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. 2013 Jul 25;288(35):25646–25657. doi: 10.1074/jbc.M113.473777

FIGURE 4.

FIGURE 4.

TACE is associated with integrins α6 and β1 via disintegrin domain and mechanical strain increases these associations. A, cells were preincubated with antibodies against different integrin subtypes as described, and cultured on full-length TACE-coated substrates in the presence of antibody and cycloheximide for 4 h. Results are from 3 different experiments performed in triplicate. *, p < 0.05 versus negative. B, type II cells were plated on wells coated with laminin (positive control), TACE fragment containing the pre-, metalloprotease and disintegrin domains (1–563), TACE fragment with the pre- and metalloprotease domains (1–477), or no substrate (negative control). Adhesion of type II cells to different substrates were quantified by optical density as described in “Experimental Procedures.” C, wild-type E17 type II cells were transfected by electroporation with plasmids encoding ADAM-17-Fc (2 μg) in combination with either integrin α6 (1 μg), integrin β1 (1 μg), or integrin α5 (1 μg) plasmids. 48 h later, cells were exposed or not to 5% cyclic strain for 30 min. Collected proteins were immunoprecipitated with TACE antibody (or negative control IgG) and immunoblotted with anti-integrin α6, β1, or α5 antibodies. Blots were reprobed with anti-TACE antibody. Results are from five independent experiments. *, p < 0.05 versus controls. C, control; S, strain. Upper panels show representative blots. Lower right panel shows a Western blot from total cell lysate demonstrating that α5 integrin is expressed in type II cells and is recognized by this antibody. D, E17 type II cells isolated from TACE knock-out mice were transfected by electroporation with plasmids encoding ADAM-17-Fc (FC) or a truncated form lacking the disintegrin domain (MP) in combination with either integrin α6 or integrin β1 plasmids. 48 h later, cells were exposed or not to 5% cyclic strain for 30 min. Collected proteins were immunoprecipitated with anti-integrin α6 or β1 antibodies (or negative control IgG) and immunoblotted with TACE antibody. Blots were reprobed with anti α6 or β1 antibodies. Blots are representative from two independent experiments. Lower panel shows a Western blot from total cell lysate demonstrating that FC and MP constructs are expressed in TACE knock-out cells.