Fig. 4

RAEC transfected with shAkt (clone #2) and then stimulated with insulin or TNFα alone or in combination demonstrates the negative regulation of Akt in VCAM-1 expression. REAC were stably transfected with shAkt (Akt KD) and then stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) alone or in combination. Total VCAM-1 protein was analyzed by SDS-PAGE and determined by Western blot analysis. (Upper Panel) “NST”, cells not stimulated with insulin or TNFα. “Ins” cells stimulated with insulin. “TNFα” cells stimulated with TNFα. “C” control RAEC not transfected with shRNA, while “sh” cells were transfected with shAkt. (Lower Panel) “C” control RAEC not transfected (open bars). Akt KD are RAEC stably transfected with shAkt (black bars). “NST” cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as the percent of controls and represents the mean ± SEM of six independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. *, P < 0.05 vs non-stimulated cells