Biospectrum 810

Transiently transfected HEK293T cells were homogenized in lysis buffer [150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA, 0.5% Triton X-100, 0.5% NP-40] mixed with protease inhibitor cocktail set III (EDTA-free, Calbiochem). Concentrations of protein samples were measured by BCA assay (Thermo Scientific, Pierce). Protein lysates (25 μg) mixed with 6X sample buffer with 1 mM DTT were resolved by SDS-PAGE and transferred onto PVDF. HA-tagged TRPV1 protein was identified by rabbit polyclonal primary antibody HA.11 (Covance PRB-101P, 1:2000 dilution). GAPDH was detected by rabbit polyclonal GAPDH antibody (Santa Cruz Biotechnology FL-335, 1:5000 dilution). Both antibodies were visualized by anti-rabbit IgG HRP-conjugated secondary antibody (Pierce #31430, 1:5000 dilution), and the blot images were acquired using BioSpectrum 810 (UVP). The images were analyzed using ImageJ. The HRP signal of each band was normalized to the rTRPV1 band on the same membrane in order to compare results from different days and different membranes.

Following transfection, HEK293T cells were lysed with lysis buffer containing 0.5% Triton X-100, 0.5% NP-40 and protease inhibitor. The cell lysates were mixed with 6× SDS non-reducing sample buffer or 6× SDS reducing sample buffer containing 2% dithiothreitol and 5% 2-mercaptoethanol. The lysates were resolved by 7.5% non-reducing SDS-PAGE before transferring the proteins onto a PVDF Transfer Membrane (Millipore). The membrane was incubated in blocking buffer (5% nonfat milk in Tris-buffered saline with 0.05% Tween 20) containing anti-rat TRPV1 antibody (GeneTex; 1:5,000 dilution) or anti-GAPDH antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:5,000 dilution). Proteins were visualized using a secondary anti-rabbit HRP-conjugated antibody (Thermo Fisher Scientific, Waltham, MA, USA; 1:20,000 dilution). PVDF membranes were visualized by supersignal West Femto chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA), and the blot images were acquired using BioSpectrum 810 (UVP).