2.9. Western blot Analysis

Western blot analysis was performed according to the standard method described by Buchmann [24]. Following treatment, the media was removed, and the cells were lysed by in RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). Total protein concentration was determined using a BCA protein assay kit according to the manufacturer protocol (Pierce, Rockford, IL, USA). Samples were mixed with 3× blue sample buffer containing 50 mM dithiothreitol (Sigma-Aldrich, St. Louis, MO, USA) (reducing agent) then boiled at 100 °C for 3–5 min to denature proteins. An equivalent volume to 20 µg of the whole cell lysates were loaded into each lane and subjected to SDS-PAGE electrophoresis, then transferred to a 0.2 µm pore size nitrocellulose membrane Whatman Protran^® (Thermo Fisher Scientific, MA, USA) using a SemiPhor semi-dry transfer system (G. E. Healthcare, Chicago, IL, USA). The membranes were blocked by incubation in TBS-T buffer (50 mM tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) containing 5% non-fat milk (Sigma-Aldrich, St. Louis, MO, USA) or BSA (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with a primary antibody against either DR4 (1:250), Cdk1, Bcl-2 and p-P38 (Santa Cruz, Dallas, Texas, USA; all 1:1000 dilution), cFLIP (Enzo, Exeter, UK; 1:500 dilution), FADD (Enzo, Exeter, UK; all 1:1000 dilution), CHOP (Cell Signalling Technology Inc., MA, USA; 1:500 dilution). Bax, JNK, P-JNK, P38, p-P38, ERK, p-ERK, and GAPDH (Cell Signalling Technology Inc., MA, USA; all 1:1000 dilution). The blots were washed with TBS-T and incubated with the appropriate species of HRP coupled secondary antibody diluted in TBS-T containing 5% BSA or non-fat milk (Cell Signalling Technology Inc., Danvers, MA, USA; 1:2000 dilution) for 1 h at room temperature. Immunodetection was performed using the SuperSignal West Pico Substrate (Thermo Scientific, Rockford, IL, USA). Following the incubation of the blots with the Chemiluminescent Substrate, blots were air-dried and transferred to a sealed cassette and exposed to autoradiographic developing film for various lengths of time depending on the strength of the signal. The film was developed in an automatic developer. Following detection, the blots were washed three times with TBS-T, and re-probed for GAPDH loading control. If the bands located within GAPDH detection range, stripping buffer was used to strip off the blots before re-probing with GAPDH primary antibody. In some cases, samples from different timepoints were run on different blots due to logistical limitations, however, all samples were normalized to their GAPDH loading controls.

ImageJ software 1.50i (National Institutes of Health, Bethesda, MD, USA) (http://imagej.nih.gov/ij) was used for greyscaling and performing densitometry analysis of the bands. All presented blots were subjected to automatic corrections for brightness.