Erk1 2 mk1

Two platinum-resistant human ovarian cancer cell lines OVCAR-3 (p53 mutant) and A2780/CP70 (p53 wild-type) were kindly provided by Dr. Jiang at West Virginia University. IOSE-364, a normal ovarian surface epithelial cell line, was a gift from Dr. Auersperg at University of British Columbia, Canada. All cells were cultured in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37 °C in a humidified incubator with 5% CO₂. ChK, kindly provided by Dr. Cutler at the University of Mississippi, was prepared in dimethyl sulfoxide (DMSO) at 100 mM and stored at −20 °C. Cisplatin, pifithrin (PFT)-α and 2′,7′-dichlorofluorescein diacetate were purchased from Sigma-Aldrich. The primary antibodies against Bcl-xL, Bad, p21, phospho-p53 (ser15), p53, MDM2, phospho-ERK1/2, ERK1/2 (MK1) and GAPDH were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The primary antibodies against caspase-3, -8, and -9, Puma, Bax, Bcl-2, cyclin B1, phospho-cdc2 (Tyr 15), cdc2, Fas, Fas L, DR5, FADD, Phosphop38 MAPK (Thr180/Tyr182), p38 MAPK, Phospho-SAPK/JNK (Thr183/Tyr185) and SAPK/JNK were purchased from Cell Signaling Technology, Inc. (Danvers, MA).

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).