FIGURE 8.

Inhibition or depletion of mTOR augments PKC-δ- and Akt-induced ICAM-1 expression in endothelial cells. HUVEC (A) or HLMVEC (B) were transfected with pCDNA3-PKCδ-CAT or empty vector (pCDNA3) using Lipofectamine 2000 as described under “Experimental Procedures.” Cells were incubated with rapamycin (5 ng/ml) or dimethyl sulfoxide (vehicle) alone for 6–8 h. Total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-ICAM-1 antibody. Actin levels were used to monitor loading. Results are representative of two separate experiments. C, HUVEC were co-transfected with pCDNA3-Akt-CAT and pCDNA3.1-mTOR shRNA using Lipofectamine 2000. Total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-ICAM-1 antibody. Actin levels were used to monitor loading (upper panel). The same lysates were re-electrophoresed and immunoblotted with an anti-mTOR antibody to assess the depletion of mTOR (lower panel). Results are representative of two separate experiments.