Pi 1000

After homogenization and sonication, samples were centrifuged at 10,000 g for 10 minutes. Supernatants were collected and protein concentrations were determined using a standard bicinchoninic acid assay. Prior to electrophoresis, samples were heated for 5 min at 95°C and 50 μg protein was applied per lane. Samples subjected to SDS polyacrylamide gel electrophoresis under standard conditions were transferred onto polyvinylidene difluride (PVDF) membrane in a buffer containing 0.2 mol/L glycine, 25 mM Tris and 20% methanol, overnight at 4°C. After blocking with 5% non-fat dry milk, membranes were incubated with the following primary antibodies FoxO1 (1:300 dilution; Cell Signaling; Cat no:880S) and GAPDH (1:1000 dilution; Cell Signaling; Cat no:2118S) overnight at 4°C. After washing, membranes were incubated for 1 h at room temperature with horseradish peroxidase conjugated secondary anti-rabbit IgG diluted by 1:5000 (Vector; PI-1000). The reaction was visualized using chemiluminescence based Super Signal CL HRP Substrate System (Thermo Scientific; Cat no:34080) and the membranes were exposed to Hyperfilm. As an internal standard to confirm the equal loading of the proteins, GAPDH was loaded to the gels. ImageJ (NIH, Bethesda, MD, USA) was used to quantify the density of the western blot bands.

vHip samples underwent subcellular fractionation protocols as in [33 (link)]. Cellular and membrane samples (50 µg/subject) were then subjected to SDS-PAGE and Western Blot protocols using TRPV1 primary antibody (Thermo Fisher PA1-29421; 1:1000). Primary antibodies for Beta-actin (Thermo Fisher PA1-183; 1:1000) and Na⁺/K⁺ ATPase (MilliporeSigma 05-382; 1:1000) and associated secondaries (Vector PI-1000 and PI-2000; 1:1000) were used as respective loading controls for cellular and membrane samples. Optical density of all bands was imaged and quantified using ChemiDoc XRS (Bio-Rad) and Image Lab software. Bands were normalized by calculating the ratio of TRPV1 to loading control optical density for each animal. All data reflect the average of samples run in duplicate.