FIGURE 5.

ADAM10 expression is repressed by the G-quadruplex motif. A, HEK293 cells were transiently transfected with the indicated ADAM10 cDNA constructs, and lysates were analyzed by immunoblotting for V5-tagged ADAM10, endogenous APP, β-actin as loading control, and GFP as transfection control. Supernatants were analyzed for APPsα secretion using antibody 2D8. Cellular APP is present in low molecular weight immature forms (im) and high molecular weight mature form (m). ADAM10 is present as a mature (m) form and predominantly as an immature (im) form. B, quantification of ADAM10 protein (black bars) and mRNA levels (white bars) from cells transfected with ADAM10 cDNA constructs shown in A. ADAM10 protein levels were normalized to GFP and actin levels. The signal for ADAM10 with the wild-type G-quadruplex GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments made in triplicate. ADAM10 mRNA was normalized to glycerolaldehyde-3-phosphate-dehydrogenase mRNA levels, and the signal for GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments. C, quantification of secreted APPsα from cells transfected with the indicated ADAM10 variants were shown in A. The signal for APPsα from GQ-WT ADAM10 transfected cells was set to 100%. Results are expressed as the means ± S.D. from three experiments. Asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post test) relative to GQ-WT ADAM10 transfected cells (*, p < 0.05; **, p < 0.01).