Figure 3.

TEV-mediated removal of cytosolic tail increases the constitutive shedding. (A) Expected TEV-mediated Cyt-TEVcs ADAM10 resultant cleavage fragments. (B) Representative immunofluorescent analysis of Cyt-TEVcs ADAM10 expressing cells using Myc antibody. Scale bar, 25 μm. (C) Representative WB analysis of cell lysates evaluating the expression and cleavage of Cyt-TEVcs ADAM10 using antibodies against the C-terminal Myc and C-terminal HA tags of the expressed proteins. Immunoblots with V5 and β-tubulin antibodies were used to evaluate the presence of TEV and the loading amount of lysates, respectively. (D) Cells were analyzed for BTC-AP release in the presence EV and TEV. The Cyt-TEVcs ADAM10 analog cleaved by TEV had significantly higher shedding activity. Star indicates the sites of TEV-induced cleavage in the ADAM10 analogs. Data are expressed as mean ± SEM. *** p < 0.0001; unpaired two-tailed Student’s t-test.