Fig. 3

Agonist-induced β-arrestin 2 recruitment to CXCR4 in presence and absence of US28wt and mutants. a BRET-based approach. CXCR4 C-terminally tagged with mVenus, β-arrestin 2 N-terminally fused to RlucII and US28, US28 mutants or empty vector (mock) were transiently expressed in HEK293T cells. β-arrestin 2 recruitment was measured 5 min post-ligand addition. ΔBRET was calculated by subtracting BRET ratio detected in vehicle stimulated cells from BRET ratio detected in CXCL12-stiumulated cells for each receptor-combination. Curves represent means ± SEM from at least three independent experiments (n = 3–6), each performed in triplicates. b BiLC-based approach. CXCR4 C-terminally tagged with ElucC, β-arrestin2 N-terminally fused to ElucN and US28, US28 mutants or empty vector (mock) were transiently expressed in HEK293T cells. Luminescence was measured following 10 min stimulation with 100nM CXCL12 or vehicle (no filters, 2 s recording). ΔLuminescence was calculated by subtracting luminescence detected in vehicle stimulated cells from luminescence detected in cells stimulated with 100nM CXCL12 for each transfection-combination and normalized on ΔLuminescence of mock-cotransfected cells. Columns represent means ± SEM from three independent experiments, each performed in triplicates