Ecl detection system

Samples were made up in dissociation buffer (1× dissociation buffer (100 mm Tris-HCl, 2% (w/v) SDS, 10% (v/v) glycerol, 100 mm DTT, 0.02% (w/v) bromphenol blue, pH 6.8) and heated at 95 °C for 5 min. Proteins were resolved by SDS-PAGE on 7–17% acrylamide Tris-glycine gels and then transferred to Hybond polyvinylidene difluoride membranes (GE Healthcare). Following electrotransfer, the membranes were blocked for 1 h in PBS with 0.1% Tween 20 (PBST) and 5% (w/v) nonfat milk and then incubated with primary antibody overnight at 4 °C. Antigens were probed using the following primary antibodies: anti-APP (22C11, Millipore), SAF32 (anti-PrP N terminus, Cayman Chemical), 8H4 (anti-PrP residues 175–185), anti-ADAM10 antibody (Abcam), 6E10 (anti-Aβ(1–17), Merck Biosciences), AC15 (anti-β-actin) and synapsin 1 (Sigma), 2B3 (anti-human sAPPα, Immuno-Biological Laboratories), and anti-phospho-Src family kinase (Tyr-416; Cell Signaling Technology). Primary antibodies were detected by incubation with horseradish peroxidase–conjugated secondary antibody, both in PBST containing 2% BSA. Bound horseradish peroxidase conjugates were visualized using the ECL® detection system with a Syngene Gbox XT4 (Syngene). Densitometric analysis was performed using Genetools analysis software (Syngene).

The total proteins from the cardiac tissues were homogenized in RIPA lysis solution that contained the phosphatase inhibitors and protease. The protein content was detected by performing BCA protein quantitation assay. Next, SDS-PAGE was used to isolate the protein samples (40 µg/lane), which were then loaded onto a PVDF membrane. Following the blocking of the membrane using 5% non-fatty milk for 1h, it was subjected to incubation with the corresponding primary antibodies overnight at 4°C, as follows: GAPDH (1:1000), PRR (1:1,000), RAC1 (1:1,000), NOX2 (1:1,000), NOX4 (1:1,000), Cleaved-caspase 3 (1:1000), and thioredoxin 2 (1:1000). After incubation with secondary antibodies for 1h, the proteins were visualized on an ECL detection system (Syngene, UK). GAPDH was utilized to normalize the band intensity values.

The treated cells were lysed in RIPA lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF). The samples were centrifuged (4 °C, 12,000 rpm, 20 min), and the protein concentration of the supernatant was quantified using the BCA protein assay kit. The samples were subjected to 10% SDS-PAGE and transferred to NC membranes. The membranes were activated with ddH₂O and blocked with 5% non-fat milk in TBST for 4 h. The membranes were incubated with primary antibodies (1:400 in TBST) overnight at 4 °C, after which they were washed four times (10 min each wash) with TBST. The membranes were incubated with secondary antibodies (1:4000) for 2 h and then washed four times with TBST. The membranes were visualized with an enhanced chemiluminescence (ECL) detection system (Syngene, Cambridge, UK).