3h rtx

48 h post transfection, TRPV1- and TRPV1/ARMS-expressing HEK 293 were prepared using either the plasma membrane protein extraction kit (Abcam, ab65400, Great Britain) to separate total (including plasma and organelle membrane proteins, TMF) and plasma membrane fractions (PMF) according to the manufacturer's instructions or membrane preparations according to a modified protocol for transfected HEK 293 cells (Szallasi et al., 1999 ; Szallasi et al., 1992 (link)).
The TRPV1 expression was determined using the radioactively labeled TRPV1 agonist [³H] resiniferatoxin (RTX). The experiment was carried out according to a modified protocol (Szallasi et al., 1999 ; Szallasi et al., 1992 (link)). Briefly, appropriate concentrations of membranes (100-200 μg) were prepared and incubated in assay buffer (50 mM Trizma, 0.25 mg/ml bovine serum albumin, pH 7.4) with 1.1 nM [³H] RTX (39.2 Ci/mmol) (Perkin Elmer, Waltham, USA) in the absence or presence of 10 μM unlabeled RTX to assess affinity by displacement (Spahn et al., 2013 (link)).