Xpert protease inhibitor cocktail solution

Cells were seeded in a six-well plate at a density of 4–6 × 10⁴ cells/well and allowed to incubate for 24 h. Extract was added to each well and incubated for 48 h. Cells were harvested and lysed in RIPA buffer (GenDEPOT) with protease inhibitors (Xpert protease inhibitor cocktail solution, GenDEPOT) and phosphatase inhibitors (Xpert phosphatase inhibitor cocktail solution, GenDEPOT). Cell lysates were boiled in 5x sample buffer and separated by 10% SDS-PAGE. Proteins were transferred onto PVDF membranes (Millipore) using a semidry electroblotter (Peqlab, Germany). Membranes were blocked with 5% skim milk in TBS-T (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) and sequentially incubated overnight with primary antibodies at 4°C. Membranes underwent additional incubation at room temperature and were then probed with secondary antibody. Immunoreactive proteins were visualized using ECL reagents and developed with X-ray film. Antibodies and dilutions are as follows: p53, p21 (1 : 2000, Millipore), c-Jun N-terminal kinase (JNK, 1 : 500, Santa Cruz Biotechnology), Bax (1 : 1000, Cell Signaling), caspase-3, caspase-8, caspase-9 (1 : 1000, Cell Signaling), β-actin (1 : 5000, Sigma-Aldrich), anti-mouse IgG (H+L) horseradish peroxidase conjugate, and anti-rabbit IgG (H+L) horseradish peroxidase conjugate (1 : 3000, Bio-Rad).

The cell lines A549 and H1299 were harvested and washed twice with PBS. Cells were lysed in a RIPA buffer containing Xpert phosphatase inhibitor cocktail solution and Xpert protease inhibitor cocktail solution (GenDEPOT, Katy, TX, USA) on ice for 10 min and centrifuged at 15,000 rpm for 15 min at 4 °C [21 (link)]. Total protein concentrations of the supernatants were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The protein lysates were loaded onto an 8~10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in 5% skim milk for 1 h and incubated with primary antibodies at 4 °C for overnight. After incubation of the primary antibodies, the membranes were washed with TBST, followed by the HRP-conjugated secondary antibody (1:5000) at room temperature for 1 h. The bands were detected using West-Q ECL solution (GenDEPOT, Katy, TX, USA).

Cells were treated with cold sample lysis buffer (1% Triton X-100, Xpert Protease Inhibitor Cocktail Solution (GenDEPOT, Katy, TX, USA), 5 mM Ethylenediaminetetraacetic acid (EDTA, Thermo Fisher Scientific, Waltham, MA, USA), and 1 mM Phenylmethanesulfonyl fluoride (PMSF, Thermo Fisher Scientific, Waltham, MA, USA)). Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used for determining the protein concentration. An equal amount of protein was loaded for SDS-PAGE and transferred onto PVDF membrane using Wet/Tank Blotting Systems (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membranes were incubated with 5% Difco^TM Skim Milk (BD Biosciences, San Jose, CA, USA) for 30 min at room temperature. The membranes were incubated with cleaved caspase-3 (Asp175) antibody (Cat. 9661S, Cell Signaling Technology, Danvers, MA, USA) and GAPDH antibody (Cat. sc-47724, Santacruz Biotechnology Inc., Dallas, TX, USA) overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). We added ECL^TM Select Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) on the membrane for the detection of the signals from HRP. The images of protein bands were obtained by a LAS-3000 imaging system (Fujifilm, Minato, Tokyo, Japan).