Cells were plated on MatTek dishes coated overnight with poly-ornithine (0.002%), transfected with calcium phosphate, and allowed to express for 48 hours before treatment. Cells were then fixed in 4% paraformaldehyde. Confocal images were collected on a Zeiss LSM-510 Meta inverted microscope using either a 40× water immersion or a 63× oil 1.4 numerical aperture oil immersion lens. Venus/YFP was visualized by excitation with an argon laser (514 nm), and emission was detected using a long-pass 530-nm filter. Enhanced GFP was visualized by excitation at 488 nm. Image acquisition was performed with identical gain, contrast, laser excitation, pinhole aperture, and laser scanning speed.

Immunostaining of β-arrestin2 in DRG neurons was done using the monoclonal β-arrestin2 (SC-13140, Santa Cruz Biotechnology). Alexa Fluor 594 antibody was visualized by excitation with a HeNe laser (543 nm), and emission was detected using a 585- to 615-nm band-pass filter.

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