Fig. 3

RAEC transfected with shERK2 (clone #2) and then stimulated with insulin or TNFα alone or in combination demonstrates the negative regulation of ERK2 in VCAM-1 expression. RAEC were stably transfected with shERK2 (ERK2 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 cells not transfected with shRNA, while “sh” cells were transfected with shERK2. (Lower Panel) “C” control RAEC not transfected (open bars). ERK2 KD are RAEC stably transfected with shERK2 (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