TRPV1-YFP and TRPV1-Rluc have been previously described (14), TRPA1-YFP was constructed by cloning the TRPA1 coding sequence (a gift from A. Patapoutian) into pEYFP-N1. TRPV1-mCherry and TRPA1-mCherry were produced by cloning the mCherry sequence in place of YFP in TRPV1-pEYFP and TRPA1-pEYFP using (Not I and Kpn I). TRPV1-CFP was generated by replacing the YFP coding sequence of TRPV1-YFP with CFP from pECFP (Clontech). β-Arrestin2–YFP and β2ARR-rLucII have been previously described (64). Rat MOR1-YFP, Cav2.2, Cavβ2a, and α2-δ1 subunits were gifts from G. W. Zamponi. The MOR-YFP plasmid was used to generate MOR1-HA by cloning sticky-ended oligos containing the HA tag (YPYDVPDYA) in place of YFP. MOR-Rluc8 was produced by using polymerase chain reaction (PCR) to introduce Age I and Not I sites to RLuc8 (a gift from M. Bruchas) to clone in place of YFP. β-Arrestin2–Venus was generated as previously described (65). PKC-βII–GFP was provided by S. S. G. Ferguson. β-Arrestin2 lentiCRISPR-V2 plasmids were a gift from M. Hollenberg and contained guide sequences identified in a genome-wide screening (65). Humanized Renilla GFP (25) was synthesized (GenScript) and cloned into pcDNA3.1. We added the SV40 large T antigen NLS PKKKRKVEDPKS (66) by PCR, using an oligonucleotide containing the C-terminal 24 bp of rGFP (25), a 9-bp linker (GGTGGATCC), and the coding sequence of the NLS: CCGAAGAAAAAAAGGAAGGTTGAAGATCCGAAATCG. Nuclear localization of the resulting construct was confirmed by colocalization with DAPI. The FYVE domain of endofin that localizes to Rab5+ early endosomes was fused to the C terminus of rGFP (rGFP-FYVE) (25). Constructs were confirmed with DNA sequencing.

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