FIGURE 6.

Overexpression of mTOR suppresses PKC-δ- and Akt-induced ICAM-1 expression in endothelial cells. A, HUVEC were transfected with pcDNA3-PKCδ-CAT, pcDNA3-Akt-CAT, or pRK5-mTOR-WT using Lipofectamine 2000 as described under “Experimental Procedures.” After 24 h, total cell lysates were prepared and analyzed by immunoblotting using an anti-PKC-δ, anti-HA, or anti-mTOR antibody to verify the expression of PKCδ-CAT, Akt-CAT, or mTOR, respectively. PKC-δ-CAT corresponds to ∼47 kDa as it contains only the catalytic domain and therefore can be distinguished from the endogenous PKC-δ (76 kDa). Anti-HA antibody allowed the detection of Akt-CAT but not the endogenous Akt. Increased expression of mTOR in mTOR-WT-transduced cells was due to overexpressed mTOR. Actin levels were used to monitor loading. B, HUVEC were transfected with pCDNA3-PKCδ-CAT in combination with pRK5-mTOR-WT using Lipofectamine 2000 as described under “Experimental Procedures.” Total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody to ICAM-1. Actin levels were used to monitor loading. Results are representative of two separate experiments. C, HUVEC were transfected with pCDNA3-Akt-CAT in combination with pRK5-mTOR-WT using Lipofectamine 2000 as described under “Experimental Procedures.” Total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody to ICAM-1. Actin levels were used to monitor loading. Results are representative of two separate experiments.