2.7 |. Western blot analysis

At the end of 7-day salt loading protocol, the rats were anaesthetised with inactin (100 mg kg⁻¹ i.p.) and decapitated. Punches containing the SON were collected from a 1-mm coronal section from each brain as described previously.^18,28 Protein was extracted from the SON punches using RIPA lysis buffer containing dithiotreitol, chelators and protease phosphatase inhibitor cocktail. Protein concentration was determined by the BCA assay with varying concentrations of bovine serum albumin (BSA) as reference standards. Total lysate (20–25 μg) was loaded onto a 4%−15% acrylamide sodium dodecyl sulphate (SDS) gel and separated by electrophoresis in Tris-glycine buffer with denaturing conditions. The protein was transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore) in Tris-glycine buffer (25 mmol L⁻¹ Tris, 192 mmol L⁻¹ glycine, 0.1% SDS; pH 8.3) with 20% (v/v) methanol. Membranes were blocked with 5% BSA in Tris-buffered saline-Tween 20 (25 mmol L⁻¹ Tris base, 125 mmol L⁻¹ NaCl, 0.1% Tween 20) for 30 minutes at room temperature. The membranes were incubated with primary antibodies made in 5% BSA overnight at 4°C. The primary antibodies used were: phosphorylated TrkB (Y515; rabbit polyclonal; dilution 1:1000; ab109684; Abcam, Cambridge, MA, USA); total TrkB (goat; dilution 1:1000; GT15080; Neuromics, Edina, MN, USA); phosphorylated KCC2 (Ser940; rabbit polyclonal; dilution 1:500; 612–401-E15; Rockland Immunochemicals, Inc., Pottstown, PA, USA); mCherry (rabbit polyclonal; dilution 1:500; ab167453; Abcam); and GAPDH (mouse monoclonal; dilution 1:2000; MAB374; Millipore).

Membranes were rinsed three times at 10-minute intervals with TBS-Tween followed by a 2-hour incubation at room temperature with a horseradish peroxidase-conjugated secondary antibody against the primary antibody host species (anti-rabbit, anti-goat, or anti-mouse; dilution 1:1000; Sigma, St Louis, MO, USA). The membranes were washed three times at 5-minute intervals with TBS-Tween. Proteins were visualised using an enhanced chemiluminescence substrate kit (Supersignal West Femto Maximum Sensitivity kit; Thermo Fisher Scientific Inc.). Blots were developed, and the digital image was obtained using gbox in genesnap (Syngene, Fredrick, MD, USA) and densitometry analysis of the bands was performed using imagej (NIH, Bethesda, MD, USA). Densitometry measurements of the immunoreactive bands were normalised using GAPDH as a loading control.