Anti ha

For the Co-IP assay, HEK293T cells were harvested 36 h after transfection and lysed in lysis buffer (RIPA). Lysates were clarified for 10 min at 12,000 rpm at 4°C, incubated with anti-HA, anti-FLAG, or anti-Myc magnetic beads (Bimake, TX, USA), washed three times, and then incubated at 4°C overnight. Immunoprecipitates were eluted by boiling in 5× SDS loading buffer containing 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 100 mM Tris-HCl (pH 6.8), 10% (w/v) SDS, and 1% (v/v) 2-mercaptoethanol.
For protein mass spectrometry, immunoprecipitates were digested with trypsin, and the mass spectrum of the peptide mixture was acquired using MALDI (EASY-nLC 1200, USA). The target peptides were identified by searching against the UniProt database of human proteins.
For Immunoblotting, samples are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes, blocked with 5% (w/v) nonfat milk for 30 min, incubated with the indicated specific antibodies and corresponding secondary antibodies, then visualized with ECL western blotting detection reagent (Tanon, Beijing, China).

1×10⁷ Cells were harvested by cytology brush, and lysed with RIPA lysis buffer (Yeasen Biotech, 20118ES60) for protein supernatant, followed by adding immune magnetic beads (Anti-Myc, Anti-HA and Anti-Flag, Bimake) for continuous slight mixing in 4°C for 24h. And then gain immune magnetic beads with Magnetic frame (Bimake), followed by TBS washing. Finally, products were boiled before dissolved in 5x SDS (Yeasen) for 5-10 minutes for western blot assay.

For immunoblotting, RIPA buffer supplemented with phosphatase and protease inhibitors (#B15003 and #B14002, Bimake, Texas, America) was used to lyse cells and tissues. After quantification using a bicinchoninic acid kit (#20201ES90, Yeasen, Shanghai, China), equal quantities of proteins were separated and then transferred to PVDF membranes (#IPVH00010, Millipore, Bedford, MA, USA). After incubation with antibodies, the protein signals were detected using electrochemiluminescence reagents (#36208ES76, Yeasen, Shanghai, China). For IP assays, cellular extracts were incubated with anti-HA (#B26202, Bimake, Texas, America) or anti-Flag beads (#B26102, Biotool, Beijing, China) at 4 °C, followed by immunoblotting. The information on antibodies is listed in Supplementary Table S4.