Fig. 4

AKT1 activation is required for SC79-induced RAGE ectodomain shedding. (A) HAECs express all three AKT isoforms, and AKT1-, AKT2-, and AKT3-siRNAs selectively deplete each AKT isoform. HAECs were transfected with AKT1-, AKT2-, AKT3-siRNAs, or control siRNAs, and the cell lysates were immunoblotted with antibodies to AKT1, AKT2, AKT3, or actin. (n = 3, ^*p < 0.05 vs. control). (B) SC79 activates AKT1. HAECs were incubated with 10 µM SC79 for various times (1, 5, 10, and 30 min) (upper panel) or different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min (lower panel). The cell lysates were immunoblotted with antibodies to p-AKT1 (Ser473) and AKT1. (n = 3, ^*p < 0.05 vs. control). (C) AKT1 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with AKT1-siRNA or control siRNA and then incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and antibodies to AKT1 and actin. (n = 3, ^*p < 0.05 vs. control cells transfected with control siRNA). (D) AKT1 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with AKT1-siRNA or control siRNA were treated for 30 min with or without SC79 (10 µM). The cells were then treated with AGE-BSA (100 µg/ml) for 24 h. The cell lysates were immunoblotted with antibodies to ICAM-1, AKT1, and actin. (n = 3, ^*p < 0.05 vs. control; ^#p < 0.05 vs. AGE-BSA; ^†p < 0.05 vs. control cells transfected with AKT1-siRNA)