Ecl western blotting detection kit

Western blot analyses were performed as previously described [11 (link)]. The frozen tissues were cut into small pieces and homogenized in RIPA buffer and then centrifuged at 12,000g for 10 min at 4°C to separate soluble from insoluble fractions. Equal amounts of proteins for each group mixed with loading buffer were separated by 10% SDS-polyacrylamine gel and transferred to nitrocellulose filter membranes.
Each membrane was incubated with specific antibodies as follows: MMP-9, ZO-1, occludin, claudin-5, HDAC4, NOX4, and GAPDH. Immunoreactive proteins were visualized using the ECL western blotting detection kit (Thermo Fisher Scientific Inc., Boston, USA), according to the manufacturer's instructions. To measure the expression of each protein, the relative intensity was calculated by comparing with the intensity of GAPDH using densitometry (Bio-Rad, USA).

The cells were lysed with RIPA on ice for 10 min and then centrifuged at 10,000 rpm at 4 °C for 15 min. The concentrations of protein samples were detected by BCA assay (P0012, Beyotime, China). The protein samples were separated through SDS-polyacrylamide gel electrophoresis and then transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% skimmed milk for 1 h and then incubated on the shaker overnight at 4 °C with the primary antibody, and the secondary antibody was incubated at room temperature for about 1 h. Signals were detected by using an ECL Western blotting detection kit (Thermo Fisher Scientific).

Murine hearts were processed as previously described (Tocchetti et al., 2014 (link)). Anti-caspase 3, anti-cleaved caspase 3, anti-GAPDH (Cell Signaling Technology), or anti-Actin antibody (Sigma), followed by anti-rabbit, HRP-conjugated IgGs from goat antiserum (Thermo Scientific) were used to detect proteins involved in the apoptotic pathway. The signal from secondary antibodies was visualized by enhanced chemiluminescence detection (ECL western blotting detection kit, Thermo Scientific). The signal intensity of reactive bands was quantitatively measured with a phosphorimager (GS-710, Biorad) or by the open source software ImageJ (NIH, USA).

The cells were lysed using a Western blot and an IP lysis buffer (Beyotime, Shanghai, China). The lysates were homogenized on ice and centrifuged at 12,000× g for 15 min at 4 °C. The supernatants were collected and the concentrations were analyzed using a bicinchoninic acid (BCA) protein assay kit. Equal amounts (15 μg) of the protein samples were separated by 10% SDS-PAGE and transferred to pure nitrocellulose membranes blocked with 5% non-fat milk in a TBST buffer. The membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies at room temperature for 1 h. Chemiluminescent detection was performed using an ECL Western blotting detection kit (Thermo Fisher, Rockford, IL, USA). The images were analyzed using Quantity One software V4.6.7 (Bio-Rad, Shanghai, China) for the density ratio of the target proteins relative to histone H1 or GAPDH.

Polysaccharide intercellular adhesin was detected by performing a lectin dot-blot assay with horseradish peroxidase (HRP)-conjugated wheat germ agglutinin (WGA-HRP, Biotium, Inc. Hayward, CA, United States) as previously described (Wu et al., 2015 (link)). Briefly, overnight cultures of S. epidermidis RP62A were diluted 1:200 in TSB containing 160 μg/mL YycG_ex mAbs and incubated at 37°C with shaking at 220 rpm for 6 h. The bacteria were harvested and resuspended in 50 μL of 0.5 M EDTA (pH 8.0) and then incubated for 5 min at 100°C. After centrifugation (13,000 × g, 5 min), the supernatant was treated with proteinase K (20 mg/mL; Roche) at 37°C for 2 h, after which the enzyme was inactivated at 100°C for 10 min. Next, 1-μL aliquots of serially diluted PIA extracts were spotted onto nitrocellulose membranes (Millipore) by using a 96-well dot-blot vacuum manifold (Biometra GmbH, Niedersachsen, Germany), and after air-drying, the membranes were blocked for 2 h with 3% (w/v) skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). Subsequently, the membranes were incubated with WGA-HRP (2 μg/mL) for 1 h at room temperature, and after washing thrice with TBST, the PIA signal was visualized using an enhanced chemiluminescence (ECL) Western blotting detection kit (Thermo Fisher Scientific, Waltham, MA, United States).