2.6. Western Blot Analysis

Protein samples were prepared by cell lysis with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 M EDTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor cocktail (Cat: 89900, 87786, 78420, Thermo, Waltham, MA, USA)) and were quantified using the Bio-Rad DC Protein Assay Kit II (Cat: 500-0113, 500-0114, 500-0115, Bio-Rad, Hercules, CA, USA). Quantified protein samples were loaded onto 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and were transferred at 250 mA for 2 h onto a Hybond ECL transfer membrane (Cat: 10600002, 10600001, Amersham Pharmacia, Piscataway, NJ, USA). Non-specific binding on membranes was blocked in fresh 5% non-fat dry milk (Cat: DI-232100, BD, Franklin Lakes, NJ, USA), after which the membranes were incubated with primary antibodies against SPHK-1 (ratio 1:1000, Cat: 3297, Cell Signaling, Danvers, MA, USA), HIF-1α (ratio 1:500, Cat: NB100-105, NOVUS, Littleton, CO, USA), AKT (ratio 1:1000, Cat: SC-8312, Santa Cruz, Dallas, TX, USA), p-AKT (ratio 1:1000, Cat: SC-7985, Santa Cruz, Dallas, TX, USA), GSK-3β (ratio 1:1000, Cat: 9832, Cell Signaling, Danvers, MA, USA), p-GSK-3β (ratio 1:1000, Cat: 9331, Cell Signaling, Danvers, MA, USA), Cyclin D1 (ratio 1:500, Cat: SC-8396, Santa Cruz, Dallas, TX, USA), VEGF (ratio 1:1000, Cat: SC-7269, Santa Cruz, Dallas, TX, USA), PARP (ratio 1:1000, Cat: SC-8007, Santa Cruz, Dallas, TX, USA), Bcl-2 (ratio 1:1000, Cat: SC-492, Santa Cruz, Dallas, TX, USA), cleaved caspase-3 (ratio 1:1000, Cat: 9664, Cell Signaling, Danvers, MA, USA), PCNA (ratio 1:3000, Cat: M0879, DAKO, Santa Clara, CA, USA), VE-cadherin (ratio 1:1000, Cat: ap2724a, Abcepta, San Diego, CA, USA), and β-actin (ratio 1:10000, Cat: A5316, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C. For the incubation secondary antibody on membranes, horseradish peroxidase (HRP)-conjugated anti-mouse (ratio 1:10000, Cat: 115-035-003, Jackson, West Grove, PA, USA) or anti-rabbit secondary antibodies (ratio 1:10000, Cat: 111-035-003, Jackson, West Grove, PA, USA) were used. An enhanced chemiluminescence (ECL) system (Cat: RPN2209, Amersham Pharmacia, Piscataway, NJ, USA) was used for protein expression. Densitometry data were obtained by quantifying each protein band using Image J 1.53k software (National Institutes of Health, Bethesda, MD, USA). The quantification of the protein levels was calculated from a duplicate analysis of each sample. The obtained protein levels of interest were normalized to β-actin.