P zap syk

Sorted M-MDSC or PMN-MDSC stimulated with or without particulate β-glucan (100 µg/ml) for indicated times were lysed in Triton X-100 lysis buffer in the presence of protease and phosphatase inhibitor. The whole cell extracts were subjected to SDS-PAGE and electro-transferred to PDVF membrane. The membranes were blocked and probed overnight at 4°C with the relevant primary and then incubated with the secondary Abs. The blots were developed with ECL Plus Western Blotting Detection Reagents (GE Healthcare). The primary Abs included: p-Erk1/2 (Thr202/Tyr204, Cell Signaling). Erk1/2 (MK1, Santa Cruz), p-Stat3 (Tyr705, Cell Signaling), p-AKT (Ser473, Cell Signaling), p-p38 (Thr180/Tyr182, Cell Signaling), p-Zap/Syk (Tyr319/Tyr352, Cell Signaling), STAT3 (C-20, Santa Cruz), p-SAPK/JNK (Thr183/Tyr185, Cell Signaling) and β-actin (Sigma-Aldrich).

For immunoblot analysis, BMM and TAM stimulated with or without WGP β-glucan were lysed in Triton X-100 lysis buffer containing protease and phosphatase inhibitors. In some experiments, the Syk inhibitor piceatannol (30 µg/ml, Sigma) was added. The whole cell extracts were separated by SDS-PAGE and electro-transferred to PDVF membrane. After blocking, the membranes were probed overnight at 4°C with appropriate primary Abs and then secondary Ab. The primary Abs included p-Erk1/2 (Thr202/Tyr204, Cell Signaling), Erk1/2 (MK1, Santa Cruz), p-Stat3 (Tyr705, Cell Signaling), p-AKT (Ser473, Cell Signaling), p-p38 (Thr180/Tyr182, Cell Signaling), and pZap/Syk (Try352, Cell Signaling). The blots were developed using ECL Plus Western Blotting Detection Reagents (GE Healthcare).