TGF-beta Pan Specific Antibody

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	-Rabbit (1:500)

Protocol tips
-Rabbit (1:500)

Publication protocol

Tumor tissues were homogenized and lysed in a custom-made total protein lysis buffer (50 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% deoxycholate, 1% Triton X-100) containing protease and phosphatase inhibitors (Roche). Protein concentration was measured using a bicinchoninic acid (BCA) assay (Pierce) and 30 μg protein was loaded per lane for all immunoblots. The protein lysates were resolved using NuPAGE Novex 4%–12% Bis-Tris Gels (Invitrogen). Protein transfer was assessed by Ponceau S solution (Sigma-Aldrich), and then membranes were blocked with 5% fat-free dry milk for 1 hour. Immobilized antibody was detected using the horseradish peroxidase–conjugated secondary antibodies and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The immune reaction was visualized with an LAS-3000 imaging system (FujiFilm). Primary antibodies used were: rat anti–GM-CSF (1:500, catalog ab13789-100, Abcam), rabbit anti–MCP-1 (1:2,000; catalog NB110-2000; Novus Biologicals), rabbit anti–IFN-γ (1:500, catalog AAM29, AbD Serotec), rabbit anti–TNF-α (1:100, catalog ACC-250844, BioSite), rabbit anti-NOS2 (1:1,000; catalog 2982S; Cell Signaling Technology), goat anti–IL-1β (1:500, AF-401-NA, R&D Systems), rat anti–IL-10 (1:500, catalog ab33471, Abcam), rabbit anti-VEGF (1:500, catalog ab46154, Abcam), goat anti-MCSF (1:500, catalog af416-sp, Novus Biologicals), rabbit anti–TGF-β (1:500, catalog AB-100NA, R&D Systems) and rabbit anti–β actin (1:500, catalog ab8227, Abcam).

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Manufacturer protocol

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