Co-immunoprecipitation analysis

Whole-cell lysates were prepared from native lysis buffer containing a complete protease inhibitor cocktail. The supernatant was collected after centrifugation at 14,000 g for 15 min and incubated with specific antibodies for 12 h at 4 °C with constant rotation, followed by the addition of 50 μL of Protein A+G agarose beads (P2055, BIOTEK, China) and incubation with constant rotation at 4 °C for 4 h. The lysate was washed three times for 5 min each time, and then washed with the lysis buffer for 5 min each time. The beads were washed with lysis buffer 3 times for 5 min each. The precipitated proteins were then eluted from the beads by adding SDS-PAGE Sampling Buffer and boiling at 100 °C for 10 min. Immunoprecipitate or cell lysate samples are used for WB analysis.