Immunosuppressive Factors in Cancer

RPMI-1640 medium, foetal bovine serum, supplements as well as basic laboratory chemicals were purchased from Sigma (Suffolk, UK). Maxisorp™ microtitre plates were obtained from Nunc (Roskilde, Denmark) and Oxley Hughes Ltd (London, UK). Mouse monoclonal antibodies directed against HIF-1α, mTOR and β-actin, as well as rabbit polyclonal Antibodies against phospho-S2448 mTOR, RAGE and HRP-labelled rabbit anti-mouse secondary antibody were purchased from Abcam (Cambridge, UK). Antibodies against phospho-S65 and non-phosphorylated (total) eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) were obtained from Cell Signaling Technology (Danvers, MA USA). Goat anti-mouse and goat anti-rabbit fluorescence dye-labelled antibodies were obtained from LI-COR (Lincoln, Nebraska USA). ELISA-based assay kits for the detection of TNFα, IL-1β, SCF and VEGF were purchased from Bio-Techne (R&D Systems, Abingdon, UK). Anti-Tim-3 mouse monoclonal antibody, its single chain variant as well as human Ig-like V-type domain of Tim-3 (amino acid residues 22–124) and human HMGB1 expressed and purified from E. coli (see below for more details) were used in our experiments.¹¹ (link)^,15 (link) All other chemicals purchased were of the highest grade of purity commercially available.