Alphaview software 3

Twenty micrograms of protein from heart homogenate were separated on 15% (wt/vol) polyacrylamide denaturing gels and electroblotted onto polyvinylidene fluoride membranes. The semiquantitative analysis (AlphaView Software 3.3, Cell Biosciences, Santa Clara, CA) results were expressed as the optical volume density (OD × mm²) normalized to β-actin (1:5,000, Servicebio, Wuhan, China). The antibodies used were goat anti-granzyme B (1:500, BAF1865, R&D, Minneapolis, MN) and rabbit anti-IL-6 (1:1,000, ab229381, Abcam, Cambridge, UK). The details are described in our previous study (13 (link)).

Twenty μg protein from kidney homogenate was separated on 15% (wt/vol) poly acrylamide denaturing gels and electro-blotted onto Hybond-C membranes. These membranes were blocked with 5% (wt/vol) milk, separately probed with anti-active caspase-3 (1:1,000 dilution, Cell Signaling Technology, Boston, USA), anti-soluble FGL2 (1:10,000 dilution, Abcam) or anti-FcγRIIB (1:10,000 dilution, LifeSpan BioSciences, Seattle, USA) antibody. For the loading control, the same membranes were probed with anti-β-actin antibody (1:10,000 dilution, Abcam), and then incubated with peroxidase-conjugated secondary antibodies (1:10,000 dilution, Jackson ImmunoResearch, West Grove, USA) at room temperature for 1 h. Immunoreactive bands were visualized using ECL substrate (Thermo Fisher Scientific, Rockford, USA) and a Bio-Image Analysis System (Cell Biosciences, Inc., Santa Clara, USA). The semi-quantitative analysis results were expressed as optical volume density (OD × mm²) and normalized by β-actin for loading (AlphaView Software 3.3, Cell Biosciences, Inc.).

Briefly, total proteins from MSCs were separated on SDS–polyacrylamide gels, transferred onto nitrocellulose membranes, blocked and incubated with anti-Nrf2, anti-Sirt3, anti-total FoxO3a, and phosphorylated-FoxO3a (p-FoxO3a, Ser253 and Ser318/321) antibodies (1:1000, Cell Signaling Technology) and anti-tubulin antibodies (1:10000, Abcam) overnight at 4 °C. Membranes were then washed 3 times and incubated with secondary antibodies for 1 h at room temperature. The semi-quantitative analysis (AlphaView Software 3.3, Cell Biosciences, Inc.) results were expressed as the optical volume densities (OD × mm²) normalized to tubulin.