and stained with anti‐VE‐cadherin antibody followed by Alexa 488‐conjugated goat anti‐mouse IgG. Scale bar = 20 μm. (c) After HUVECs were treated with 20, 40, 60, 80, or 100 nM MLN4924 for 72 h, VE‐cadherin proteins were detected by Western blotting. Anti‐β‐actin antibody was used as an internal control. (d) Endogenous protein levels of VE‐cadherin were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). **P < 0.01; ***P < 0.001. (e) VE‐cadherin mRNA levels were quantified by quantitative RT‐PCR in MLN4924 (20, 40, 60, 80, or 100 nM)‐treated HUVECs, and normalized to GAPDH mRNA levels. n.s., not significant.