Odyssey double color infrared laser imaging system

Sorted CD4+ T lymphocytes were treated with 100 μl of protein lysis buffer and centrifuged at 12,000 r/min for 20 min at 4°C. Supernatants were transferred to clean centrifuge tubes and tested for protein concentration using the bicinchoninic acid assay, with GAPDH as internal control (Beyotime Institute of Biotechnology, China). Equal amounts of protein were separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with 5% stacking gel, and transferred onto PVDF membranes by the semi-dry method for 40 min. The membranes were blocked in Tris-buffered saline-Tween containing 5% skimmed milk for 1 h, and incubated with the primary antibody (PDCD4, Abcam, USA; 1:2500) at 4°C overnight. After five washes of 5 min with TBST, the samples were incubated with 1:5000 infrared fluorescent secondary antibody (LI-COR, USA) at room temperature for 2 h. The membrane was then scanned on an Odyssey double-color infrared laser imaging system.

Total protein was obtained from the ventricular myocardial tissues using tissue homogenates, centrifugation and heat denaturation. The protein lysates were electrophoresed and separated by 6–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore Immobion-P; BioSharp, Anhui, China). The membranes were blocked with 5% skim milk at room temperature for 1 h, and then incubated overnight at 4°C with primary antibodies, including rabbit anti-Bcl-2 (BA0412; 1:500), rabbit anti-Bax (BA0315-2; 1:500), rabbit anti-Calr (BM1798; 1:500), rabbit anti-GRP78 (BA2042; 1:500) (all from Boster Biotechnology, Inc., Wuhan, China), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (AP0063; 1:5,000; BioWorld, Inc., Jiangsu, China). The membranes were then incubated with IRDye800-conjugated secondary antibodies (1:20,000; Rockland, Inc., Gilbertsville, PE, USA) at room temperature for 1 h. The Odyssey double color infrared laser imaging system (LI-COR; Lincoln, NE, USA) was used to detect the antigen-antibody complexes in a western blotting detection system (Bio-Rad). The results were expressed as density values normalized to GAPDH.

Cell lysates were prepared in RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (Roche, Germany) by incubating for 20 min at 4°C. The protein concentrations were determined using a BCA Protein Assay kit (Thermo Fisher Scientific., Rockford, IL, U.S.A.). SDS/PAGE separation gel (10%) was prepared. Equal amount of total proteins was separated by SDS/PAGE (10% gels) transferred on to a PVDF membrane (PerkinElmer, Boston, MA) incubated with primary antibodies overnight. The blots were subsequently incubated with HRP-conjugated secondary antibodies. An Odyssey double color infrared laser imaging system (LI-COR, Lincoln, NE, U.S.A.) was used for scanning and data analyses. GAPDH was used as the internal control for the normalization of the data. The related primary antibodies were anti-GAPDH (dilution 1:2000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-p53 (dilution 1:100, Abcam, Cambridge, MA, U.S.A.; ab28), anti-NF-κB p65 (dilution 1:1000, Abcam, ab76026), anti-mTOR (dilution 1:1000, Abcam, ab2732), anti-β-catenin (dilution 1:500, Abcam, ab6302), anti- caspase-3 (dilution 1:1000, Abcam, ab13586), anti-Bcl-2 (dilution 1:1000, Abcam, ab32124), anti-LC3 (dilution 1:1000, Abcam, ab128025).