Fig. 6.

Cleavage experiments using TACE to determine unmasked S2 site specificity and whether mechanical force can allow for S2 cleavage. (A) Cleavage is observed when TACE is incubated with hN2 S2 site fluorescent peptide. Increase in fluorescence over time after addition of TACE shows cleavage occurring at S2 site. Western blot (Inset) probed for the C-terminal His-tag of recombinant protein shows a loss of signal in the denatured protein (d) containing TACE (+) highlighting it’s specificity for the unfolded form of the protein. Controls lacking TACE (-) or containing nondenatured protein (n) show no cleavage. (B) Raw data from AFM cleavage experiments. The height of the AFM tip is measured over time; when protein is held, the tip is maintained close to the surface; however, when cleavage occurs tension across the tip is lost and retraction from the surface is observed. Point of TACE injection is marked by arrowhead. (C) Graph showing percentage frequency of cleavage events when zinc chloride containing buffer (B, n = 65), ADAM10 (A, n = 56), TACE in a zinc chloride buffer (T+, n = 61), TACE without the necessary zinc ions (T-, n = 54), and denatured TACE (dT, n = 72) are injected. (D) Graph showing time taken postinjection for cleavage events to occur when TACE was injected (x axis values represent the upper limit of the histogram interval).