Statistical parameters, including the number of sampled units, n, and methods used for conducting statistical tests, are indicated in the figure legends and table legends and described in Results. For Fig. 2, MFIs from at least three experiments performed in duplicate were used in the statistical analysis. An ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was used to calculate statistical significance (table S2) (Prism 7, GraphPad). Statistical significance of RAMP Ab validation (fig. S3) was determined using a Kruskal-Wallis ANOVA, with experiments performed in duplicate on at least 200 individually prepared lysates (R package). Statistical significance of GPCR Ab validation, and GPCR-RAMP complex detection captured by anti-GPCR Abs (Fig. 3 and figs. S4 and S5), was determined using a single-tail Z test comparing signal from all Abs for each lysate, with at least three experiments performed in duplicate (Excel, Microsoft). A one-way ANOVA followed by Dunnett’s multiple comparisons test was used to compare the mean PLA puncta count of the positive condition (CALCRL-RAMP2, both primary Abs) to that of each of the controls and to compare CXCR3/RAMP2 and mock to the other GPCR/RAMP2 pairs (Fig. 5) (Prism 8, GraphPad). Outliers from each GPCR-RAMP pair were determined in Prism via the ROUT method with Q = 1%. Two outliers were removed from the PTH1R/RAMP2 dataset and three from the CXCR3/RAMP2 dataset (Fig. 5). A two-tailed P test in Prism was used to compare the numbers of PLA puncta from methanol-fixed and paraformaldehyde-fixed cells (fig. S7). Significance was determined by P < 0.05. The alpha level used for each test was 0.05.

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