Extended Data Fig 10. Cell biological characterization of Ptchd1 actions.

a, Representative immunofluorescence images examining co-localization of MOR with Na⁺/K⁺ ATPase. n=8 cells from each genotype.

b, Quantification of the Ptchd1 effect on co-localization based on Pearson’s correlation coefficient in confocal images of fixed cells. 0x and 2x refer to the amount of DNA transfected, where x = 0.21 μg. n=12 cells from each genotype. Significance determined using unpaired two tailed Student’s t-test. ***p<0.001

c, Western blot of HEK293T cells expressing HiBiT-MOR alone or with PTCHD1-flag.

d, Schematic of the assay design to study the effect of C. elegans protein PTR-25 on internalization of MOR. Addition of LgBit to cells expressing HiBit-MOR forms a nanoluciferase (nLuc) enzyme. DAMGO treatment causes internalization of the complex and quenching of the luminescence.

e, Quantification of DAMGO-induced internalization of HiBiT-MOR in HEK293T cells with or without PTR-25 co-expression. 0, 1x, and 2x refer to the amount of DNA transfected, where x = 0.21 μg. n=5 biologically independent experiments. Significance determined using one-way ANOVA and Dunnett’s post-hoc. *p<0.05

f, Schematic of assay design to study the effect of Ptchd1 on internalization of β2-adrenergic receptor (ADRB2). Addition of LgBit to cells expressing HiBit-ADR2B forms a nanoluciferase (nLuc) enzyme. Isoproterenol treatment causes internalization of the complex and quenching of the luminescence.

g, Quantification of isoproterenol induced internalization of HiBit-ADRB2 in HEK293T cells with or without PTCHD1 co-expression. 0x, 1x, and 2x refer to the amount of DNA transfected, where x = 0.21 μg. n=4 biologically independent experiments. Significance determined using one-way ANOVA and Dunnett’s post-hoc. n.s. is p>0.05

h, Experimental design of β-arrestin recruitment assay to study the effect of Ptchd1 on type 2 vasopressin receptor (VPR2). Addition of arginine vasopressin (AVP) induces recruitment of LgBiT-tagged β-arr2 to VPR2-SmBiT to form a functional nLuc enzyme.

i, Time course of Ptchd1 effect on recruitment of β-arr2 to VPR2 determined in n=5 biologically independent experiments.

j, Quantification of the effect of Ptchd1 on the maximum fold-change of β-arr2 recruitment from data in panel i. Significance determined using one-way ANOVA. n.s. is p>0.05

k, Quantification of the initial rate of β-arr2 recruitment to MOR from data in b. 0x, 1x, and 2x refer to the amount of DNA transfected, where x = 0.21 μg. n=5 biologically independent experiments. Significance determined using one-way ANOVA and Dunnett’s post-hoc. n.s.,p>0.05

l, Schematic of the assay design of β-arrestin recruitment assay. Addition of DAMGO induces recruitment of LgBiT-tagged β-arr2 to MOR-SmBiT to form a functional nLuc enzyme.

m, Modulation of DAMGO mediated- βarr2-LgBiT recruitment to MOR-SmBiT following incubation with 4 mM MBCD or 200 μg / mL cholesterol. n=7 biologically independent experiments for control and MβCD and n=6 biologically independent experiments for cholesterol.

n, Quantification of the maximum fold change and o, the initial recruitment rate from panel n. Significance was determined using a one-way ANOVA and Dunnett’s post-hoc. ns, p>0.05, *p<0.05, ***p<0.001, ****p<0.0001.

In all graphs mean values with S.E.M. errors are shown.