Anti myc magnetic beads

The flag-ATP11B protein was purified using anti-Flag magnetic beads (B26101, Bimake), and then incubated with the flag peptide to release the protein from the anti-Flag magnetic beads. The eluate was then incubated with purified Myc-PD-L1 using anti-Myc magnetic beads (B26301, Bimake) for an additional 2 hours at 4°C. The samples were washed three times with washing buffer, boiled in 1×NuPAGE LDS Sample Buffer (NP0007, Thermo Fisher Scientific) for 5 min, and subjected to western blot.

For Co-immunoprecipitation (Co-IP) assay, cells were collected 24 h after transfection and lysed in lysis buffer [1.0% (v/v) NP-40, 50 mM Tris-HCl, pH 7.4, 50 mM EDTA, 0.15 M NaCl] supplemented with a protease inhibitor cocktail (Sigma), and a phosphatase inhibitor cocktail (Sigma). After centrifugation for 10 min at 14,000 × g, supernatants were collected and incubated with the indicated antibodies followed by the addition of protein A/G beads (Santa Cruz), with Anti-Flag magnetic beads (Bimake), anti-Myc magnetic beads (Bimake), or anti-HA magnetic beads (Bimake). After incubation overnight at 4°, beads were washed four times with lysis buffer. Immunoprecipitates were eluted by boiling with 2×SDS loading buffer containing 100 mM Tris-HCl pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 0.2% (w/v) bromophenol blue, and 1% (v/v) 2-mercaptoethanol.

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).