Western blot detection kit

The cells were scraped from the plates using RIPA Lysis Buffer (Merck Millipore, Darmstadt, Germany) containing a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). After incubation on ice for 30 min, the cell lysates were centrifuged at 14,000 rpm for 20 min at 4°C. Proteins were quantified using BCA protein assay (Thermo Scientific, Waltham, MA, USA). The protein lysates were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane. Anti-COX-2, anti-phospho-cPLA₂, anti-phospho-Lyn, anti-phospho-Syk, anti-phospho-Fyn, anti-phospho-PLCγ1, anti-phospho-ERK, anti-phospho-Akt, and anti-α-tubulin antibodies (Cell Signaling Technology, Boston, MA, USA) were used to detect COX-2, p-cPLA₂, p-Lyn, p-Syk, p-Fyn, p-PLCγ1, p-ERK, p-Akt, and α-tubulin, respectively. α-Tubulin was used as protein loading control. Blots were observed using a western blot detection kit (Thermo Scientific, Waltham, MA, USA). Protein bands were quantified using Image Lab software (Bio-Rad Laboratories, Richmond, CA, USA).

The RBL-2H3 mast cells were sensitized with IgE for 10 min or 4 hr. Total proteins were extracted using RIPA buffer (Merck Millipore, Darmstadt, Germany) containing a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Proteins were quantified using the bicinchoninic acid assay and were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto an activated polyvinylidene difluoride membrane for 100 min. The blots were blocked with 5% BSA and incubated with primary antibodies (1 : 1000) at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature. Protein expressions were detected using a western blot detection kit (Thermo Fisher Scientific, MA, USA) and ChemiDoc™ Touch Imaging System (Bio-Rad, CA, USA).

After various combined treatments based on our treatment groups, the PBMC and BMMC samples were resuspended in the RIPA lysis buffer. Subsequently, using the BCA protein analysis kit (Thermo Fisher), the concentration of cell lysate suspensions was determined, and an equal quantity of protein and protein markers were separated on 12% Bis–Tris acrylamide gels (stacking gel/separator) and transferred to nitrocellulose membranes (PVDF). Membranes were blocked with 5% skimmed milk in TBS buffer (170 mm NaCl, 70 mM Tris, pH 7.7) for 1 h at room temperature or overnight at 4 °C. Afterward, membranes were incubated with monoclonal antibodies against the Nrf2 gene (1: 1000), bcl2 (1: 400), Bax (1: 1000), and polyclonal antibodies against β-actin (1: 2000) overnight. After three washes with PBST and 1 h of incubation with anti-rabbit IgG-HRP antibodies, the western blot detection kit (Thermo Fisher Scientific) was used to measure the relative amounts of targeted proteins.