Sphk2

ABC294640 was purchased from DC Chemicals (Shanghai, China). SKI-II (4-[[4-(4-Chlorophenyl)-2-thiazolyl]amino]phenol) was purchased from Tocris Bioscience (Ellisville, Mo). Cell permeable short-chain C6 ceramide was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). S1P was purchased from Cayman Chemical Co. (Ann Arbor, MI). 5-fluorouracil (5-FU) and cisplatin were purchased from Sigma (Shanghai, China). SP600125 and JNK inhibitor II (JNKi-II) were obtained from Selleck (Shanghai, China). Antibodies against phospho (p)-AKT (Ser 473), p-AKT (Thr 308), p-ribosomal protein S6 kinase 1 (S6K1) (Thr-389), p-JNK1/2 (Thr 183/Tyr 185) and p-c-Jun (Ser73) were purchased form Cell Signaling Tech (Denver MA). Anti-AKT1, SphK2, c-Jun and tubulin antibodies were obtained from Santa Cruz (Santa Cruz, CA).

Whole cell lysates were prepared using RIPA lysis buffer (Sigma-Aldrich) containing protease and phosphatase inhibitor cocktail (Roche). Protein concentrations of cell lysates were determined by the BCA protein assay (Pierce). 30-50 μg of lysate were loaded onto a 4-20% gradient polyacrylamide gel (Bio-Rad) and subjected to gel electrophoresis. Resolved proteins were transferred to a PVDF membrane (Millipore) using the Trans-Blot® Turbo^™ transfer system (Bio-Rad) at 25V for 10 minutes. Membranes were then blocked in Odyssey Blocking Buffer (Li-Cor) or 5% non-fat dry milk in TBS-T for 1 hour and then incubated with the appropriate antibody overnight at 4°C. Antibodies included: αSMA (Abcam ab5694), FAP (Abcam ab53066), FN detecting total fibronectin (Santa Cruz Biotechnology sc-9068), FN [IST-9] detecting only FN-EDA (Abcam ab6328), GAPDH (Fitzgerald Industries 10R-G109a; Acton, MA), p38α MAPK (Cell Signaling Technology (CST) #9217), phospho-p38 MAPK (CST #9211), SPHK1 (CST #12071), and SPHK2 (Santa Cruz Biotechnology sc-22704). Following overnight incubation at 4°C, the membranes were washed with TBS-T and then incubated with the appropriate IRDye ® secondary antibody (Li-Cor) for 1 hour at room temperature. Membranes were washed with TBS-T and the membrane signal was subsequently analyzed by the Li-Cor Odyssey system.