Wet blotter system

The method has been previously described [31 (link)]. Briefly, cells were gently washed with phosphate-buffered saline three times and lysed with lysis solution (5 mM EGTA, 150 mM NaCl, 50 mM Tris/HCl, PH 7.4, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 unit protease inhibitor cocktail III (EDTA-free)). Cell lysates were then centrifuged at 12,000× g for 12 min at 4 °C, and the protein concentration of the supernatants was determined using the BCA Protein Assay Kit. Samples for immunoblot (18–25 µg of protein/lane) were analyzed by 10% SDS-polyacrylamide gel electrophoresis with the Bio-Rad mini-gel system. Then, the proteins were blotted onto PVDF membranes with the Bio-Rad wet blotter system. After electro-transfer, the membranes were treated with 5% non-fat milk in the Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h. Subsequently, the membranes were washed three times with TBST, and were incubated overnight at 4 °C with appropriate antibodies (anti-NPFFR2 was 1:1000, anti-Actin was 1:5000, and anti-second antibody was 1:10,000). After three rinses with TBST, membranes were treated with horseradish peroxidase-conjugated secondary antibodies for two h at room temperature. Finally, membranes were analyzed using an ECL detection kit. The analysis of the band intensity was conducted with ChemDocTM XRS (Bio-Rad, Hercules, CA, USA) and Image J [44 (link)].

This approach has been previously described [82 (link)]. In brief, cells were washed with phosphate-buffered saline3 times and lysed by lysis solution (150 mM NaCl, 5 mM EGTA, 1% Nonidet P-40, 50 mM Tris/HCl, 0.5% sodium deoxycholate, 1 unit protease inhibitor cocktail III (EDTA-free), 0.1% SDS, PH 7.4). Then, cell lysates were centrifuged (12,000× g for 12 min, 4 °C), and the protein concentration of the supernatants was evaluated by the BCA Protein Assay Kit. Samples for immunoblot (18–24 µg/lane) were analyzed with the Bio-Rad mini-gel system by 10% SDS-polyacrylamide gel electrophoresis. Next, the proteins were blotted onto PVDF membranes using the Bio-Rad wet blotter system. The membranes were blocked with the solution (5% non-fat milk in the tris-buffered saline containing 0.05% tween-20) for 1 h. Then, the membranes were rinsed three times with TBST and were treated at 4 °C with appropriate antibodies (anti-GPR10 was 1:1000, and anti-Rabbit antibody was 1:10,000, and anti-Actin was 1:5000) for 12 h. The membranes were rinsed with TBST 3 times, followed by treated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Subsequently, the PVDF membranes were detected using an ECL detection kit. The band intensity was analyzed by the ChemDocTM XRS (Bio-Rad, Hercules, CA, USA) and Image J [83 (link)].