Polyclonal anti cleaved caspase 3

After removal of cell culture medium from each well, the cells were washed with ice-cold PBS, lysed in RIPA buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium-deoxycholate, 1 mM sodium orthovanadate, 5 mM sodium fluoride, 1 mM PMSF, Protease Inhibitor Cocktail, from Roche Molecular Biochemicals, Mannheim, Germany) and dislodged using a sterile cell scraper. Homogenates were placed on ice for 30 min and centrifuged at 16,000× g for 15 min at 4 °C. The protein concentration of each extract sample was determined using the Micro BCA Protein Assay Kit (Pierce Biochemicals, Rockford, IL, USA). Proteins were separated on 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes at 35 V overnight [28 (link)]. The membranes were incubated for 1h at room temperature or overnight at 4 °C with the following primary antibodies: polyclonal anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, MA, USA), monoclonal anti-β-actin (Calbiochem Oncogene Research Products, Cambridge, MA, USA). The immunoreactive bands were detected by chemiluminescence, coupled to peroxidase activity (ECL kit, Thermo Scientific, Rockford, IL, USA) and imaged with a Bio-Rad ChemiDoc XRS system (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Monoclonal anti-YAP, monoclonal anti-cyclin D1, polyclonal anti-cleaved caspase 3 and polyclonal anti-pYAP antibodies were obtained from Cell Signalling Technology (Danvers, MA). Polyclonal anti-CCN1 (Cyr61), anti-CCN2 (CTGF) and monoclonal anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories (Hercules, CA). S1P was obtained from Avanti Polar Lipids (Alabaster, AL). Verteporfin (VP) was obtained from VWR International (Randor, PA). For the VP experiments, cells were treated with 10 μm of VP for 10 min, washed and then treated with 300 nM of S1P for additional 2 h. Cycloheximide (#1041) was obtained from BioVision (Mountain View, CA).

The SDS PAGE (7.5%–12%) electrophoresis was conducted at 100 V as instructed by the manufacturer (Life Science, CA, USA). The 100 μg of sample protein was loaded in each well. The primary antibodies of monoclonal anti-βactin, polyclonal anti-Bax, polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), polyclonal anti-cleaved caspase-12, and polyclonal anti-BcL-2 (BioVision Inc., Milpitas, CA, USA) were applied as 1/1000 of dilution and left in a 4 °C ice box overnight. The PVDF membranes were rinsed with TBST buffer once for 30 min. The secondary antibodies Gt × Ms IgG(H + L) HRP and goat anti-rabbit IgG pAb HRP (Stressgen Biotechnologies Corporation Corporate, San Diego, CA, USA) were applied as 1/5000 of dilution and left in 4 °C ice box for 30 min. while shaken. The membranes were rinsed with TBST buffer twice, each time for 30 min. Enhanced chemiluminescence (ECL) was added and mixed well for 5 min to facilitate the reaction. The emitted chemiluminescence was taken by the Hansor LIS02 photo system, and the intensity was quantified with Image J (NIH Image, Bethesda, MD, USA).