Ip beads

Purified protein complex eluates were concentrated using IP beads (Bimake), resolved by SDS-PAGE, and stained with Coomassie Brilliant Blue (Beyotime). Stained bands that differed from the control were excised, subjected to in-gel reduction, alkylated, and digested with trypsin (Gibco) at 37 °C overnight. The digested peptides were dried and resuspended in an MS-compatible buffer, and the mixture was analyzed using the LTQ Orbitrap Velos MS (Thermo Fisher) in combination with the UltiMate RSLC Nano LC system (Dionex). The Proteome Discoverer 1.4 software (Thermo Fisher) was used to identify the protein, and the files were imported and used to explore the UniProtKB/Swiss-Prot database. The mass tolerances of precursors and fragments were set to 10 ppm and 0.8 Da, respectively. Data on the peptides with a false discovery rate of <1% (q < 0.01) were discarded.

Cells were cultured in 10 cm dishes for 48 h after transfection and were harvested using an IP lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40, 0.1% SDS) containing a proteinase inhibitor cocktail (Keygen). Protein lysates were incubated on a rotator with 1 µg of primary antibodies for 2 h at room temperature, followed by the addition of 30 µL of IP beads (Bimake, China) and incubation on a rotator at 4 °C overnight. The beads and immune complexes were subjected to washing steps with IP lysis buffer 5 times, with each wash involving rotation at 20 s per round for 5 min at room temperature. The samples were boiled in SDS loading buffer at 95 °C for 5 min. The immunoprecipitated samples were detected via western blotting.