Anti mt1 mmp

The PLA was performed using a Duolink PLA kit (Sigma-Aldrich) according to the manufacturer's instructions and as previously described (Arasaki et al., 2015 (link)) except for the use of anti–MT1-MMP (R&D Systems) and anti-GFP (Thermo Fisher Scientific), or anti-mCherry (GeneTex) and anti-FLAG M2 (Sigma-Aldrich). Cholesterol depletion was performed as described previously (Ohtani et al., 1989 (link); Rothberg et al., 1992 (link)). Briefly, cells were spread on fibronectin-coated coverslips and left for 7 h, and then treated with 5 μM methyl-β-cyclodextrin (Wako Pure Chemicals) or 50 μg/ml nystatin (Sigma-Aldrich) for 30 min at 37°C. After that, the cells were fixed and subjected to PLA reactions. PLA signals were imaged by confocal microscopy and quantified by using ImageJ software.

The commercial mouse monoclonal antibodies used here were anti-FLAG M2 (Sigma-Aldrich), anti–MT1-MMP (R&D Systems), anti-cortactin (clone 4F11; EMD Millipore), anti-Bet1 (clone 17; Santa Cruz Biotechnology), anti-calnexin (clone 37; BD), anti-GM130 (clone 35; BD), anti-EEA1 (clone 14; BD), anti-LAMP1 (clone H4A3; Developmental Studies Hybridoma Bank), anti-α-tubulin (clone B-5-1-2; Sigma-Aldrich), and anti-integrin β1 (clone 12G10; Santa Cruz Biotechnology). The commercial rabbit polyclonal antibodies used were anti-FLAG (Sigma-Aldrich), anti–MT1-MMP (EMD Millipore), anti-STX16 (Synaptic Systems), anti-GFP (Thermo Fisher Scientific), and anti-GPP130 (Covance). The rabbit polyclonal antibodies to STX4, STX5, STX7, STX17, and STX18 were as described previously (Hatsuzawa et al., 2000 (link); Mizoguchi et al., 2000 (link); Arasaki et al., 2015 (link); Inoue et al., 2015 (link)). Anti-STX2, -STX3, -VAMP7, and -Vti1b polyclonal antibodies were raised by immunizing rabbits with the purified recombinant His-tagged cytoplasmic domains of human STX2 (aa 1–264), STX3 (aa 1–263), VAMP7 (aa 1–188), and Vti1b (aa 1–209), respectively, and then the antisera were affinity-purified. Secondary antibodies labeled with HRP and fluorochrome were purchased from BioRad Laboratories and Life Technologies, respectively.