Bcl 2

Cells were washed twice in PBS and lysed using M-PER mammalian protein extraction reagent (Thermoscientific) according to the manufacturer’s recommendations. Protein concentrations were quantified using the Pierce 660-nm protein assay reagent (Thermoscientific) according to the manufacturer’s suggested protocol. Whole-cell lysates (20 μg) were resolved by SDS-polyacrylamide gel electrophoresis on 10%–15% gels, and transferred to polyvinylidene difluoride membranes (Immobilon; Amersham, Arlington Heights, IL). Membranes were then probed sequentially with antibodies against Bcl-2 (1∶1,000; Epitomics, Burlingame, CA), Bcl-xL (1∶1,000; Cell Signaling Technology), Bax (1∶1,000; Cell Signaling Technology), cleaved caspase-3 (1∶1000; Cell Signaling Technology), and GAPDH (1∶10,000; Abcam, Cambridge, UK). The blots were developed using an enhanced chemiluminescence substrate (Western Lightning ECL Pro) kit (PerkinElmer, San Jose, CA).

Equal amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membranes were blocked in 5% non-fat skim milk/TBST [20 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 0.1%Tween-20] at room temperature for 1 h. Thereafter, they were detected with primary antibodies at 4°C overnight. Then, they were blotted for 1 h at room temperature with the help of an appropriate secondary antibody. Thereafter, enhanced chemiluminescence detection reagents were used (Amersham Pharmacia Biotech, USA). The primary antibody PRRX1was purchased from Abcam(UK). Caspase-3, Bcl-2, andγ-H2AX were purchased from Epitomics(UK). E-cadherin, ZO-1, Vimentin, N-cadherin were purchased from Becton, Dickinson and Company(USA). GAPDH was purchased from BOSTER(China).

Cell pellets were homogenized in extraction buffer (50 mmol/L Tris-HCl, pH 6.8, 0.1% SDS, 150 μmol/L NaCl, 100 mg/L phenylmethylsulfonyl fluoride, 1 mg/L aprotinin, 1% NP-40 and 0.5% sodium orthovanadate), incubated at 4°C for 30 min, and centrifuged for 20 min at 12000 g/min. Total protein in the cell lysate was measured with use of the Bio-Rad colorimetric kit (Bio-Rad, Hercules, CA, USA). For western blot analysis, total protein was separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes (0.45 μm, Millipore, Billerica, MA, USA), which were incubated for 24 h at 4°C with the antibodies for α5-nAChR (1:500, ab41173 or ab166718), AKT(1:500, Epitomics Cat no:1085–1), P-AKT(1:500, Epitomics Cat no:2118–1), Caspase-3 (1:500, Epitomics Cat no:1087–1), Bcl-2 (1:500, Epitomics Cat no:1017–1), Survivin (1:500, Epitomics Cat no:2463–1) and GAPDH (1:1000; ab37168), then horseradish peroxidase-conjugated anti-mouse/rabbit IgG antibody (Santa Cruz Biotechnology) after a final wash. Signals were detected with use of an enhanced chemiluminescence kit (Amersham Pharmacia, Buckinghamshire, UK). GAPDH level was an internal standard.

Rabbit antibodies against Phospho-eIF2α (Ser51), eIF2α, PKR were purchased from Cell Signaling Technology (New England Biolabs, Pickering, ON). Phospho-PKR (pT446), P-PKR (Thr 446), PKR (B-10), PACT, Bcl-2, Bax antibodies were from Epitomics (Burlingame, CA) or Santa Cruz (Santa Cruz, CA). Affinity purified goat polyclonal anti-GFP (EU4) (Eusera, Edmonton, AB) was utilized for immunoprecipitation and mouse monoclonal anti-GFP (B-2) (Santa Cruz) for immunoblotting using GFP-tagged plasmids. Rabbit (A2066, Sigma-Aldrich Canada, Oakville, ON) or mouse anti-β-actin (C4, Santa Cruz), goat anti-FLAG (Santa Cruz) and mouse monoclonal anti-HSP60 (H-1) (Santa Cruz) antibodies were used for loading controls. Secondary antibodies were purchased from GE Healthcare (Baie d’Urfe, Quebec) or Santa Cruz. Tm and IFN-α were from Sigma-Aldrich Canada.

Cells and tissue samples were lysed in RIPA buffer with PMSF as protease inhibitor (Beyotime, Shanghai, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on NC or PVDF membrane (Millipore, Bedford, MA). After blocking with 5% dry milk in TBST for 2 h at room temperature, the membranes were incubated with the primary antibodies against SDHB (1:1000, Epitomics), β-tubulin (1:4000, Epitomics), β-actin (1:500, Abmart), caspase 3 (1:1000, Cell Signalling Technology), Bcl-2 (1:4000, Epitomics), MMP-2 (1:500, Abcam), FAK (1:1000, CST), p-FAK (1:1000, CST), AMPKα (1:1000,CST), p-AMPKα (1:1000, CST), GAPDH (1:4000, Abmart), P38 (1:1000, CST), p-P38 (1:1000, CST), ERK (1:1000, CST), p-ERK (1:1000, CST), HIF-1α (1:1000, Epitomics) in dilution buffer overnight at 4°C. Membranes were washed for three times with TBST, then were incubated with IRDye 800CW conjugated goat (polyclonal) anti-Rabbit IgG or anti-Mouse IgG (1:10000) antibodies for 1 h at room temperature. The expression of specific proteins was detected through the use of Odyssey system following the manufacturer's instructions.