Extended Data Fig. 2: Additional demonstration that multiple GPCRs can activate the discrete β-arrestin trafficking mechanism.

a, Live-cell TIRF microscopy images showing Flag–MOR (blue), β-arrestin-2–GFP (green) and clathrin light chain–DsRed (red) before and after treatment with the opioid peptide DAMGO (10 µM). b, Mean Flag–MOR and β-arrestin-2–GFP enrichment at CCSs after treatment with 10 µM DAMGO (n = 12 cells). c, Maximum β-arrestin-2–GFP enrichment at CCSs for HEK 293 cells expressing Flag–MOR or empty vector and treated with 10 µM DAMGO (n = 12 cells per condition from three independent experiments; P < 0.0001 using a two-tailed unpaired t-test with Welch’s correction). d, Live-cell TIRF microscopy images showing Flag–KOR (blue), β-arrestin-2–GFP (green) and clathrin light chain–DsRed (red) before and after treatment with 10 µM dynorphin. e, Enrichment into CCSs after bath application of 10 µM dynorphin (n = 18 cells). f, Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells expressing Flag–KOR or empty vector and treated with 10 µM dynorphin (n = 18 and 13 cells, respectively, from three independent experiments; P = 0.0028 using a two-tailed unpaired t-test with Welch’s correction). g, Live-cell TIRF microscopy images showing Flag–DRD2 (blue), β-arrestin-2–GFP (green) and clathrin light chain–DsRed (red), before and after treatment with 10 µM quinpirole. h, Enrichment into CCSs after bath application of 10 µM quinpirole (n = 12 cells). i, Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing Flag–DRD2 or untransfected and treated with 10 µM quinpirole (n = 11 and 12 cells from three independent experiments; P = 0.0095 using a two-tailed unpaired t-test with Welch’s correction). a, d and g show representative images from three independent experiments. b, e and h show mean ± s.e.m. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 µm. *P < 0.05; **P < 0.01; ***P < 0.001.