Hrp donkey anti rabbit igg

Cell monolayers were washed with PBS and lysed in ice-cold extraction buffer (0.5% Triton X-100, 150 mM NaCl, 25 mM KCl, 25 mM Tris, pH 7.4, 1 mM EDTA) supplemented with a protease inhibitor mixture (Roche Applied Science). Extracts were clarified by centrifugation (12,000 × g for 10 min at 4°C). The sample protein concentration was determined by Bradford assay (BD Biosciences) using standard protocols, and 10 μg denatured by boiling for 5 minutes in SDS sample buffer. Proteins in the extracts were resolved by SDS-PAGE using 12% acrylamide gels, transferred to PVDF membranes, and probed by immunoblotting using mouse anti-Actin (Sigma-Aldrich), mouse anti-AP2α (BD Biosciences), mouse anti-AP1γ (Sigma-Aldrich), rabbit anti-BST-2 (NIH AIDS Reagent Program), rabbit anti-CD8 (Santa Cruz Biotechnology), mouse anti-Clathrin HC (Cell Signaling Technology), mouse anti-FLAG (Sigma-Aldrich), and horseradish peroxidase-conjugated goat anti-Mouse IgG (BioRad) or HRP-donkey anti-Rabbit IgG (BioRad) and Western Clarity detection reagent (BioRad). Apparent molecular mass was estimated using commercial protein standards (PageRulePlus, Thermo Scientific). Chemiluminescence was detected using a BioRad Chemi Doc imager and analyzed using BioRad Image Lab v5.1 software.