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. 2023 Jan 4;24(2):917. doi: 10.3390/ijms24020917

Figure 2.

Figure 2

TEV-mediated removal of prodomain does not induce ADAM10 activation. (A) ADAM10 protein after TEV activity, wt ADAM10 lacking TEVcs was not a substrate of TEV. HEK293T cells were co-transfected with a ratio of 1:1:1 of plasmids encoding wt ADAM10:BTC-AP:EV (empty vector) or constitutive active TEV. (B) Representative WB analysis of cell lysates evaluating the expression and cleavage of wt ADAM10 using an antibody against the C-terminal Myc tag of the expressed proteins. V5 antibody was used to show the presence of TEV. Anti-β-tubulin was used as a loading control. (C) Shedding levels in cells expressing EV or TEV were similar. (D) US-BS-TEVcs ADAM10 analog after TEV activity. (E) Immunoblot analysis revealing TEV-mediated cleavage of US-BS-TEVcs. (F) Shedding was unaffected by TEV-mediated cleavage in correspondence of US and BS sites. (G) US-TEVcs ADAM10 analog after TEV activity. (H) WB of the expression of US-TEVcs analog in the presence of EV or TEV. (I) Shedding levels in cells expressing TEV was significantly lower than those in cells expressing EV. (J) BS-TEVcs ADAM10 analog after TEV activity. (K) Immunoblot analysis of BS-TEVcs analog in the presence of EV or TEV. (L) Shedding levels in cells expressing EV or TEV was similar. 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. (M) Representative immunofluorescent analysis of ADAM10 analogs in HEK293T cells using antibodies against the C-terminal Myc tag. Scale bar, 25 μm.