Ab8245

Following the manufacturer's steps to extract the total protein (Sigma) from the cell line, all steps are carried out on ice. BCA protein assay kit determinate the concentration of total protein. The electrophoresis protein was heated to 100°C, incubated for 5 min, then using electrophoretic with SDS- polyacrylamide gel (120 v, 100 min). Afterwards, the protein separated from SDS gel was transferred to PVDF membrane (300 mA, 80 min). After the membrane transfer was completed, the target band was sealed with 5% TBS, and membrane was incubated with an anti-rabbit monoclonal anti-human GAPDH antibody (abcam catalog number ab8245), diluted overnight at 4°C with a dilution of 1:1,500. After incubation on the second day, the membrane was incubated with horseradish peroxidase to incubate the (HRP)-coupled polyclonal Goat anti-Rabbit IgG-HRP (Bioworld) second antibody (1:10,000). It was placed at room temperature for 1 h. The film has an enhanced chemiluminescence system and is imaged by X-ray film. The image is then quantized by Image J (NIH). Information about first antibody GAPDH (ab8245; abcam); PTEN (ab170941; abcam); AKT (ab8805; abcam); p-AKT (4060T; Cell Signaling Technology); PI3K (ab151549; abcam); p-PI3K (17366S; Cell Signaling Technology); Caspase-3 (ab13847; abcam); Caspase-9 (ab32539; abcam). The image is then quantized with Image J (NIH).

Total proteins were extracted by RIPA buffer (i.e., 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and 0.1% SDS, Thermo Fisher Scientific, USA) containing proteinase inhibitor cocktail (Roche, Germany) and phosSTOP phosphatase inhibitor cocktail (Roche, Germany) from homogenised liver tissues. Then the proteins were separated by SDS - PAGE electrophoresis, and were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Germany). The membranes were blocked with 5% slim milk and incubated with primary antibodies including p-PI3K (ab245781, Abcam, USA; 1:1,000), PI3K (ab191606, Abcam, USA; 1:1,000), p-Akt (ab81283, Abcam, USA; 1:1,000), Akt (ab8805, Abcam, USA; 1:1,000) and GAPDH (ab8245, Abcam, USA; 1:1,000). After incubation with secondary antibodies, protein bands were visualised using an ECL detection kit (Thermo Fisher Scientific, USA). Protein expression levels were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalised to GAPDH (ab8245, Abcam, USA; 1:1000).

VSMCs from each group were collected and lysed on ice using lysis buffer (Solarbio, Beijing, China). The mixture was centrifuged at 12,000 rpm at 4°C for 15 minutes. After discarding the supernatant, the protein concentration was determined using a BCA kit (Merck, Kenilworth, NJ, USA). Equal amounts of protein were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane, which was then blocked with 5% skimmed milk at 25°C. The membrane was then incubated at 4°C overnight with the following primary antibodies, which were all purchased from Abcam: anti-Bax (ab3191, 1 : 2000), anti-Bcl-2 (ab196495, 1 : 1000), anti-vascular cell adhesion molecule 1 (VCAM-1; ab174279, 1 : 2000), anti- intercellular cell adhesion molecule-1 (ICAM-1; ab109361, 1 : 2000), anti-E-selectin (ab137732, 1 : 2000), anti-GBP-1 (ab22604, 1 : 2000), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, 1 : 5000). The next day, the membrane was rinsed and incubated with the secondary antibody at 37°C for 30 minutes. Enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) was used to visualize the protein bands and GAPDH was used to normalize the protein levels.

Transfected COV434 cells were harvested and homogenized with a RIPA buffer, pH 7.4 (BioBasic, Toronto, Canada) containing phosphatase and protease inhibitors (Roche, Basel, Switzerland). A Bicinchoninic Acid Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) was to calculate protein concentrations, and plates were read in a Varioskan multimode plate reader (Thermo Scientific). Criterion XT precast gels (4–20% bis–tris) (Biorad, Hercules, CA, USA) were loaded with 50 µg of total protein per well and transferred to a nitrocellulose membrane. Primary antibodies were anti-AMH (Ab229212, 1:2000, Abcam) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Ab8245; 1:10,000, mouse monoclonal, RRID: AB2107448, Abcam). ChemiDocTM XRS + imager was used to scan the membrane, and Image Lab software (Biorad) assisted in quantifying the intensity of the bands of interest.