Western Blot Analysis of Cells and Liposomes

For western blot analysis, cells or liposomes were removed from solution via centrifugation at 300g for 10 min or 10,000 × g for 20 min, respectively, supernatants aspirated and then pellets solubilized in 50 to 100 μl of CHAPS cell extract buffer (Cell Signaling Technologies, Danvers, MA) supplemented with Halt protease inhibitor mixture (Thermo Fisher Scientific) and incubated on ice for 20 min. Debris was removed by centrifugation at 20,000 × g for 20 min, and supernatants were collected. Polyacrylamide gel electrophoresis, transfer to nitrocellulose membranes, antibody incubation, and detection were performed as described using lysates from 1 × 10⁶ cell equivalents or 0.1 μmol total lipid equivalents of lysosomes [57 (link)].