![Figure 6. CCRL2 and CXCR2 form homodimers and heterodimers in living cells. FRET saturation curves for (A) CXCR2/CXCR2, (B) CCRL2/CCRL2, and (C) CXCR2/CCRL2 complexes in HEK293 T cells. Curves were obtained by using cells transiently cotransfected with either the vector encoding CXCR2-CFP and increasing amounts of CXCR2-YFP plasmid or the CCRL2-CFP plasmid and increasing amounts of CCRL2-YFP plasmid. For heterodimer evaluation, we used CXCR2-CFP plasmid and increasing amounts of CCRL2-YFP plasmid. For negative controls, cells were transfected with CXCR2-CFP or CCRL2-CFP plasmid and increasing amounts of H3R-YFP plasmid. By using ImageJ 1.43u software (National Institutes of Health), FRET efficiency was determined on a pixel-by-pixel basis (E) and calculated in percent as E = [(ICFPpost – ICFPpre)/ICFPpost] × 100, where ICFPpre and ICFPpost are the background-corrected CFP fluorescence intensities before and after YFP photobleaching, respectively. FRET efficiency was calculated from ≥20 images from each of 3 independent experiments. Data are expressed as the mean ± SEM of 5 independent experiments performed in duplicate. FRET50 and FRETmax value were calculated by using a nonlinear regression equation for a single binding site model and are expressed as mean ± SEM (n = 5). (D) FRET analysis by acceptor photobleaching of CXCR2/CCRL2 heterodimers. Shown are representative images of CFP and YFP staining before photobleaching (CFP-Pre, YFP-Pre) and of CFP and YFP after photobleaching (CFP-Post, YFP-Post) and a zoom image of FRET at the photobleached areas (1 and 2) using a false color scale (inset). Only areas with a ∼2:1 YFP:CFP ratio were selected for bleaching analysis (white outline). Areas in which the YFP:CFP ratio was unsuitable were not included in the analysis. In area 2, the red dashed line indicates the position of the cell membrane. Percentage of FRET efficiency ± SEM is shown for each photobleached area. (E) Membrane expression of CXCR2 in HEK293 T cells transfected with CXCR2 alone (empty vector) or with CCRL2 was determined by flow cytometry analysis using specific anti-CXCR2 moAb. Data are expressed as the mean ± SEM of 3 independent experiments performed in duplicate. (F) Membrane expression of CXCR2 on CD11b+Ly6G+ purified BM neutrophils from WT and CCRL2-deficient mice. Data are expressed as relative MFI (mean ± SEM of 6 mice per group). *P < .05 by Student t test. ND, not determined.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/130/10/10.1182_blood-2017-04-777680/4/blood777680f6.jpeg?Expires=1735562920&Signature=E9uLweaHJKnoVJfEhB1ipVmn7Jh0okHefyuMUFoFnWV50cZJoXF4qBCNlMsZ6PZQDSw~O1PlrDUcQAk8FQt9Bu41aiCvH86u~G3HnSoaVayY7Yj2PYy31~WhI~Gf9fA9Ez9ypnCjLRSOyRK4rovopTg8NAO5fWUJXwTEdECiNClRbarv2wMkSuDbB~OlNg6CXGwbxdQisjF7eGEWhBVfqQsTgv6U0wdMY-mUGtayC2Xf2c4FaRJmvNkqCUO2p5SEztVR0F7Bx3hUSu5FaOD-xJ2rRqOGwarZLgPKTL8~HUPEbKRT0A54YpYjx9FD98l3TnIl0gchjR4MojT7BID6uA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CCRL2 and CXCR2 form homodimers and heterodimers in living cells. FRET saturation curves for (A) CXCR2/CXCR2, (B) CCRL2/CCRL2, and (C) CXCR2/CCRL2 complexes in HEK293 T cells. Curves were obtained by using cells transiently cotransfected with either the vector encoding CXCR2-CFP and increasing amounts of CXCR2-YFP plasmid or the CCRL2-CFP plasmid and increasing amounts of CCRL2-YFP plasmid. For heterodimer evaluation, we used CXCR2-CFP plasmid and increasing amounts of CCRL2-YFP plasmid. For negative controls, cells were transfected with CXCR2-CFP or CCRL2-CFP plasmid and increasing amounts of H3R-YFP plasmid. By using ImageJ 1.43u software (National Institutes of Health), FRET efficiency was determined on a pixel-by-pixel basis (E) and calculated in percent as E = [(ICFPpost – ICFPpre)/ICFPpost] × 100, where ICFPpre and ICFPpost are the background-corrected CFP fluorescence intensities before and after YFP photobleaching, respectively. FRET efficiency was calculated from ≥20 images from each of 3 independent experiments. Data are expressed as the mean ± SEM of 5 independent experiments performed in duplicate. FRET50 and FRETmax value were calculated by using a nonlinear regression equation for a single binding site model and are expressed as mean ± SEM (n = 5). (D) FRET analysis by acceptor photobleaching of CXCR2/CCRL2 heterodimers. Shown are representative images of CFP and YFP staining before photobleaching (CFP-Pre, YFP-Pre) and of CFP and YFP after photobleaching (CFP-Post, YFP-Post) and a zoom image of FRET at the photobleached areas (1 and 2) using a false color scale (inset). Only areas with a ∼2:1 YFP:CFP ratio were selected for bleaching analysis (white outline). Areas in which the YFP:CFP ratio was unsuitable were not included in the analysis. In area 2, the red dashed line indicates the position of the cell membrane. Percentage of FRET efficiency ± SEM is shown for each photobleached area. (E) Membrane expression of CXCR2 in HEK293 T cells transfected with CXCR2 alone (empty vector) or with CCRL2 was determined by flow cytometry analysis using specific anti-CXCR2 moAb. Data are expressed as the mean ± SEM of 3 independent experiments performed in duplicate. (F) Membrane expression of CXCR2 on CD11b+Ly6G+ purified BM neutrophils from WT and CCRL2-deficient mice. Data are expressed as relative MFI (mean ± SEM of 6 mice per group). *P < .05 by Student t test. ND, not determined.
