Nanoluc complementation assay

RTK dimerization and interaction with LRIG1 were monitored by NanoLuc complementation assay (NanoBiT, Promega).^{24–26 (link)} U87 cells were transfected with pNBe vectors containing human EGFR, AXL, or LRIG1, C-terminally fused to LgBiT or SmBiT. For competition experiments, pIRES plasmids containing untagged EGFR, AXL, LRIG1, or sLRIG1 were co-transfected. Forty-eight hours posttransfection, cells were harvested and distributed into white 96-well plates, incubated with ligands of interest, and then with Nano-Glo Live Cell substrate. RTK dimerization or interaction with LRIG1 were evaluated with a ClarioStar luminometer (BMG LabTech). The signal is reported as a ratio to “untreated” control condition (without ligand), being set to 1 (Supplementary Methods).

β-arrestin recruitment to chemokine receptors in response to CCL19, CXCL14 or positive control chemokines was monitored by NanoLuc complementation assay [NanoBiT, Promega, Madison, WI, United States (35 (link))] as previously described (36 (link)). In brief, 6.5 × 10⁵ HEK293T cells were plated per well in a 12-well dish (6 × 10⁶ per 10 cm dish for concentration response curves) and 24 h later co-transfected with pNBe vectors encoding a chemokine receptor C-terminally tagged to the luciferase fragment SmBiT and human β-arrestin-2 N-terminally fused to LgBiT. 24 h post-transfection cells were harvested, incubated for 25 min at 37°C with Nano-Glo Live Cell substrate diluted 200-fold and distributed into white 96-well plates (1 × 10⁵ cells per well). Ligand-induced, β-arrestin recruitment to chemokine receptors was evaluated with a Mithras LB940 luminometer (Berthold Technologies, Bad Wildbad, Germany) for 20 min. For concentration-response curves, 3 nM CCL19 was co-incubated with increasing concentrations of CXCL14 for 15 min at room temperature before treatment. For screening experiments, 200 nM of one known agonist chemokine listed in the IUPHAR repository of chemokine receptor ligands was added as positive control to each receptor.

Recruitment of β-arrestin-1 to human and mouse CCR5 (hCCR5 and mCCR5) induced by human and mouse CCL5 chemokines (hCCL5 and mCCL5) was monitored by NanoLuc complementation assay (NanoBiT, Promega, Madison, WI, USA), as previously described [35 (link),36 (link),37 (link)]. Briefly, 5 × 10⁶ HEK293T cells were seeded in 10 cm culture dishes and, 24 h later, co-transfected with pNBe vectors encoding human or mouse CCR5 C-terminally fused to SmBiT and β-arrestin-1 N-terminally fused to LgBiT. Twenty-four hours post transfection cells were harvested, incubated for 25 min at 37 °C with 200-fold diluted Nano-Glo Live Cell substrate and distributed into white 96-well plates (5 × 10⁴ cells per well). Cells were then treated with MVC for 20 min at room temperature at concentrations ranging from 0.05 nM to 1.11 µM for hCCR5 and from 4.57 nM to 50 µM for mCCR5 then stimulated with human or mouse CCL5 (5 nM). β-arrestin recruitment to hCCR5 and mCCR5 was evaluated by measuring bioluminescence with a Mithras LB940 luminometer (Berthold Technologies, Bad Wildbad, Germany).