Fig. 8

Depletion of AKT1, AKT2, or AKT3 impairs SC79-induced ADAM10 cell surface translocation. (A) AKT inhibition prevents SC79-induced ADAM10 cell surface translocation. HAECs were preincubated with or without MK-2206 (1 µM) or DMSO (vehicle) for 60 min. Following this, they were further incubated for 20 min with or without SC79 (10 µM). Cells were immunostained with an antibody to an extracellular portion of ADAM10. Representative photos and the relative fluorescence intensities are shown (scale bar: 100 μm). (n = 3, ^*p < 0.05 vs. control; ^#p < 0.05 vs. SC79 treatment alone). (B–D) AKT1, AKT2, and AKT3 knockdowns prevent SC79-induced ADAM10 cell surface translocation. HAECs grown in culture dishes with a coverslip were transfected with AKT1-siRNA, AKT2-siRNA, AKT3-siRNA, or control siRNA, and then incubated for 20 min with DMSO or SC79 (10 µM). (a) Cells grown on the coverslip were immunostained for ADAM10. Representative photos and the relative fluorescence intensities are shown (scale bar: 100 μm). (b) Cell lysates from cells that were not on the coverslip in the same culture plate were immunoblotted with antibodies to AKT1, AKT2, AKT3, or actin. (n = 3, ^*p < 0.05 vs. control cells transfected with control siRNA)