αenac

The cell lysates were separated by SDS-PAGE (10% polyacrylamide gels) and transferred onto PVDF membrane (Invitrogen, Waltham, MA, USA). After blocking with 5% nonfat dried milk in Tris-buffered saline containing 0.05% Tween 20, the membranes were incubated with primary antibodies α-ENaC (1:2000, PA1-920A, Thermo Fisher, Waltham, MA, USA), γ-ENaC (1:2000, ab3468, Abcam, Cambridge, MA, USA), PKGII (1:2000, sc-393126, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (1:1000, sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. The membranes were washed three times with TBST for 10 min each time and then reacted with horseradish peroxidase-conjugated secondary antibody, goat-anti-rabbit or goat-anti-mouse secondary antibody (1:5000, ZSGB-BIO, Beijing, China) at room temperature for 1 h. The protein bands were visualized using ECL kit on a Tanon-5200 chemiluminescence detection system (Tanon, Shanghai, China), and the intensity of each specific band was quantified with Image J program.

The ERR granulation tissues were frozen and sliced, and then dried at room temperature. After washed with PBS, these cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 5% bovine serum albumin (BSA), and then incubated with primary antibodies overnight at 4 °C. Primary antibodies included αENaC (Thermo Fisher Scientific), and Fibronectin (Thermo Fisher Scientific). The secondary antibodies were respectively Cy3-labeled Goat Anti-Rabbit IgG (Servicebio, Wuhan, China) and Alexa Flour-labeled Goat Anti-Mouse IgG (Servicebio). Nuclei were counterstained with DAPI (Cell Signaling Technology). Finally, the slides were imaged using a fluorescence microscope (Echo Revolve, San Diego, CA, USA) or a laser scanning confocal microscope (Leica-SP8, Wetzlar, Germany), and the colocalization of αENaC with Fibronectin was observed.

Total RNA from either STC-1 cells or mucosal cells obtained from the small intestine from C57/B6 mice was purified by using the TRIzol reagent (cat# 15596018, Thermo Fisher Scientific, MA, USA) and reverse transcripted by using High-Capacity cDNA Reverse Transcription Kit (cat# 4368814, Thermo Fisher Scientific, MA, USA). RT–PCRs for the detection of nAChR subunits and the other taste receptors were carried out by using MyTaq red mix (Bioline, Luckenwalde, Germany). Briefly, 2 μg total RNA was mixed with 2✕ Reverse Transcription Master Mix in a total volume of 20 μl per reaction. Reverse transcription were performed at 25°C for 10 min, then 37°C for 120 min, followed by 85°C for 5 sec and cooled to 4°C. Subsequently, 200 ng total cDNA was used as template, 35 to 40 cycles of PCR amplification were performed (initial denaturation at 95°C for 1 min, denaturation at 95°C for 15 sec, annealing for 15 sec at 53–60°C, and extension for 10 sec at 72°C). RT-PCR products were subjected to electrophoresis on a 1% agarose gel to determine the expression of nAChR subunits and other taste receptors. The mouse primers used to detect the presence of mRNAs for the nAChR subunits (chrna3, chrna4, chrna5, chrna6, chrna7, chrnb2, chrnb4), TRPM5, αENaC, TRPV1, GAPDH, BDNF and β-actin are shown in Table 1 and were synthesized by Thermo Fisher Scientific.

Whole-cell lysates were prepared using RIPA containing protease and phosphatase inhibitors (Beyotime, Shanghai, China). The protein was quantified with BCA kit (Solarbio, Beijing, China), separated on a 10% SDS-polyacrylamide gel electrophoresis and transferred onto 0.45 μm PVDF membranes (Invitrogen, Carlsbad, USA). Membranes were blocked using 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight After washed three times by TBST for 10 min intervals, membranes were incubated with diluted secondary antibodies for 1 h at room temperature. The expression of proteins was assessed using an ECL kit (Tanon, Shanghai, China), and quantified by the image J software. The expression of β-actin did not change significantly either under hypoxia or after drug treatments, which was used as an internal parameter in our experiment accordingly.
The primary antibodies used were antibodies against α-ENaC (1:2000, Invitrogen, Carlsbad, USA), γ-ENaC (1:2000, Abcam, Cambridge, USA), β-actin (1:2000, Proteintech, Chicago, USA), HIF-1α (1:1000, Affinity Biosciences, Cincinnati, USA), ERK1/2, p-ERK1/2 (1:1000, Cell Signaling, Mass, USA), inhibitor κBα (IκBα), p-IκBα, p65, and p-p65 (1:1000, Abmart, Shanghai, China). The secondary antibodies used were goat anti-rabbit or goat anti-mouse antibodies (1:5000, ZSGB-bio, Beijing, China), respectively.