NF-κB/p65 is degraded by ubiquitination. (A) HPAECs were transfected with either the CON plasmid (empty VEC) or HA-ubiquitin (HA-Ubi; 1 µg) plasmid for 24 hours. HPAECs were collected and assayed for HA, p65, and β-actin (loading CON, ACTB) by immunoblotting. (B-C) HPAECs were transfected with either the CON plasmid (VEC) or HUWE1 (1 μg, oxHUWE1) plasmid for 6 hours; Media were replaced with EGM containing 5% FBS and then incubated for an additional 72 hours. (D) CHX treatment was carried out at a concentration of 40 to 100 μg/mL at varying time points (0-8 hours) in 0% FBS medium. (E) HPAECs were pretreated with DMSO or MG132, and leupeptin (Leup) for 2 hours and 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. ACTB, actin beta.