Fig. 3.

CXCL12 induces rearrangements between CXCR4 and the G_i1 protein. (A and B) Schematic depicting the settings employed to investigate the interaction between the receptor and the G_i1 protein. HEK293 cells were transfected with CXCR4-YFP and Gα_i1/Gβ₁/Gγ₂-CFP (A) or Gα_i1-CFP/Gβ₁/Gγ₂ (B). (C and D) Representative traces of the FRET response from a single HEK293 cell expressing CXCR4-YFP and the G protein CFP-labeled at the Gγ₂ (C) or Gα_i1 subunit (D) and stimulated with 30 µM CXCL12 for the indicated period of time (black line). Upper panels show corrected YFP (yellow) and CFP (cyan) emissions. Lower panels show corrected and normalized FRET ratios. (E) On-kinetics of the interaction of CXCR4 with the G_i1 protein upon CXCL12 stimulation as measured in the two settings. τ values from individual experiments are represented in a scatter plot. Data show median and IQR of 12 cells for each setting, measured on at least 3 independent experimental days. Statistical significance was tested using Mann-Whitney test. a.u.,arbitrary units; n.s., nonsignificant.