Anti mpd 1 rpmi 30

Zymosan A from Saccharomyces cerevisiae was purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-mouse mAbs, such as CD11b-FITC, F4/80-PECy7, CD11c-PerCP-Cy5.5, CD19-BV650, MHC II (I-A/I-E)-PECy5, Siglec-F-PE, LY6G-APC, LY6C-APC-CY7, IL-6-PE and MCP-1-APC were purchased from eBioscience (San Diego, CA, USA). Recombinant mouse B7-H1/Fc chimera (1019-B7) was purchased from R&D Systems (Minneapolis, MN, USA). Human IgG for control experiments was purchased from Sigma–Aldrich. Anti-mouse PD-1 (clone J43) were purchased from eBioscience. LY294002, PD98059, SP600125 and SB203580 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA). The following mAbs were used for western blotting and immunoprecipitation: anti mPD-1 (RPMI-30; eBioscience), anti-phosphotyrosine Ab (Cell Signaling Technology, Beverly, MA, USA), anti-SHP-1 (sc-287), anti-SHP-2 (sc-280), p-STAT1, STAT1, p-STAT6 and STAT6 (Cell Signaling Technology), anti-mouse IgG-HRP, anti-rabbit IgG-HRP Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

For immunoprecipitation, BMDMs stimulated with zymosan (20 μg/ml) for 12 h were washed twice with cold PBS and resuspended in 0.5 ml of PBS. The zymosan-stimulated BMDMs were stimulated with rmB7-H1 at 37 °C for 5 and 15 min. Cells were lysed in 0.5 ml of 1% NP-40 lysis buffer for 30 min and pelleted at 14 000 r.p.m. for 10 min at 4 °C. To detect PD-1 phosphorylation and recruitment of SHP-1 and SHP-2, whole cell lysates were incubated overnight with 5 μg/ml anti-mPD-1 (RPMI-30; eBioscience). The next day the samples were incubated with protein A/G-agarose (sc-6244; Santa Cruz Biotechnology) for an additional 3 h at 4 °C, then washed five times with 1% NP-40 lysis buffer and finally resuspended in SDS loading buffer. The immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose. Blots were probed with appropriate primary and secondary antibody combinations, and proteins were visualized by using an ECL kit (Pierce, Rockford, CA, USA).