Chemidic xrs imaging system

Total protein was extracted from skeletal muscle using protein extraction reagents. Western blot analyses were performed with the use of primary antibodies against myogenin (1:500), MyHC (1:500), Akt, p-Akt (1:500), mTOR, p-mTOR (1:1000), or actin (1:1000) as previously described [18 (link)]. The protein bands were visualized and analyzed using the ChemiDicTM XRS+ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

Tissue protein samples were prepared with protein extraction reagents from renal tissues. Protein concentrations were measured with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples with equal amount of proteins were separated with SDS-PAGE and then transferred onto a PVDF membrane for Western blot analysis with specified antibodies. The ChemiDic TM XRS+ imaging system (Bio-Rad Laboratories, Hercules, United States) was used to analyze the signals and the band densities were quantified with Multi Gauge software of science Lab 2010 (FUJIFILM Corporation, Tokyo, Japan).

Western blotting was performed using routine protocols. Treated cells were isolated using radioimmunoprecipitation assay buffer containing 1 mM phenylmethanesulfonyl fluoride, and protein concentration was measured using a Bicinchoninic Acid Protein Assay Kit (Beyotime). Protein samples (30 μg) were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane (Life Technologies, Gaithersburg, MD, USA), and blocked. The membranes were incubated overnight at 4 °C with one of the following primary antibodies: LC3, beclin-1, cleaved caspase-3, Bax, Bcl-2, caspase-9, cytochrome c, p-mTOR, mTOR, p-4EBP1, 4EBP1, p-AMPKα, AMPKα, p-p70S6K, p70S6K, or P62. The densities of the protein bands were detected using the ChemiDicTM XRS + Imaging System (Bio-Rad, Hercules, CA, USA). Densitometric quantification of the membranes was performed using ImageJ.

The samples were subjected to 10% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, United States). The membranes were blocked with 5% fat-free milk at room temperature for 2 h and then incubated with the following primary antibodies: anti-GAPDH (1:10000), anti-NF-κB (1:1000), anti-Nrf2 (1:1000), anti-occludin (1:1000), anti-p-120catenin (1:1000), and anti-cleaved caspase-3 (1:1000) at 4°C overnight. The membranes were then washed with TBST and incubated with a secondary horseradish peroxidase-conjugated antibody at room temperature for 1 h. The signals were visualized with the ChemiDicTM XRS+ Imaging System (BioRad Laboratories, Hercules, CA, United States), and the densities of the immunoreactive bands were analyzed using ImageJ software (NIH, Bethesda, MD, United States).

The total cell lysates of NIH 3T3 fibroblasts after different treatments were collected with RIPA lysis buffer (with 1% PMSF) for 15 min on ice. After centrifugation (12,000 × g, 10 min, 4°C), the supernatant was collected and the protein concentration of which was then quantified using BCA reagents. After that, equal amounts of protein samples from each group were separated through a Bis-Tris polyacrylamide gel (12%) under 80 V and transferred to a PVDF blotting membrane, which was then blocked with 5% skimmed milk in TBST (10 mM Tris–HCl, 100 mM NaCl and 0.1% Tween 20) for 1.5 h at room temperature on a rotary shaker and then incubated with a rabbit monoclonal antibody to HO-1 (1:1000) diluted in TBST overnight at 4°C. The membrane was washed with TBST three times and incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:8000) for 2 h at room temperature on a rotary shaker. The protein bands on the PVDF membrane were visualized using ChemiDicTM XRS + Imaging System (Bio-Rad), and the signal intensities of which were quantified using ImageJ software. Band intensities were normalized to GAPDH.