Ecl chemiluminescent reagent

HEK293 and SH-SY5Y cells were lysed and the proteins concentration was quantified with a BCA Analysis Kit (KeyGEN, Nanjing, China). Proteins were separated on 10% SDS-PAGE gels and subsequently transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk at room temperature for 1 h and then incubated with a primary antibody (1:1000) against KOR, B2R, or GAPDH in TBST at 4°C overnight. After washing three times with TBST for 10 min, the membrane was incubated with a HRP-conjugated secondary antibody (1:5000) at room temperature for 1 h and washed three times for 10 min with TBST. The immunoreactive proteins were visualized by ECL chemiluminescent reagent (Pierce Biotechnology). An anti-GAPDH antibody was used as a loading control.
To assay CREB activation, HEK293 cells expressing KOR, B2R, or KOR/B2R cells were pre-treated with or without protein kinase A (PKA) inhibitor H89 (10 µM, 30 min) or phospholipase C (PLC) inhibitor U73122 (10 µM, 30 min), followed by stimulation with Dyn

The A549 cells were washed twice with cold PBS and lysed in 200 μl RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate; Beyotime Co., China) containing 100 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime Co., China) for 30 min on ice. The lysates were centrifuged at 14, 000×g for 10 min at 4°C, and the supernatants were collected. Twenty micrograms of protein/well was loaded onto 10% gels for separation using sodium dodecyl sulfatepolyacrylamide gels electrophoresis (SDS-PAGE). The gels were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 or 0.20 µm pore size; Millipore, Billerica, MA, USA). The blotted membranes were blocked with 5% nonfat dry milk in a Tris-buffered saline solution (25 mM Tris, pH 7.5, and 150 mM NaCl) containing 0.05% Tween 20 (TBST) for 2 h at room temperature, followed by incubation with the diluted primary antibody against target protein for 4 h at room temperature. After washing for 10 min in TBST solution, the membranes were incubated with properly diluted secondary antibody conjugated with horseradish peroxidase for 2 h at room temperature. Western signals were developed using ECL chemiluminescent reagents from Thermo Scientific (Waltham, MA, USA). The β-actin levels were used as loading controls.