Fig. 10

Rab14 is required for SC79-induced ADAM10 cell surface translocation. (A) Rab14 knockdown prevents SC79-induced ADAM10 cell surface translocation. HAECs grown in culture dishes with a coverslip were transfected with Rab14-siRNA or control siRNA and then incubated for 20 min with DMSO or SC79 (10 µM). (a) Cells grown on the coverslip were immunostained with an antibody to an extracellular portion of 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 Rab14 and actin. (n = 3, ^*p < 0.05 vs. control cells transfected with control siRNA). (B) Rab14 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with Rab14-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 Rab14 and actin. (n = 3, ^*p < 0.05 vs. control cells transfected with control siRNA). (C) Rab14 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with Rab14-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, Rab14, and actin. (n = 4, ^*p < 0.05 vs. control; ^#p < 0.05 vs. AGE-BSA; ^†p < 0.05 vs. control cells transfected with Rab14-siRNA)