Apaf 1

The proteins were extracted from the cells using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) and their concentration levels were quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). The proteins (40 µg per lane) were separated by SDS-PAGE on 10% gels, and were transferred onto 0.45 µm PVDF membranes (Thermo Fisher Scientific, Inc.). Following blocking with 5% non-fat milk at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Anti-Bax (Abcam; cat. no. ab32503; 1:1,000), anti-Bcl-2 (Abcam; cat. no. ab32124; 1:1,000), anti-cleaved caspase 3 (Abcam; cat. no. ab2302; 1:1,000), anti-cytochrome c (Cyto C; Abcam; cat. no. ab13575; 1:1,000), anti-apoptotic protease activating factor-1 (Apaf 1; Abcam; cat. no. ab2001; 1:1,000), anti-cleaved caspase 9 (Abcam; cat. no. ab2324; 1:1,000) and anti-β-actin (Abcam; cat. no. ab8227; 1:1,000). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Abcam; cat. no. ab150077; 1:200) for 1 h at room temperature. Finally, an image of the protein band was detected using ECL reagent (Santa Cruz Biotechnology, Inc.). Image-Pro Plus software (version 7, Media Cybernetics, Inc.) was used for the targets that were normalized to β-actin.

Total protein was obtained from cells using a protein extraction kit (Solarbio, Beijing, China). The protein sample concentrations were measured using a BCA kit (Solarbio) according to the manufacturer’s instructions. A total of 50 μL of cell lysate was loaded and run on 8% SDS-PAGE gel and then transferred onto a polyvinylidene difluoride membrane. After blocking with 5% nonfat dry milk, the membrane was incubated using the indicated antibodies at 4°C overnight. After washing with Tris-buffered saline-Tween 20 (TBST) 3 times, the membrane was incubated with horseradish peroxidase–-conjugated secondary antibodies at 37°C for 1 hr and washed with TBST 3 times. Finally, the proteins were visualized using an ECL Western blotting detection kit (Amersham Biosciences, Little Chalfont, UK) according to the manufacturer’s instructions. The authors used the following antibodies and dilutions: APAF1 (1:1000, ab2001; Abcam, UK); β-actin (1:3,000; ab124964; Abcam, Cambridge, UK). Each experiment was performed in triplicate.

After incubation with 4.58×10³ µM fasudil for 6 h, protein was extracted from Hep-2 cells using a protein extraction reagent (Beyotime, Shanghai, China) and protein concentration was measured using the bicinchoninic acid Protein Assay kit (Beyotime). The aliquots of protein (50 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride membrane. The membrane was then blocked with 5% non-fat milk overnight at 4°C, and incubated with apoptotic protease activity factor-1 (APAF-1) (1:500 dilution; Abcam, Cambridge, UK), caspase-3 (1:500 dilution; Abcam), caspase-9 (1:500 dilution; Abcam), MMP-2 (1:500 dilution; Abcam), MMP-9 (1:500 dilution; Abcam), and β-actin (1:500 dilution; ZhongShan, Beijing, China) antibodies for 2 h, followed by incubation with horseradish peroxidase-coupled goat anti-rabbit antibody (1:2,000 dilution; ZhongShan) or horseradish peroxidase-coupled goat anti-mouse antibody (1:10,000 dilution; ZhongShan) for 1 h. Detection was performed by enhanced chemi-luminescence using a Western blotting luminological reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer’s instructions. β-actin was used as a reference protein. Relative densitometry was performed using a computerized software package (NIH Image 1.63 software; https://imagej.nih.gov/ij/).