Protein a conjugated agarose beads

Immunoprecipitation was performed following standard procedures. In brief, cells were washed in PBS and lysed in cold NP-40 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40) supplemented with a protease/phosphatase inhibitor cocktail (Cell Signaling Technology). The lysate was briefly sonicated and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was then collected and 1 mg of total protein lysate was incubated with protein A-conjugated agarose beads (Beyotime technology, P2051) and primary antibody overnight at 4°C. Rabbit anti-FLAG antibody (Beyotime biotechnology, AF0036, 1: 200 dilution) was used. The beads were spun down, washed with PBS buffer, and denatured with 2 × Laemmli sample buffer (Bio-Rad), followed by western blotting for validation.

Immunoprecipitation was performed following standard procedures. In brief, cells were washed in PBS and lysed in cold NP-40 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40) supplemented with a protease/phosphatase inhibitor cocktail (Cell Signaling Technology). The lysate was briefly sonicated and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was then collected and 1 mg of total protein lysate was incubated with protein A-conjugated agarose beads (Beyotime technology, P2051) and primary antibody overnight at 4°C. Rabbit anti-FLAG antibody (Beyotime biotechnology, AF0036, 1: 200 dilution) was used. The beads were spun down, washed with PBS buffer, and denatured with 2 × Laemmli sample buffer (Bio-Rad), followed by western blotting for validation.