Eyfp n1

TurboID was a kind gift of A. Ting (Addgene #107171)^{29 (link)}. PMLIVa^WT and PMLIVa^3MAS were previously described^{21 (link)}. SUMO1, SUMO2, Ub, RANGAP1 and UBC9 ORFs were amplified from U2OS cell cDNA by high-fidelity PCR (Platinum SuperFi DNA Polymerase; Invitrogen). A GSQ linker (GGGSSGGGQISYASRG) was placed between the C-terminal part of TurboID and the UbLs as well as between the N-terminal part and the substrates. All constructs were generated by standard cloning or by Gibson Assembly (NEBuilder HiFi Assembly, NEB) using XL10-Gold bacteria (Agilent). Depending on the construction, plasmid backbones derived from EYFP-N1 (Clontech/Takara), Lenti-Cas9-blast (a kind gift of F. Zhang; Addgene #52962) or TRIPZ (Open Biosystems/Horizon) were used. After assembly, all vectors were validated by sequencing. Additional details for constructs are described in Supplementary Table 1. Oligonucleotides sequences are shown in Supplementary Table 2. The sequences of representative constructs are in the Source Data file. Cloning details about other constructs are available upon request.

The rat TRPV1 cDNA (in pcDNA3) was generously provided by Prof. David Julius. Site-directed mutagenesis was performed on rTRPV1-pcDNA3 to generate the phospho-disruptive alanine substitution at PKC phosphorylation sites, S502, T704 and S800 (rTRPV1-S502A/T704A/S800A or rTRPV1-TM as denoted in the figure), as described previously (Mohapatra et al., 2003 (link); Loo et al., 2012 (link)). Mutations at only these three residues were confirmed by DNA sequencing of the full cDNA. Human embryonic kidney cells stably expressing the T-antigen (HEK293T) were cultured in DMEM (with Glutamax), 10% FBS and penicillin/streptomycin. Cells were co-transfected with plasmids containing eYFP-N1 (Clontech) and wild-type (WT) or PKC-triple mutant TRPV1, and/or YFP-tagged rat PTH1 (generously provided by Prof. Matthew Mahon) cDNAs using the Lipofectamine-2000™ reagent per the manufacturer’s instructions, as detailed previously (Mohapatra and Nau, 2003 (link), 2005 (link); Loo et al., 2012 (link)). Transfected cells were used for recordings 1–2 days post-transfection.