Figure 1.

Specific tetraspanins associating with MT1-MMP. (A) MCF-7-MT1 and MDA-MB-231 cells were lysed in 1% Brij 99 lysis buffer, and the indicated tetraspanins were immunoprecipitated (TSPAN, cocktail of tetraspanin antibodies against CD9, CD63, CD81, CD82, CD151, and TSPAN4). Eluates were analyzed by gelatin zymography and by Western blotting for MT1-MMP, using the anti-catalytic domain LEM2/15.8 antibody. Std, MMP-2 standard; pMMP-2, pro-MMP-2; aMMP-2, active MMP-2; n.s., nonspecific. All antibodies used in these and other immunoprecipitation experiments have been previously shown to immunoprecipitate their target antigens (Barnstable et al., 1978; Tachibana et al., 1997; Yang et al., 2002). (B) HT1080 cells stably expressing TSPAN12 and MDA-MB-231 cells were lysed in 1% Brij 99, cell surface proteins were immunoprecipitated, and eluates were analyzed by gelatin zymography and by MT1-MMP Western blotting using the anti-hinge AB815 antibody. cMT1, cleaved MT1. MT1 Standards are from MCF-7 cells (negative for MT1) or MCF-7 cells stably expressing MT1-MMP. (C) FLAG-MT1-MMP, MHC1, and CD147 were immunoprecipitated from MCF-7-MT1 cells (lysed in 1% Brij 99) and eluates were Western blotted for CD9 and CD81.