Phosphatase inhibitor mix 1

Cells grown to 60% confluency were lysed using RIPA buffer supplemented with phosphatase and protease inhibitors (Protease Inhibitor Mix G and Phosphatase Inhibitor Mix I, Serva, Heidelberg, Germany). Samples were separated by SDS-PAGE on 12% polyacrylamide gel and then transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). After blocking in Tween-PBS with 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) for at least 1.5 h, membranes were incubated with GM3 synthase and GM2/GD2 synthase antibody (1:2000; Santa Cruz sc-365329 and sc-376505, Dallas, TX, USA), or β-actin (1:2000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After washing, membranes were incubated with anti-mouse m-IgGκ BP-HRP (Santa Cruz sc-516102, Dallas, TX, USA) for 1 h. Immunocomplexes on the membranes were visualized with ECL Western Blotting Detection Reagents (Cell Signaling Technology) using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

At 48 h post-transfection, Huh7 cells were washed with phosphate-buffered saline (PBS) and lysed under denaturing conditions in lysis buffer (2% SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0) supplemented with 10 mM NEM (N-ethylmaleimide, all Sigma-Aldrich, St. Louis, MO, USA), protease inhibitors (Protease inhibitor cocktail, Sigma-Aldrich, St. Louis, MO, USA), and phosphatase inhibitors (Phosphatase-Inhibitor-Mix I, Serva Electrophoresis GmbH, Heidelberg, Germany) [37 (link)]. The cell lysates were boiled for 10 min and each sample was 10x diluted with the dilution buffer (10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, pH 8.0) supplemented with 10 mM NEM, protease, and phosphatase inhibitors. The samples were incubated at 4 °C for 30 min with rotation followed by centrifugation at 20,000× g for 30 min at 4 °C. The precleared supernatants were used for co-immunoprecipitation, BCA protein concentration measurement (Pierce™ BCA Protein Assay Kit, ThermoFisher Scientific, Waltham, MA, USA), and sample preparation for SDS-PAGE in protein loading buffer (100 mM Tris-HCl, 4% SDS, 3% beta-mercaptoethanol, 0.2% bromophenol blue, 20% glycerol, pH 6.8).

Cells grown at 50% confluency were lysed in RIPA buffer which was supplemented with phosphatase and protease inhibitors (Protease-Inhibitor Mix G and Phosphatase-Inhibitor-Mix I, Serva, Heidelberg, Germany). The cell lysates were clarified by centrifugation, separated by SDS-PAGE, blotted to 0.45 μm nitrocellulose membranes, blocked in 5% nonfat milk in PBS, and probed with primary antibodies. Fluorescent labeled anti-rabbit IRDye 700 and anti-mouse IRDye 800 secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) were used for visualization using an Odyssey infrared imaging system (LI-COR Biosciences). The rabbit phospho-p44/42 MAPK (Erk1/2), rabbit phospho-Akt (Ser473), mouse p44/42 MAPK (Erk1/2), and rabbit AKT primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The mouse p120RasGAP antibody was obtained from ECM Biosciences LLC (Versailles, KY, USA).