Bio spectrum gel imaging system

Protein extracts of liver tissues were loaded on sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA, USA) after electrophoresis. Protein concentration was determined with a bicinchoninic acid protein assay kit (Beyotime Biotechnology, Shanghai, China). The proteins were marked with anti-Connexin 43 (Cell Signaling Technology; Danvers, Massachusetts, USA, #3512S, 1:1,000), N-cadherin (Cell Signaling Technology; Danvers, Massachusetts, USA, #13116S, 1:1,000), GAPDH (Cell Signaling Technology; Danvers, Massachusetts, USA, #2118s, 1:1,000), TOM20 (Proteintech; Wuhan, China, #11802-1-AP, 1:1,000), or collagen I (Abcam; Cambridge, CB2 0AX, UK, #ab117119,1:1,000). Enhanced chemiluminescence was used for final detection. Secondary antibodies were used at a dilution of 1:10,000. Gels were imaged using a Bio-Spectrum Gel Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and band intensity was measured with Photoshop CS6 software (Adobe, San Jose, CA, USA). Cx43 protein bands were normalized to TOM2. Mitochondria from LX-2 cells were collected with a mitochondria isolation kit (Beyotime Biotechnology, Shanghai, China).

The VSMCs and HUVECs were grown in the 6-well plate. Total proteins were extracted by radioimmunoprecipitation assay buffer lysis buffer containing phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluorid (Beyotime, Beijing, China). Bicinchoninic acid assay (BCA) assay was used to determine supernatant protein levels. Then, sodium dodecyl sulfate (SDS) loading buffer was added for 5 minutes denaturation at 95°C. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted before transferring membrane, followed by the addition of 5% skim milk for closure overnight at 4°C. After membrane washing with Tris-HCl buffer solution-Tween (TBST), the primary antibodies including PI3K, Akt, mTOR, phosphorylated-PI3K (p-PI3K), phosphorylated-Akt (p-Akt) phosphorylated-mTOR (p-mTOR), bcl-2, and internal reference β-actin (all were purchased from CST Company, USA) were added respectively and incubated overnight at 4°C. Then membranes were washed with TBST and the marked secondary antibody horseradish peroxidase (HRP) was added to incubate for 1 hour at room temperature. The protein bands were revealed by electrochemiluminescence (ECL) method and imaged using a BioSpectrum Gel Imaging System (Bio-Rad, Hercules, CA).

Cells were collected and lysed with RIPA buffer containing a fresh protease inhibitor mixture (50 μg/mL aprotinin, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 10 mM NaF, and 10 mM glycerol phosphate). Protein concentrations were quantified using BCA assay kit. Equal amounts of proteins were separated by SDS-PAGE (12%) and electrotransferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBST buffer (20 mM Trisbuffered saline and 0.5% Tween 20) for 1 h at room temperature followed by incubation with the corresponding primary antibodies overnight at 4 °C. Primary antibodies are listed in Table S5. After washing with TBST buffer, secondary antibodies were used at 1:2000 dilutions for 1 h at room temperature. Protein detection was performed based on an enhanced chemiluminescence (ECL) method and photographed by using a BioSpectrum Gel Imaging System (Bio-Rad, Hercules, USA).