3 n morpholino propanesulfonic acid buffer

Proteins were separated by gel electrophoresis using NuPAGE gels and 3-(N-morpholino)propanesulfonic acid buffer (Life Technologies). Proteins were transferred onto nitrocellulose membranes for 120 min at 100 V at 4°C. The membranes were blocked with phosphate-buffered saline (PBS) containing 5% (wt/vol) blotting-grade nonfat dry milk blocker (Rockland, Limerick, PA) and 0.05% (wt/vol) Tween 20. Proteins were detected by probing the membranes with the indicated primary antibodies at appropriate dilutions and using a detection system consisting of horseradish peroxidase–conjugated secondary antibodies (Bio-Rad Laboratories, Hercules, CA) and the chemiluminescence substrates SuperSignal, WestPico, and WestFemto (Thermo Scientific) and then visualized using Hyperfilm (Denville Scientific, Holliston, MA). For immunoprecipitation assay, cells were lysed either by nitrogen cavitation in relaxation buffer supplemented with 1% NP-40 or directly using RIPA buffer (50 mM Tris/HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40), the samples were cleared by centrifugation, and the supernatants were then incubated with anti–M2 agarose beads (Sigma-Aldrich, St. Louis, MO) at 4°C with rotation overnight. After three washes with lysis buffer, the immunoprecipitates were subjected to Western blotting.