Ecl plus western blotting substrate

The full-length human recombinant interleukin-23 receptor (rIL-23R, 10 nM) (Abnova, Jhouzih St. Taipei, Taiwan), embedded in proteoliposomes, was incubated at 37 °C in the presence or absence of Der p 1 (2.5 nM, preactivated with 1 mM DTT), Der p 3 (0.25 nM) or Der p 6 (0.5 nM) in PBS pH 7.5 for different periods of time (from 0 to 24 h). At the appropriate time points, proteolysis was stopped by the addition of protease inhibitors (10 µM E-64 or PMSF). rIL-23R degradation was analyzed by SDS-PAGE and revealed using the Sypro Ruby protein gel stain (Bio-Rad, Temse, Belgium) and by western blot analysis using a rabbit anti-human IL-23R (residues 62–75) monoclonal primary antibody (SAB1104999, dilution 1/1000) (Sigma-Aldrich) and a goat HRP/anti-rabbit IgG monoclonal conjugate as secondary antibody (1706515, dilution 1:3000) revealed using the ECL plus western blotting substrate (Bio-Rad).

The PGSP-treated cells (2 × 10⁶ cells/mL) were extracted in 120 μL of lysis buffer containing radioimmunoprecipitation assay (RIPA) buffer, 0.5 mM ethylenediaminetetraacetic acid (EDTA), and an inhibitor cocktail (protease and phosphatase). The cell lysates were incubated at 4°C for 30 min and then centrifuged at 13,000 rpm for 20 min. The protein concentration in the supernatant was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and estimated using the standard curve, as directed by the manufacturer. Subsequently, equal volumes of proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene membranes. The membranes were blocked for 1 h in 5% skim milk in 1 × Tris-buffered saline containing 1% Tween-20 (1 × TBST). After incubation, the membranes were washed with 1 × TBST and incubated overnight with the primary antibody (1:2000). Then, the membranes were washed and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) (1:2000). The protein bands were detected by Pierce^® ECL Plus Western Blotting Substrate and visualized with the ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA).