CD151 forms ternary complexes with MT1-MMP and α3β1 integrin at intercellular contacts between primary human endothelial cells. (A) HUVECs seeded at confluence on collagen I–coated coverslips were double-stained for MT1-MMP and 1 of tetraspanin CD151, tetraspanin CD9, or the tetraspanin-associated integrin α3. Samples were analyzed by confocal microscopy. The maximal projection is shown together with the cytofluorogram, in which colocalization is depicted as pixel accumulation along the central diagonal. (B) HUVEC monolayers were lysed in buffer containing 1% Brij96. Samples were immunoprecipitated with the following mAbs: anti–VE-cadherin (TEA1/31), anti–MT1-MMP (LEM-2/63), anti-CD151 (11B1 and LIA1/1), anti-CD9 (VJ1/20), anti-α3 integrin (VJ1/6), and anti-β1 integrin (TS2/16). Immunoprecipitates were analyzed by Western blot against VE-cadherin, MT1-MMP, CD151, and CD9. (C) HUVEC monolayers were lysed in 1% Brij96-containing lysis buffer. Samples were immunoprecipitated with mAbs anti–VE-cadherin (TEA1/31), anti–MT1-MMP (LEM-2/63 and LEM-2/15) and anti-CD151 (LIA1/1), and with anti–MT1-MMP pAbs (Ab2 and Ab3). Immunoprecipitates were analyzed by Western blot against MT1-MMP and CD151. (D) HUVEC monolayers were lysed in buffer containing 1% digitonin. Samples were immunoprecipitated with mAbs anti–VE-cadherin (TEA1/31), anti–MT1-MMP (LEM-2/63), anti-CD151 (11B1), anti-CD9 (VJ1/20), and anti-α3 integrin (VJ1/6). Immunoprecipitates were analyzed by Western blot against MT1-MMP, CD151, and CD9. (E) HeLa cells transiently transfected with different FLAG-tagged constructs for MT1-MMP (full-length and deletion mutants lacking the cytoplasmic, hemopexin, or catalytic domains) were lysed in 1% Brij96-containing buffer and immunoprecipitated with anti-FLAG mAb and antitetraspanin antibodies against CD151 and CD9 or with negative control X63 (IgG). Anti-FLAG mAb without cellular lysates (anti-FLAG) is shown to discriminate nonspecific bands. Membranes were probed with biotinylated anti-FLAG mAb.