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. 2014 Mar 24;289(19):13308–13322. doi: 10.1074/jbc.M114.559088

FIGURE 6.

FIGURE 6.

Effect of siRNAs to ADAM9, ADAM10, and ADAM17 on meprin β shedding in meprin β and meprin αβ-expressing clones in the absence and presence of PMA or IM. A, ADAM10 expression was knocked down with siRNA pools to human ADAM10 in HEK293 or HEK cells stably transfected with meprin β or co-transfected with meprin β and meprin α. Scrambled siRNA was used as a negative control. Forty-eight hours after transfection, cells were incubated with PMA (30 ng/ml), IM (2.5 μm), or vehicle for 4 h. Cell lysates were analyzed for expression of ADAM10 by Western blot. α-Actinin served as a loading control. B, ADAM17 expression was knocked down with siRNA pools to human ADAM17 in HEK293 or HEK cells stably transfected with meprin β or co-transfected with meprin β and meprin α. Scrambled siRNA was used as a negative control. Forty-eight hours after transfection, cells were incubated with PMA (30 ng/ml), IM (2.5 μm), or vehicle for 4 h. Cell lysates were analyzed for expression of ADAM10 by Western blot; α-actinin served as a loading control. C, ADAM9, ADAM10, or ADAM17 expression was knocked down with siRNA pools to the indicated ADAMs in HEK293 cells stably transfected with meprin β. Scrambled siRNA was used as a negative control. Cells (48 h after transfection) were incubated with PMA (30 ng/ml) or vehicle for 4 h. Medium supernatants were harvested, and aliquots of equal protein amount were analyzed by Western blot for the presence of meprin β (left panels). Albumin detected by Ponceau S stain is shown as a loading control. This panel shows a representative Western blot from three independent experiments. Western band intensities were evaluated by densitometry normalized to those observed in medium supernatant of the corresponding clone treated with scrambled siRNA (right panel). Numbers in the diagrams represent the average band intensities, and bars represent the S.E. obtained from 3 independent experiments. *, p < 0.05 was considered statistically significant. D, ADAM9, ADAM10, or ADAM17 expression was knocked down with siRNA pools to the indicated ADAMs in HEK293 cells stably co-transfected with meprin α and β. Cells (48 h after transfection) were incubated with PMA (30 ng/ml) or vehicle for 4 h. Medium supernatants were analyzed by Western blot for the presence of meprin β as described for panel B (left panel). Albumin detected by Ponceau S stain is shown as a loading control. This panel shows a representative Western blot from three independent experiments. Western band intensities were evaluated by densitometry normalized to those observed in medium supernatant of the corresponding clone treated with scrambled siRNA (right panel). Numbers in the diagrams represent the average band intensities, and bars represent the S.E. obtained from 3 independent experiments. **, p < 0.01, ***, p < 0.001.