Figure 4.

HUWE1, E3 ligase induces the degradation of NF-κB/p65. (A-B) HPAECs were treated with scrambled (SCR) or HUWE1 (10 or 20 nM) siRNAs for 6 hours and then incubated for an additional 72 hours. Real-time qPCR or western blotting was performed for HUWE1 mRNA (A) or p65 protein (B). Each bar represents mean ± SEM HUWE1 or p65 level relative to GAPDH expressed as fold change vs cells treated with scrambled siRNA (SCR). *P < .05 vs SCR, n = 4 to 6. (C) HPAECs were transfected with scrambled siRNA or HUWE1 siRNA for 72 hours and were treated with CHX (40 µg/mL) for 0, 2, 4, and 8 hours to inhibit de novo protein synthesis and harvested for western blotting. The levels of p65 at time 0 was set as 100% and the percent p65 protein remaining following CHX treatment at each time point was calculated accordingly. (D-F) HPAECs were transfected with either the CON plasmid (VEC or HEM/oxHUWE1[−]) or HUWE1 (1 μg, oxHUWE1 or HEM/oxHUWE1[+]) plasmid for 6 hours and then treated with DMSO vehicle (CON) or HEM (5 µM) for 72 hours. Mean HPAEC HUWE1 (D), p65 (E), ET-1 (F), and VCAM1 (G) mRNA levels were measured with real-time qPCR or western blotting. Each bar represents the mean mRNA level ± SEM relative to GAPDH expressed as fold change vs CON. *P < .05 vs VEC or HEM/oxHUWE1−, n = 6.