Protein extraction kit

Total protein of different groups of cells was extracted with the protein extraction kit (Beijing Applygen Technologies Inc., China, P1250-50/100), and the bicinchoninic acid (BCA) method was adopted for concentration determination of proteins. Total protein (50 μg) was loaded on each well after isolation using the sodium dodecyl sulfate polyacrylamide gel electropheresis. Two hours after electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane using the wetting transfer method, followed by 1 h of blocking with 5% skimmed milk, and the subsequent overnight cultivation with primary antibodies BCL2-associated X (Bax) (ab32503), B-cell lymphoma-2 (Bcl2) (ab59348), cleaved Caspase-3 (ab2302), E Cadherin (ab40772), Vimentin (ab92547), SNAIL (ab53519), and ZEB1 (ab185228) all diluted at the ratio of 1 : 1000 and purchased from Abcam, USA. The next morning, the TBS+Tween (TBST; Beijing Yita Biotechnology Co., Ltd., China, YT8022) rinsed membrane was placed into the horseradish peroxidase-labeled secondary antibody goat antirabbit (ab6721, 1 : 3000, USA) for 1 h of incubation at 37°C. This was followed by development with enhanced chemiluminescence (ECL), image acquisition and optical density measurement using the Quantity One (EasyBio (Beijing) Technology Co., Ltd., China). The experiment was repeatedly determined three times.

Briefly, a protein extraction kit (Applygen Technologies, Beijing, China) was used to extract protein from VSMCs. Protein concentration was assessed by bicinchoninic acid protein assay (Applygen Technologies). 30 μg of protein was fractionated by sodium dodecyl sulfate-polyacrylamide gels and electroblotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated in 4% skim milk solution and then reacted with antibodies at 4°C overnight, including anti-β-actin (ab8226, Abcam, Cambridge, MA, USA), anti-CyclinD1 (ab16633, Abcam), anti-matrix metallopeptidase 9 (MMP9; ab76003, Abcam), anti-BCL2-Associated X (Bax; ab325033, Abcam), anti-B-cell lymphoma-2 (Bcl-2; ab196495, Abcam), and anti-IGF2 (ab177467, Abcam). After washing with Tris-buffered saline with Tween 20, the membranes were incubated with HRP-conjugated secondary antibodies (Abcam). Finally, the Alpha Innotech Imaging System (ProteinSimple, Santa Clara, CA, USA) was used to visualize protein signal. The band density was analyzed using Image J software (NIH, Bethesda, MA, USA).

Total protein was extracted using a protein extraction kit (Applygen Technologies, Beijing, China). The protein concentration was determined with a BCA protein assay kit (Applygen Technologies, Beijing, China). Western blot analysis was performed routinely, with primary antibodies against β-actin, Acc, Fas (Cell Signaling Technology, Beverly, MA, United States), Scd1 and Srebp1 (Thermo Scientific, Fremont, CA, United States). 100 μg protein was loaded in each well. The bands were detected using an ECL detection kit (Applygen Technologies, Beijing, China). For quantification, band intensity was assessed by densitometry and expressed as the mean area density using Quantity One image analyzer software (Bio-Rad, Richmond, CA, United States).

The total amount of protein from thoracic aorta samples (in vivo) and the total amount of mitochondrial proteins from HUVECs (in vitro) were extracted using a protein extraction kit (Applygen Technologies Inc., Beijing, China) and a mitochondria isolation kit (Abcam, Cambridge, UK), respectively. A total of 50 μg of protein was separated by denaturing SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were then blocked with 5% skim milk, washed, incubated with primary antibodies directed against DDAHII (1 : 1000), eNOS (1 : 1000), eNOS phospho-S1177 (1: 1000), cyt C (1 : 1000), β-actin (1 : 2000), and COX4 (1 : 1000), and then incubated with an HRP-conjugated secondary antibody. Subsequently, membranes were incubated with an enhanced chemiluminescence reagent for 2 min at room temperature, and protein bands were visualized using an enhanced chemiluminescence method and analyzed with Quantity One software (Bio-Rad, Hercules, CA, USA) [37 (link)].

Total protein was extracted from liver and skeletal muscle using Protein Extraction Kit (Applygen Technologies Inc., Beijing, China) according to the manufacturer's protocol, respectively. Total protein levels were determined by the bicinchoninic acid (BCA) method (Applygen Technologies Inc., Beijing, China). Equal amounts of protein samples were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to 0.45 um PVDF membranes. Blotted membranes were blocked with 5% skim milk in TBS with 0.1% Tween 20 and incubated at 4°C overnight, respectively, with one of the following primary antibodies: goat anti-rat adipoR1 polyclonal antibody (diluted to 1 : 1000 with TBS with 0.1% Tween 20; Novus Biologicals, Littleton, CO, USA) or goat anti-rat adipoR2 polyclonal antibody (diluted to 1 : 1000 with TBS with 0.1% Tween 20; Novus Biologicals, Littleton, CO, USA). After three washes in TBS with 0.1% Tween 20, the membranes were incubated with 1 : 5,000 secondary HRP-conjugated anti-goat antibody (MultiSciences Biotech Co., Hangzhou, China) at room temperature for 1 h. Membranes were exposed to the ECL system (Applygen Technologies Inc., Beijing, China) and the bands were quantified with the use of Adobe Photoshop CS5.0 software (Adobe Company, USA).