Chemidoc software

To analyze changes in protein expression, cell lysates were obtained by harvesting in lysis buffer containing 0.25 M mannitol, 0.05 M Tris-HCl, 1M EDTA, 1M EGTA, 1 mM DTT, 1% Triton X-100 and supplemented with Complete Mini Protease Inhibitor Cocktail and PhosSTOP (both Roche Diagnostics, Penzberg, Germany). Cell lysates were centrifuged at 10 000×g for 15 min at 4 °C to remove insoluble fragments. The total protein content was determined using the Pierce BCA Protein Assay Kit (Perbio Science, Bonn, Germany). For western blot analysis, 50 μg of protein were loaded on a 10% SDS-gel and transferred onto a PVDF membrane. Incubation with the primary antibody was performed overnight at 4 °C. The following primary antibodies were used: rabbit polyclonal anti-GRP75 (Cell Signaling, Danvers, MA, USA), mouse monoclonal anti-c-myc 9E10 (Santa Cruz Biotechnology, Heidelberg, Germany), rabbit monoclonal anti-GADPH (Cell Signaling) and mouse monoclonal anti-vinculin (Sigma-Aldrich). Following overnight incubation, PVDF membranes were washed 3 times with 0.05% TBS-Tween and incubated with corresponding secondary HRP-labeled antibodies (Vector Laboratories, Burlingame, CA, USA). Protein expression was detected by chemiluminescence using the Chemidoc software (Bio-Rad, Munich, Germany) and quantified using the Quantity One software (Bio-Rad).

The collected cells were routinely lysed in RIPA buffer on ice for 10 min, followed by ultrasonication for 30 s. After centrifugation, the supernatants were collected and quantified by Pierce™ BCA Protein Assay Kit (Thermo, 23,227). An equal amount of proteins was denaturated with SDS-Loading buffer (Fude, FD002) at 95 ℃ for 10 min. Subsequently, 40 µg proteins were loaded on a 12% SDS-PAGE gel and blotted onto a PVDF-membrane at 100 V for 80 min. After blocking, the primary antibodies were incubated overnight at 4 ℃. After being washed with TBST, membranes were incubated with proper HRP-labeled secondary antibodies for 1 h at room temperature and were washed again. The blot signals were detected by chemiluminescence with Chemidoc software (Bio-Rad, Germany), and quantified by Image J software.

Heart proteins were extracted using RIPA lysis buffer. Total protein was quantified by Bradford assay and the expression of specific proteins was determined by Western blotting as described previously (Chavali et al., 2014 (link)). The primary antibodies for TNF-α (Cell Signaling, 3707s), IL-10 (EMD Millipore, ABF13), and β-actin (Santacruz Biotech, sc-47778) were diluted 1:1000 and blots were incubated in primary antibody overnight at 4°C. HRP-linked secondary antibodies against rabbit (Santacruz Biotech, sc-2054) or mouse (Santacruz Biotech, sc-2005) were used in 1:4000 dilution. Blots were incubated in secondary antibodies for 1 h at room temperature (RT). The membranes were exposed with HRP substrate and imaged with BioRad ChemiDoc. Densitometry analyses was performed by ChemiDoc software (BioRad, CA, USA).

To determine the levels of autophagy-related protein in Ana-1 and RAW264.7 cells, total protein contents were analyzed by western blot as described previously.^{2 (link)} The protein bands were incubated with specialized antibodies, and identified with HRP-conjugated secondary antibodies. Detection was performed with enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL, USA). Intensities in the resulting bands were quantified by ChemiDoc software (Bio-Rad, CA, USA).