HEK293T cells were transfected
with 0.5
μg of WT or mutant H4R-Rluc8 in combination with
4 μg of β-arrestin1-eYFP, β-arrestin2-mVenus, or
GRK2/3/5/6-mVenus DNA, or 2 μg of Venus-Rab5a DNA. At 24 h post-transfection,
cells were transferred to poly-l -lysine-coated white 96-well
plates (Greiner Bio-one; Frickenhausen, Germany). At 48 h post-transfection,
baseline BRET was measured using 5 μM coelenterazine-H (Promega;
Madison, WI, USA) in HBSS in a Mithras LB940 multilabel plate reader
(Berthold Technologies; Bad Wildbad, Germany) followed by stimulation
with H4R ligands at 37 °C, as previously described.67 (link) The BRET ratio was calculated by dividing acceptor
light emission at 540 nm by Rluc8 light emission at 480 nm.
with 0.5
μg of WT or mutant H4R-Rluc8 in combination with
4 μg of β-arrestin1-eYFP, β-arrestin2-mVenus, or
GRK2/3/5/6-mVenus DNA, or 2 μg of Venus-Rab5a DNA. At 24 h post-transfection,
cells were transferred to poly-
plates (Greiner Bio-one; Frickenhausen, Germany). At 48 h post-transfection,
baseline BRET was measured using 5 μM coelenterazine-H (Promega;
Madison, WI, USA) in HBSS in a Mithras LB940 multilabel plate reader
(Berthold Technologies; Bad Wildbad, Germany) followed by stimulation
with H4R ligands at 37 °C, as previously described.67 (link) The BRET ratio was calculated by dividing acceptor
light emission at 540 nm by Rluc8 light emission at 480 nm.
