Mouse anti p230 clone 15

The following antibodies were used: rabbit anti-LBR (ProteinTech, 12398-1-AP), goat anti–lamin B (C-20; Santa Cruz Biotechnology), mouse anti–ERGIC-53 (G1/93; Enzo Life Sciences), rabbit anti–GMAP-210 (HPA002570, Sigma-Aldrich), mouse anti-p230 (clone 15; BD Biosciences), rabbit anti-PDI (SPA-890; Stressgen Biotechnologies), rabbit anti-Sec13 (Rainer Pepperkok, European Molecular Biology Laboratory, Heidelberg, Germany), sheep anti-ZFPL1 (70 (link)), rabbit anti-giantin (Manfred Renz, Institute of Immunology and Molecular Genetics, Karlsruhe, Germany), mouse anti-GAPDH (G-9; Santa Cruz Biotechnology), rabbit anti–LAMP-1 (D2D11, Cell Signaling Technology), and anti-LAMP2 (H4B4; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, USA). Alexa 488–, 546–, 594–, and 647–conjugated secondary antibodies were from Molecular Probes (Thermo Fisher Scientific). Horseradish peroxidase–conjugated secondary antibodies were from Sigma-Aldrich.

Cells were grown on poly-(l)-Lysine coated cover slips at a density of 4.5 × 10⁴ cells per 24-well for 24 h, fixed with 4% paraformaldehyde, permeabilized for 4 min with 0.1% Triton X-100 in PBS, blocked for 10 min with 0.5% BSA in PBS and stained with mouse anti-hCD81 (JS-81, BD Biosciences, San Jose, CA, USA, 0.2 μg/mL) or rabbit anti-HA (H6908, Sigma Aldrich, St. Louis, MO, USA, 0.5 μg/mL) and mouse anti-p230 (clone 15, BD Biosciences, 1.25 μg/mL) over night at 4 °C in PBS with 0.5% BSA. Staining with secondary antibodies (goat anti-mouse-IgG-Alexa488 and goat anti-rabbit-IgG-Alexa647, 2 μg/mL, Life Technologies, Carlsbad, CA, USA) was performed for 1 h at room temperature. Nuclei were counterstained with DAPI (300 nM, Life Technologies) and coverslips mounted in Prolong Gold (Life Technologies) followed by inverse confocal laser-scanning microscopy (Olympus Fluoview 1000), using ×60 and ×100 magnification lenses. Channels were read in sequential acquisition mode with a Kalman filter (n = 3) and single planes of at least five frames per biological replicate were analyzed.