Sodium dodecyl sulfate sample buffer

Cells were lysed at 4°C in ice-cold RIPA buffer (9806, Cell Signaling Technology) with protease inhibitor cocktail (G6521, Promega), and cell lysates were cleared by a brief centrifugation (12000g, 10 min) (Tang et al., 2010 (link)). Concentrations of proteins in the supernatant were determined by a BCA Protein Assay Kit (7780, Cell Signaling Technology). Prior to immunoprecipitation, samples containing equal amounts of proteins were pre-cleared with protein A agarose beads (9863, Cell Signaling Technology) at 4°C for 3 h, and subsequently incubated with various irrelevant IgG or specific antibodies (3–5 μg/mL) in the presence of protein A agarose beads for 2 h or overnight at 4°C with gentle shaking (Li et al., 2021c (link); Zhu et al., 2017 (link)). Following incubation, protein A agarose beads were washed extensively with phosphate-buffered saline and proteins were eluted by boiling in 2 × sodium dodecyl sulfate sample buffer (LC2676, Thermo Fisher Scientific) before electrophoresis of the sodium dodecyl sulfate polyacrylamide gel.