Anti gm130 antibody

Cells were plated on glass coverslips coated with poly-l-lysine (Sigma-Aldrich). For all experiments except where Golgi staining was performed, cells were fixed with 4% formaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM ethylene glycol tetraacetic acid [EGTA], and 4 mM MgSO₄, pH 7) for 10 min. Blocking and all antibody dilutions were performed using AbDil (20 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 3% bovine serum albumin [BSA], and 0.1% NaN₃, pH 7.5). Phosphate-buffered saline (PBS) plus 0.1% Triton X-100 (PBS-TX) was used for washes. For Golgi staining, cells were fixed with 4% formaldehyde in PBS for 15 min. Blocking and all antibody dilutions were performed using PBS plus 1% BSA and 0.3% Triton X-100. Anti-GM130 antibody (Cell Signaling Technologies) was used at a 1:3200 dilution. PBS was used for washes. For all experiments, GFP-Booster (Chromotek; 1:200 dilution) was used to amplify the fluorescence of the GFP-tagged transgenes and DM1A was used to stain the microtubules (Sigma-Aldrich; 1:3000 dilution). Anti-mouse Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) was used at 1:300. DNA was visualized by incubating cells in 1 µg/ml Hoechst-33342 (Sigma-Aldrich) in PBS-TX for 10 min. Coverslips were mounted using 0.5% p-phenylenediamine and 20 mM Tris-Cl, pH 8.8, in 90% glycerol.