C100860

Western blots were were blocked using 3% BSA (Millipore, C100860) for 1 h at room temperature. They were incubated with Aleuria Aurantia Lectin (AAL), Biotinylated (Vector Laboratories, California, USA, B-1395-1) overnight at 4 °C and High Sensitivity Streptavidin-HRP (Biolegend, California, USA, 405210) for 1 h at room temperature. Then, they were detected using chemiluminescence (GE Healthcare Life Sciences, New Jersey, USA, RPN2235).

Cells were harvested, washed twice with PBS, and lysed using lysis buffer with 20 mM HEPES (pH 7.5), 150 mM NaCl, 10% Glycerol, 5 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail (Roche, Basel, Switzerland, 11836153001). For immunoprecipitation, the cell lysate was incubated overnight at 4 °C with indicated antibody. Dynabeads Protein G (Invitrogen, 10003D) was added and incubated for another hour. The proteins were washed four times with lysis buffer, eluted in SDS sample loading buffer, separated by SDS–polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Massachusetts, USA, IPVH00010), and blocked using 3% BSA (Millipore, C100860) for 1 h. The blots were incubated with appropriate primary antibodies overnight at 4 °C and horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Then, they were detected using chemiluminescence (GE Healthcare Life Sciences, New Jersey, USA).