Protein purification and in vitro kinase assays

For purification of PD-L1, Flag-tagged PD-L1 was transfected into HEK293T cells for 24 h. Cells were lysed in 1 mL of lysis buffer (TAP) (0.5% NP-40, 1 mM Na₃VO₄, 20 mM Tris-HCl pH 7.5, 1 mM NaF, 150 mM NaCl, 1 mM EDTA) supplemented with a complete protease inhibitor cocktail (Bimake, added fresh), and incubated with anti-Flag magnetic beads (after washing the beads with PBS twice) for 6 h on a rotating wheel at 4 °C. The beads were washed with TAP buffer three times and treated with CIP (#M0290, Biolabs) at 37 °C for 30 min. The kinase assays were performed with recombinant human GSK3α proteins (#Ab42597, Abcam). The purified PD-L1 or synthetic peptides (PD-L1-WT/2SA) were incubated in 30 μL of kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na₃VO₄, 10 mM MgCl₂) supplemented with phosphatase inhibitor cocktail (#b15001, Bimake), with or without 100 μM ATP for 2 h at 37 °C. The reactions were stopped by adding SDS-PAGE 2× loading buffer (100 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.1% Bromophenol blue 0.2 M DTT) and heating at 100 °C for 10 min. Kinase activity was evaluated by dot blot or western blotting with anti-phospho-human PD-L1 Ser279 antibody.