Ecl detection system

Western blot analysis was performed using frozen PBMCs from N = 12 PAH patients vs. N = 5 CTRLs. The membranes were incubated with both mouse anti-human anti-SOCS3 (OriGene, #TA502991) at a dilution 1:1000 and mouse anti-human anti-GAPDH at a dilution 1:5000 (Santa-Cruz #sc-365062) overnight at 4 °C and next incubated with peroxidase-labeled secondary antibody and visualized using the ECL detection system (Data Supplement). GAPDH protein served as loading control and was detected on the same membranes for normalizing the signals.

Total cell protein was prepared using a Whole Protein Extraction Kit (KeyGEN, China). Protein concentrations were determined using a BCA Assay Kit (KeyGEN, China). Equal amounts of proteins from each sample were subjected to SDS-PAGE followed by transfer of proteins to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5% skimmed milk and incubated with a primary antibody overnight at 4°C. After washing with TBST, membranes were incubated with secondary antibody linked to HRP. The blots were then developed with an ECL detection system (Santa Cruz, USA).

Lysates were separated on 10% SDS-PAGE gels and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% defatted milk in Tris-buffered saline for 2 h at room temperature, followed by an overnight incubation at 4 ℃ with a primary anti-Foxp3 antibody (Abcam, Cambridge, UK) at a 1:1000 dilution. After three washes, the membranes were probed with an HRP-conjugated secondary antibody (1:5000) for 2 h at room temperature, and the visualized protein bands were detected with an enhanced chemiluminescence (ECL) detection system (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Normalization was performed against β-actin expression.

Pancreatic tissue lysates were prepared in saline-containing protease inhibitor and/or complete phosphatase inhibitor cocktail. The nucleoprotein was extracted using the reagents according to the instructions. The protein concentration was measured using a BCA kit and subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE), and the proteins were transferred to a PVDF membrane, blocked with 5% skim milk at room temperature for 2 h, and then incubated overnight at 4°C with primary antibodies against Nrf2 (1 : 1000 dilution), HO-1 (1 : 1000 dilution), Lamin B1 (1 : 1000 dilution), and β-actin (1 : 2000 dilution) in blocking buffer. Membranes were washed with TBST (3^∗10 min) the next day and incubated with a secondary goat antimouse or goat antirabbit IgG horseradish peroxidase (HRP) antibody (1 : 10000 dilution) diluted in 5% (w/v) dry nonfat milk in TBST for 1 h at room temperature. Finally, membranes were washed with TBST (3^∗10 min), developed using the ECL detection system (Santa Cruz Biotechnology), quickly dried, and exposed to ECL film. Image intensity was analyzed with ImageJ software.

For immunoblot analysis, homogenates of fresh liver and renal tissue prepared in sucrose buffer were used. Twenty micrograms of liver and kidney protein fractions were separated onto 4% stacking/12% separating slab gels. After electrophoresis, proteins were transferred to PVDF membranes (Hybond-P, Amersham Pharmacia Biotech) and immunoblot analysis was performed using rabbit polyclonal antibodies to O-linked β-N-acetylglucosamine (O-GlcNAc), N-(carboxymethyl)lysine (CML), RAGE, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, β-actin; goat polyclonal anti-bodies to lamin B (Santa Cruz Biotechnology, Santa Cruz, CA, United States). Appropriate horseradish peroxidase-conjugated anti-rabbit, anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA, United States), and anti-mouse (Cell Signaling) immunoglobulins were used as a secondary antibody. Each PVDF membrane was reprobed according to the supplier’s protocol for reprobing. Immunoreactive bands were identified by an enhanced chemiluminescence (ECL) detection system (Santa Cruz Biotechnology) according to the manufacturer’s instructions. The blots were scanned, and the intensities of the signals were quantified using TotalLab (Phoretix, Newcastle Upon Tyne, England) electrophoresis software (ver. 1.10).

Western blot analysis was performed on frozen tissues and cultured cells. Tissues or cells were homogenized in lysis buffer (5 mmol/L DTT, 5 mg/mL aprotinin, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 5 mg/mL leupeptin in 10 mmol/L Tris buffer) on ice for 10‐20 minutes and ultrasonically dispersed. Then, mixture was separated by centrifugation at 18750g and the supernatant was collected. Total protein was quantified using a bicinchoninic acid (BCA) kit. After SDS‐PAGE, isolated proteins were transferred onto nitrocellulose membranes. After blocked in 5% BSA for 1 hour, membranes were incubated with primary monoclonal antibodies against target at 4°C overnight. On the next day, membranes were incubated in appropriate horseradish peroxidase‐conjugated secondary antibodies at 37°C for 1 hour after washing with Tris‐buffered saline containing 0.1% Tween‐20 thrice. GAPDH or β‐actin was used as an internal control. The proteins were visualized using an ECL detection system (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and the protein bands were quantified using the Quantity‐One software (Bio‐Rad, Hercules, CA, USA).