Rabbit anti mouse cox2

Equal amount of protein from each sample was separated in 10% SDS-PAGE and transferred to the PVDF membrane overnight at 4°C. Membrane was blocked in 5% non-fat milk in PBS (pH 7.4) for 2h at RT. Blot was incubated overnight at 4°C with rabbit anti-mouse p53 (1:500 dilution, Imgenix) or rabbit anti-mouse iNOS (1:200 dilution, Cayman) or rabbit anti-mouse COX2 (1:500 dilution, Cayman) in 1% BSA and 0.05% Tween-20 in PBS (pH 7.4). Blot was washed and incubated with HRP-conjugated goat anti-rabbit IgG (1:2500 dilution, Bangalore Genei) in PBS (pH 7.4) containing 1% BSA and 0.05% Tween-20 for 2 h at RT. Immunoreactive proteins were detected with ECL super signal kit (Pierce Biotechnology) in X-ray film. Band density values were normalized with β-actin.

The primary antibodies that were used for immunohistochemistry and immunoblotting included rabbit anti-mouse COX-2 from Cayman Chemicals, cyclin D1 and c-Myc from Santa Cruz Biotechnology; rat anti-mouse F4/80 (marker of macrophages/dendritic cells) from AbD Serotec; rabbit anti-p-ERK, p-p70 S6K (Thr389), p-PI3K p85 (Tyr458), p-PDK1 (Ser241), p-AKT (Thr308), p-mTOR (Ser2448), p-raptor (Ser792), p-elF-4B(Ser422), p-S6 ribosomal protein (p-rpS6, Ser240/244), and rabbit anti-cleaved caspase-3 (9661) from Cell Signaling Technology; rabbit anti-iNOS, rabbit-anti-Ki67 (ab15580), and goat anti-arginase 1 from Abcam, mouse anti-interleukin 4 receptor α (IL-4Rα) and mannose receptor (MR, CD206) from R&D.