HEK cells cotransfected with a DNA mix of TRPV1-mCherry and MOR-YFP (500 ng each) were treated with vehicle or DAMGO (1 μM). To assess the effect of TRPV1 activation, cells were pretreated with capsaicin (100 nM) for 20 min before DAMGO treatment. Cells were imaged on a Zeiss LSM-510 Meta inverted confocal microscope. Images were then analyzed with ImageJ software, using a sequence of events including background subtraction and transformation of MOR-YFP signal to an 8-bit image, adjusting threshold and noise reduction. Vesicles within the cells were then counted using ImageJ macro. The same sequences of events using identical settings were consistently applied to each image.

To quantify the extent of β-arrestin2 translocation to the nucleus, we transfected HEK cells on coverslips with YFP-βarr2 with or without TRPV1 and allowed the plasmids to express for 48 hours. Cells were then washed in HBSS and stimulated with 1 μM capsaicin for 15 min at 37°C. The 15-min–time point coverslips were fixed, medium was replaced, and then the remaining coverslips were fixed at 30, 60, and 120 min from the beginning of stimulation. Coverslips were mounted on slides using Prolong Gold Antifade with DAPI mountant. Wide-field fluorescent images were acquired on a Zeiss LSM-510 Meta confocal microscope, and we quantified the number of cells in the field of view that showed YFP-βarr2 in the nucleus (indicated by DAPI) and the total number of YFP-βarr2–positive cells.

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