Sb216763

DCs were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10 mM sodium pyruvate, 10 μg/mL penicillin and streptomycin, HEPES buffer, 2 g/L of NaHCO₃, 100 µM β-mercaptoethanol, and 10% fetal bovine serum (FBS) (DC culture medium, hereafter). DC culture medium was used in all in vitro experiments, unless otherwise stated. Recombinant mouse IL-4 and recombinant mouse granulocyte/macrophage colony-stimulation factors (GM-CSF) were purchased from JW Creagene (Seoul, South Korea). Monoclonal antibodies against MHC-I, MHC-II, CD80, CD86, and CD40 were obtained from eBioscience (San Diego, CA, USA). LPS from Escherichia coli (O111:b4), lanosterol, SB203580, PD98059, and LiCl were purchased from Sigma-Aldrich. Wortmannin, SB415286, SB216763, and BMS345541 were purchased from R&D Systems (Minneapolis, MN, USA).

Antibodies used were: anti-p-Src Y416, anti-Src 36D10, anti-LC3B, anti-FAK, anti-p-PDK1 S241, anti-PDK1, anti-p-Akt S473, anti-Akt, anti-GAPDH, anti-p-mTOR S2448, anti-p-mTOR S2481, anti-mTOR, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6, anti-p-p70S6K T389, anti-p70S6K, anti-mouse and anti-rabbit IgG-peroxidase conjugated secondary antibodies (all New England Biolabs, Herts, UK), anti-LC3 (MBL, MA, USA), and anti-paxillin (BD Transduction Laboratories, Oxford, UK). Alexa 488 and Alexa 594 conjugated secondary antibodies, Deep Red Cell Mask and Vectashield microscope mounting medium with DAPI were from Invitrogen (Paisley, UK).
Tocriscreen Kinase Inhibitor Toolbox, SB218078, SB216763, ZM306416, PD407824, BIO, Ki8751 and PF4708671 were all from R & D (Abington, UK), hygromycin B from Merck Biosciences (Nottingham, UK), dasatinib from Bristol Myers Squibb (Middlesex, UK) and 3MA from Sigma (Poole, UK). The Micro BCA Protein Assay Kit was from Pierce Ltd. (Northumbria, UK), Lipofectamine2000 from Invitrogen (Paisley, UK) and HiPerFect from Qiagen (Crawley, UK). siRNA was from Dharmacon (Colorado, USA).