Figure 3.

Integrin β1 binds to hensin and integrin αV and gets tyrosine phosphorylated in high density cells. (A) In the first panel, 140 μg of DTSSP–cross-linked cell protein from LD and HD phenotypes were immunoprecipitated with anti–integrin β1 CSAT antibodies, and the blots were probed with guinea pig anti-hensin antibody. In control experiment, membrane containing integrin β1 immunoprecipitates described in the first panel was stripped and reprobed with a different anti–integrin β1 antibody MAB1965 (second panel). Chemically cross-linked cell lysates from LD and HD cells were immunoprecipitated with goat anti-hensin/DMBT1 antibody and blotted with anti–integrin β1 MAB1965 antibody (third panel). Control experiment in which the same samples were probed with guinea pig anti-hensin antibody (fourth panel). (B) Tyrosine phosphorylation of integrin β1. (Left) Anti-phosphotyrosine Western blot of integrin β1 immunoprecipitates (described in A) from LD and HD. (Middle) Control Western blot with integrin β1 antibody. (Right) A streptavidin Western blot of biotinylated basolateral membrane proteins (from HD) that were immunoprecipitated with anti–integrin β1 antibody. (C) Interaction of integrin β1 with integrin α subunits was examined by Western blotting of integrin β1 immunoprecipitates with antibodies to integrin αv, α6, and α1 (left lane in first three panels) and α3 and α5 (last two lanes). Interaction of hensin with integrin α subunits was examined by Western blotting of hensin immunoprecipitates with antibodies to integrin αv, α6, and α1 (right lane in first three panels) from HD cells. (D) Interaction of integrin αv with integrin β subunits was examined by Western blotting of integrin αv immunoprecipitates with integrin β1 (first lane), integrin β3 (second lane), and integrin β5 (third lane) antibodies.