Figure 5
Figure 5. Loss- and gain-of-function experiments indicate a role for ADAM17 in JAM-A shedding. (A) Overexpression of ADAM10 and 17 in human HEK293 cells. Cells were transiently transfected with ADAM10 and ADAM17 or the empty expression vector as a control. Overexpression was controlled by flow cytometric analysis of ADAM10/17 surface expression (top panel). Conditioned media (middle panel) were analyzed for the presence of JAM-A by Western blotting, and signals were quantified by densitometry and calculated as percentage of the mock-transfected control (bottom panel). (B) Down-regulation of endogenous ADAM10/17 using siRNA. HUVECs were transiently transfected with ADAM10- ADAM17- or control-siRNA. Down-regulation of ADAM10 and ADAM17 surface expression was analyzed by flow cytometry (top panel). Soluble JAM-A released into the conditioned media was detected Western blotting using an anti–hJAM-A monoclonal antibody and quantified by densitometry (bottom panels). (C) JAM-A shedding in adam17−/− fibroblasts. Total cell extracts from adam17−/− and wild-typet MEFs were controlled for the absence and presence of ADAM17 by Western blotting (top panel). Adam17−/− and wild-type MEFs were transiently transfected with JAM-A. After 48 hours, conditioned media and cell lysates were harvested and analyzed by Western blotting for the presence of sJAM-A and flJAM-A (bottom panels, left and right, respectively). (D) Recombinant JAM-A.Fc was incubated with the recombinant catalytic domain of ADAM17 for 3 hours, and subsequently cleavage products were analyzed by Western blotting using a monoclonal antibody against JAM-A. (Top panel) HEK293 cells were incubated with the recombinant catalytic domain of ADAM17 for 3 hours, and conditioned media were analyzed for the presence of JAM-A by Western blotting. (Bottom panel) All Western blots were shown as representative experiments, and quantified signals were calculated as mean plus or minus SD for 3 independent experiments. *Statistically significant changes in JAM-A expression compared with the control (P < .05).

Loss- and gain-of-function experiments indicate a role for ADAM17 in JAM-A shedding. (A) Overexpression of ADAM10 and 17 in human HEK293 cells. Cells were transiently transfected with ADAM10 and ADAM17 or the empty expression vector as a control. Overexpression was controlled by flow cytometric analysis of ADAM10/17 surface expression (top panel). Conditioned media (middle panel) were analyzed for the presence of JAM-A by Western blotting, and signals were quantified by densitometry and calculated as percentage of the mock-transfected control (bottom panel). (B) Down-regulation of endogenous ADAM10/17 using siRNA. HUVECs were transiently transfected with ADAM10- ADAM17- or control-siRNA. Down-regulation of ADAM10 and ADAM17 surface expression was analyzed by flow cytometry (top panel). Soluble JAM-A released into the conditioned media was detected Western blotting using an anti–hJAM-A monoclonal antibody and quantified by densitometry (bottom panels). (C) JAM-A shedding in adam17−/− fibroblasts. Total cell extracts from adam17−/− and wild-typet MEFs were controlled for the absence and presence of ADAM17 by Western blotting (top panel). Adam17−/− and wild-type MEFs were transiently transfected with JAM-A. After 48 hours, conditioned media and cell lysates were harvested and analyzed by Western blotting for the presence of sJAM-A and flJAM-A (bottom panels, left and right, respectively). (D) Recombinant JAM-A.Fc was incubated with the recombinant catalytic domain of ADAM17 for 3 hours, and subsequently cleavage products were analyzed by Western blotting using a monoclonal antibody against JAM-A. (Top panel) HEK293 cells were incubated with the recombinant catalytic domain of ADAM17 for 3 hours, and conditioned media were analyzed for the presence of JAM-A by Western blotting. (Bottom panel) All Western blots were shown as representative experiments, and quantified signals were calculated as mean plus or minus SD for 3 independent experiments. *Statistically significant changes in JAM-A expression compared with the control (P < .05).

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