Supersignal west femto maximum sensitivity substrate detection kit

Due to variability of the villin promoter on expression [35 (link)], hTHTR2 protein expression was evaluated in various tissues. Tissue extracts were prepared in RIPA lysis buffer (Thermo Fisher, Waltham, MA, USA) with dissolved Pierce protease inhibitor tablets (Thermo Fisher) followed by homogenization. After centrifuging the homogenates at 15,000× g and at 4 °C for 15 min, supernatants were collected, and the protein content was determined by Pierce BCA Protein Assay Kit (Thermo Fisher). Samples of 20 μg of protein were electrophoresed on precast Mini-protean TGX gels with 4–20% polyacrylamide (Bio-Rad, Hercules, CA, USA), transferred to a low-fluorescence PVDF membranes (Thermo Scientific), and then blocked in Tris-buffered saline with 0.1% Tween 20 (TBS-T) supplemented with 5% non-fat dry milk at room temperature for 1 h. Membranes were incubated overnight at 4 °C with the 1:1000 dilutions of rabbit anti-SLC19A3 primary antibodies (1:1000, Sigma-Aldrich, St. Louis, MO, USA) in TBS-T supplemented with 5% non-fat dry milk, then washed with TBS-T and incubated with 1:2000 dilutions of secondary horse radish peroxidase conjugated goat anti-rabbit IgG (Cell Signaling Technologies, Danvers, MA, USA). Signals were detected using the enhanced chemiluminescence SuperSignal West Femto Maximum Sensitivity Substrate detection kit (Thermo Fisher).

Immunoblotting was performed essentially as described elsewhere (Eyre et al, 2010 (link)). Briefly, membrane-bound protein was blocked with 5% skim milk in 0.1% TBS-T for 1 h and then incubated in the appropriate dilution of primary antibody in 1% skim milk overnight at 4°C. Thereafter, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h and washed before detection using either the ECL Plus Western blotting detection reagent kit (Amersham Pharmacia Biotech) or the Supersignal West Femto Maximum Sensitivity Substrate detection kit (Thermo Fisher Scientific) as per the manufacturer’s instructions. Protein bands were visualized by a Chemi DocTM MP Imaging System (Bio-Rad).