Dye reagent

The Bio-Rad protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used to measure the protein concentration of the HCE cell samples. The bovine serum albumin standards (0.1–2 µg/mL) were prepared in blank cell lysate, and the lysed samples were centrifuged at 2800 rpm for 5 min. The supernatant (5 µL) in duplicates were then transferred into 96-well plates. Dye reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) solution (Dye reagent concentrate diluted in distilled water at 1:4 ratio) was added to each well (200 µL/well) and incubated for 15 min at room temperature. The absorbance was measured at 595 nm in Victor² multilabel plate reader (PerkinElmer, Wallac, St. Paul, MN, USA)

Protein concentration was determined according to Bradford (Bradford, 1976 (link)) by utilizing a dye reagent purchased from Bio-Rad Laboratories, Inc. (Hercules, CA), and SDS-PAGE was performed according to Laemmli (1970) (link). Western blotting was performed as described previously (Mukhopadhyay et al., 1995 (link)) with the following modifications. Monoclonal ANTI-FLAG^® M2 antibody produced in mouse (catalog number, F1804; Sigma-Aldrich, Inc., St. Louis, MO) served as the primary antibody, and anti-mouse IgG (whole molecule) rabbit antibody conjugated with alkaline phosphatase (catalog number, A2418; Sigma-Aldrich) was the secondary antibody. The antibody reacting band was detected with BCIP and NBT.

Total proteins were extracted from U87-MG cells using RIPA buffer (25 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% NP-40; 1% sodium deoxycholate; and 0.1% SDS) containing a protease inhibitor cocktail (complete, Mini, EDTA-free, Roche Applied Science, Penzberg, Germany) and homogenized using an ultrasonic homogenizer. After quantitative analysis using the Bradford protein assay with a Bio-Rad Dye Reagent (500-0006, Bio-Rad, Hercules, CA, USA), the protein samples were denatured with 5× sample buffer (10% SDS, 25% beta-mercaptoethanol, 50% glycerol, 0.25 M Tris-HCl [pH 6.8], and 0.01% bromophenol blue) and incubated in a water bath at 95°C for 10 min. Cell protein lysates were loaded into 10% SDS-polyacrylamide gels. The gels were transferred onto PVDF filters and incubated with a specific primary antibody to caspase-3 (1:1000) (Novusbio Inc., USA), cleaved caspase-3 (1:1000) (Novusbio Inc., Centennial, CO, USA), PARP (1: 1000) (Cell Signaling Inc., Danvers, MA USA), cleaved PARP (1:1000) (Cell Signaling Inc.), and β-actin (1:1000) (Cell Signaling Inc.). All blots were normalized to the internal standard of β-actin. Bands of interest were visualized using ECL reagents (PerkinElmer, Waltham, MA, USA) and quantified using the UVP BioImaging system (Biospectrum AC Imaging System, Upland, CA, USA) and the ImageJ software program (National Institutes of Health, Bethesda, MD, USA).