Figure 4.

TEV-mediated shedding activity of ADAM10 analogs with mutations in the prodomain and cytosolic region. (A) ADAM10 protein after TEV activation. (A′) Representative WB analysis of cell lysates evaluating the expression and cleavage of US-BS-Cyt-TEVcs. (A″) Shedding levels in cells expressing EV were almost zero and TEV did not increase constitutive shedding. (B) US-Cyt-TEVcs ADAM10 analog after TEV activity. (B′) Immunoblot analysis revealing TEV-mediated cleavage of US-Cyt-TEVcs. (B″) Shedding was unaffected by TEV-mediated cleavage in corresponding US and Cyt sites. (C) BS-Cyt-TEVcs ADAM10 analog after TEV activation. (C′) WB of the expression of BS-Cyt-TEVcs analog in the presence of EV or TEV. (C″) Shedding levels in cells expressing TEV were significantly higher than those in cells expressing EV. (D,E) ADAM10 proteins after TEV activation; US-mut and BS-mut ADAM10 analogs lacking TEVcs were not TEV substrates. (D′,E′) Immunoblot analysis in cells expressing US-mut (D′) and BS-mut (E′) in the presence of EV and TEV. (D″,E″) Shedding levels in cells expressing EV or TEV for US-mut (D″) and BS-mut (E″). (F,G) US-mut-Cyt-TEVcs and BS-mut-Cyt-TEVcs ADAM10 proteins after TEV activation. (F′,G′) Immunoblot analysis in cells expressing US-mut-Cyt-TEVcs (F′) and BS-mut-Cyt-TEVcs (G′) in the presence of EV and TEV. (F″,G″) Shedding levels in cells expressing EV or TEV for US-mut-Cyt-TEVcs (F″) and BS-mut-Cyt-TEVcs (G″). (H,I) T719A and T719A Cyt-TEVcs ADAM10 proteins after TEV activation. (H′,I′) Representative WB analysis of cell lysates evaluating the expression and cleavage of T719A (H′) and T719A Cyt-TEVcs (I′). T719A analog lacking TEVcs was not a TEV substrate. (H″,I″) Shedding levels in cells expressing EV or TEV for T719A (H″) and T719A Cyt-TEVcs (I″). Star indicates the sites of TEV-induced cleavage in the ADAM10 analogs. Data are expressed as mean ± SEM. ns, not significant; *** p < 0.0001; unpaired two-tailed Student’s t-test.