Clarity maxtm kit

HBO cells or mouse gastric smooth muscle strips were solubilized in Triton X-100-based lysis buffer plus protease and phosphatase inhibitors. After centrifugation of the lysates at 20,000× g for 10 min at 4 °C, protein concentrations of the supernatant were determined with the DC Protein Assay kit from Bio-Rad (Hercules, CA, USA). Equal amounts of proteins were fractionated by SDS-PAGE and transferred to PVDF membranes. Blots were blocked using blocking buffer (BioRad) for 10 min at room temperature and then incubated overnight at 4 °C with various primary antibodies in a blocking buffer. After incubation for 1 h with horseradish peroxidase-conjugated corresponding secondary antibody (1:5000, GE Amersham) in the blocking buffer, immunoreactive proteins were visualized using Clarity Max^TM kit (BioRad). All washing steps were performed with TBS-T. A 10–250 kDa PageRuler^TM plus pre-stained protein ladder (Fisher Scientific #PI26620) was used to evaluate the protein expression.

Sequential immunoprecipitation and immunoblot with selective antibodies were used to determine the association of ACE2 with δ-ENAC; T1R3 with TRPV1; T1R3 with ACE2; TRPV1 with ACE2; and GPER1 with AT1R, TRPV1, ACE2, and δ-ENaC. HBO cells or gastric smooth muscle were lysed by incubation for 30 min at 4 °C in Triton X-100-based lysis buffer plus protease and phosphatase inhibitors. After centrifugation of the lysates at 20,000× g for 10 min at 4 °C, protein concentrations of the supernatant were determined with the DC Protein Assay kit from Bio-Rad (Hercules, CA, USA). A total of 100 µg of protein was precleared by incubation with 40 μL of protein A/G agarose for 4 h and then incubated overnight with antibody to ACE2, T1R3, TRPV1, or GPER1. Protein A/G agarose was then added and incubated for another 2 h, and the mixture was centrifuged at 13,000× g for 5 min. The immune-precipitates were washed four times in lysis buffer and boiled in Laemmli buffer. Samples were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibody to δ-ENAC, T1R3, TRPV1, ACE2, and AT1R. After incubation with secondary antibody, the proteins were visualized using Clarity Max^TM kit (BioRad). All washing steps were performed with TBS-T. A 10–250 kDa PageRuler^TM plus pre-stained protein ladder (Fisher Scientific #PI26620) was used to evaluate the protein expression.