Publication protocol
Cells (2×106) were treated with 100 nM RAD001 for 48 h at 37°C, and then with and without 2.5 nM YM155 1 h prior to RAD001 treatment. Control cells were treated with equivalent quantities of DMSO. Cells then were lysed by incubating in lysis buffer (1% Triton, 0.1 % sodium dodecyl sulfate-SDS, 150 mM NaCl, 50 mM Tris HCl pH 7.4, 2 mM EDTA) plus a protease inhibitor cocktail tablet (Roche Applied Science, Penzburg, Germany) for 30 min at 4°C. Lysates were then centrifuged at 16,000 × g for 15 min at 4°C and the supernatant was collected. The protein concentration was evaluated using the Bio-Rad Protein Concentration assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein lysate samples (50–100 µg) were separated by molecular weight on 10, 12 or 14% SDS-PAGE and then transferred onto nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in 5% non-fat dry milk and then incubated with primary antibodies overnight at 4°C, washed in Tris-buffered saline with 0.1% Tween-20 and then incubated with horseradish peroxidase-conjugated anti-mouse IgG (cat. no. A4416) or anti-rabbit IgG (cat. no. A0545) (both 1:5,000; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. The filters were then developed using enhanced chemiluminescence (Super Signal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using Kodak X-Omat films (Kodak, Rochester, NY, USA). The primary antibodies used were as follows: Rabbit anti-survivin (1:1,000; cat. no. NB500-201; Novus Biologicals, Littleton, CO, USA), mouse anti-poly (ADP-ribose) polymerase 1 (PARP1; 1:500; cat. no. SC-8007; Santa Cruz Biotechnology, Dallas, TX, USA), which is able to detect both cleaved and uncleaved PARP1, mouse anti-caspase-8 (1:500; cat. no. 9746; Cell Signaling Technology, Danvers, MA, USA) which is able to detect both cleaved and uncleaved caspase-8, mouse anti-caspase-9 (1:500; cat. no. 9508; Cell Signaling Technology), mouse anti-B cell lymphoma 2 (Bcl2; 1:250; cat. no. Sc7382; BD Biosciences, Franklin Lakes, NJ, USA), rabbit anti-BCL2 associated X, apoptosis regulator (Bax; 1:250; cat. no. Sc493; BD Biosciences), rabbit anti-LC3 (1:500; cat. no. ABC232; Sigma-Aldrich, St. Louis, MO, USA), which is capable of recognizing the doublet of LC3 composed of the two single bands of LC3I and LC3II; rabbit anti-beclin-1 (1:500; cat. no. 62557; Abcam, Cambridge, UK), mouse anti-β-actin (1:750; cat. no. A5060; Sigma Aldrich, St. Louis, MO, USA).
Full paper
Login or
join for free to view the full paper.