Anti gpp130

Lipofectamine 2000 and Lipofectamine RNAi MAX were purchased from Life Technologies (Carlsbad, CA). As primary antibodies, we used mouse monoclonal anti-CD59 (clone 5H8; Hirata et al., 2013 (link)), anti-FLAG (clone M2), anti-HA (clone HA7; Sigma-Aldrich, St. Louis, MO), anti-STX6 (Stressgen, San Diego, CA), anti–α-tubulin, anti-Golgin97 (clone CDF4; Life Technologies), anti-TMEM87A (clone #772807; R&D systems, Minneapolis, MN), rabbit monoclonal anti-LAMP1 (clone D2D11; Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-VPS53, VPS52 (a kind gift from Chris Schindler and Juan Bonifacino, National Institutes of Health, Bethesda, MD; Perez-Victoria et al., 2008 (link)), anti-GPP130 (Covance, Princeton, NJ), and anti-EEA1. The secondary antibodies were phycoerythrin (PE)-conjugated goat anti-mouse immunoglobulin G (IgG; BioLegend, San Diego, CA), Alexa Fluor 594–conjugated goat anti-mouse IgG, and Alexa Fluor 647–conjugated goat anti-mouse IgG (Life Technologies). Alexa Fluor 488–conjugated CTxB was purchased from Life Technologies.

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.

Immunofluorescence was carried out as described previously (Lavieu et al., 2014 (link)). Anti-Gpp130 and anti-Golgin97 antibodies were purchased from Covance and Abcam, respectively. Anti-rabbit Atto594 (Rockland), anti-mouse Atto594 (Sigma-Aldrich), and anti-mouse- and anti-rabbit 635P (Abberior) secondary antibodies were used for the STED experiments.