PD-1 Immunoprecipitation and Analysis

PD-1 IP studies were performed using Pierce Protein A/G Plus agarose beads (Invitrogen), according to the manufacturer’s instructions. Briefly, cells were lysed in ice-cold buffer (150 mM NaCl, 50 mM Tris-HCI and protease inhibitor cocktail, Roche), sonicated (three 10 s bursts), vortexed for 2 h at 4 °C in 2% Nonidet P-40 (NP-40, Sigma), and centrifuged. B16-F10 lysates were concentrated using Microcon-10 Ultracel PL-10 filter columns, as above. Cell lysates were precleared for 2 h at 4 °C by incubation with Protein A/G Plus agarose beads previously blocked in ice-cold buffer supplemented with 1% BSA for 1 h at 4 °C, incubated with anti-mouse PD-1 (4–6 µg, RMP1-14)^{53 (link)} or rat IgG2a isotype control abs for 2 h at 4 °C, and then with Protein A/G Plus agarose beads overnight at 4 °C under continuous rotation. Supernatants were kept for assessment of IP efficiency, Protein A/G Plus agarose beads washed extensively, and IP products eluted in ice-cold buffer, as above, supplemented with 1.5 × Non-reducing Lane Marker Sample Buffer (Thermo Fisher), and boiled at 100 °C for 7min^{54 (link)}. IP products were then analyzed by immunoblotting, as above, or resolved in 7.5% SDS-PAGE gels, stained with Colloidal Coomassie Blue (Bio-Rad), and subjected to MS sequencing, as described below.