C fos 9f6

Rats were perfused transcardially with 200 mL heparinized saline and 200 mL freshly prepared 4% paraformaldehyde through the ascending aorta under pentobarbital sodium anesthesia (50 mg/kg, i.p.). The brains were carefully removed, post-fixed for 12 h in 4% paraformaldehyde at room temperature, and dehydrated with 20% sucrose overnight at 4 °C. Afterward, they were placed in 30% sucrose for further dehydration overnight at 4 °C. Next, 30 μm-thick frozen coronal sections containing RVLM were cut using a cryostat (Thermo Scientific, USA). The sections were permeabilized with 0.3% Triton X-100 for 10–15 min, blocked with QuickBlock blocking buffer (Beyotime, China) for 30 min, and incubated with rabbit monoclonal c-Fos (9F6, 1:1000, Cell Signaling Technology, USA) and mouse monoclonal tyrosine hydroxylase (TH, F-11, 1:100, Santa Cruz) primary antibodies overnight at 4 °C. The next day, the sections were washed with phosphate buffered saline (PBS) and incubated with Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L; 1:400, Jackson ImmunoResearch, USA) and fluorescein isothiocyanate-conjugated AffiniPure Goat Anti-Mouse IgG (H + L; 1:200, Jackson ImmunoResearch, USA) secondary antibodies for 2 h at room temperature. A confocal laser scanning microscope (Zeiss, Germany) was used to monitor the fluorescent signals.

Samples from isolated BBMVs (30 μg), serosal nuclear fractions (30 μg), or total membrane (30 μg) were separated by SDS-PAGE and blotted onto nitrocellulose membranes. After blocking non-specific sites, total membranes or BBMVs were probed for Glut2 (cat#20436-1-AP, Proteintech, Rosemont, IL), custom purified rat Glut2 raised using the first extracellular loop (AA 40–56: SHYRHVLGVPLDDRRA; 21st Century Biochemicals, Marlboro, MA, USA), PepT1 (cat# sc-20653, Santa Cruz, Dallas, TX), SGLT1 (cat# ab14686, Abcam, Cambridge, UK) or NaKATPase (cat# ab7671, Abcam). Nuclear fractions were probed with c-fos (9F6) (cat# 2250S, Cell Signaling, Danvers, MA), p-c-fos (Ser32) (cat# 5348S, Cell Signaling), and YY1 (cat# sc281, anta Cruz). Total membrane fractions were probed with T1R3 (cat# NB10098792, Novus Biologicals, Littleton, CO) and NaKATPase. All antibodies were used with the recommended dilutions and detected by enhanced chemiluminescence using Supersignal West Pico or Femto Chemiluminescent Substrate (cat# 34080, cat#37074, Thermo Scientific, Rockford, IL). Images were taken using a Kodak Image Station 4000 mm Pro and signal intensity was quantified using ImageJ software, version 1.8 (NIH).