X2-C/X1-Y X2-C/VCAM-Y. FRET efficiency. Ratio YFP/CFP

Transcription

1 FRET efficiency X2-C/X1-Y X2-C/VCAM-Y Ratio YFP/CFP Supplemental Data 1. Analysis of / heterodimers in live cells using FRET. FRET saturation curves were obtained using cells transiently cotransfected with the vector encoding X2-C plasmid (2 μg, ~3 FU) and increasing amounts of X1-Y plasmid (~-8 FU). For negative controls, cells were transfected with X2-C (2 μg. ~4 FU) and increasing amounts of VCAM-YFP plasmid (~1-14 FU). Data are expressed as the mean ± SEM of four independent experiments performed in duplicate.

2 1 3 X1-C/X2-Y 2,% 6,% 1 3 X2-C/X1-Y 22,% 1,6% ,% 9,% 21,9% 4,6% Supplemental Data 2. and are expressed at the cell membrane of transfected cells. Cell surface expression of and in X1-C/X2-Y and X2 C/X1-Y-transiently transfected HEK293T cells was determined by flow cytometry analysis using specific mab. The percentage of positive cells is indicated.

3 X1/X2 X1-C/X1-Y X2-C/X2-Y X1-C/X2-Y X2-C/X1-Y % Response time (sec) Supplemental Data 3. and fused to fluorescent proteins are functional receptors. Calcium mobilization assays were performed in HEK293 cells stably transfected with / (X1/X2), and in cells transiently transfected with different combinations of the same receptors coupled to yellow or cyan fluorescent proteins: -CFP/-YFP (X1-C/X1-Y), - CFP/-YFP (X2-C/X2-Y), -CFP/-YFP (X1-C/X2-Y) and -CFP/-YFP (X2-C/X1-Y). Cells loaded with Fluo-3AM were stimulated with CXCL8 (2nM) and calcium flux monitored in an flow cytometer. FIgure shows a representative experiment of five performed.

4 Mr kda kda 38 kda b-actin Lysate ipp: : Lysate 293 X1 2: Neutrophils ipp: Lane 1: Lane 2: Lane 3: 293 X1/X2 293 X2 293 X1 Supplemental Data 4. Co-immunoprecipitation of / heterodimers in 293X1/X2 cells and primary neutrophils. Unstimulated 293X1/X2, 293X1 and 293X2 cells (A) or human neutrophils () were lysed, extracts immunoprecipitated with anti- mab, and analyzed by Western blot with anti- mab, as indicated; lysates from unstimulated cells were used as controls. The membranes were stripped and reprobed with anti- mab to control protein loading and with β-actin mab. Arrows indicate and bands.

5 X1-CFP X2-CFP pires-x1gfpnuc piresgfpnuc pires-x2gfpnuc piresgfpnuc DIC/GFP CFP/GFP GFP/anti-X2-Cy3 DIC/GFP CFP/GFP GFP/anti-X1-Cy3 upplemental Data. or expression at the plasma membrane of EK293 cells expressing GFP in the nucleus. (A) HEK293T cells were cotransfected ith -CFP (X1-CFP) and pires2-acgfp1-nuc or empty vector, or () with XCR2-CFP (X2-CFP) and pires2-acgfp1-nuc or empty vector. Differential intererence contrast images show nuclear GFP when present (DIC; left columns); confocal cyan luorescence images show expression of the CFP-labeled receptor (middle columns) and lso GFP. The unlabeled receptor was detected by immunostaining using specific mab and y3-goat anti-mouse antibody (right columns); GFP is also shown. Images shown are epresentative of at least five experiments.

6 X1-C/X1-Y/pIRES-Ac X2-C/X2-Y/pIRES-Ac Relative cell number Fluorescence intensity Relative cell number Fluorescence intensity GFPNUC X2-GFPNuc control GFPNUC X1-GFPNuc control Supplemental Data 6. The expression of pires constructs does not modify neither nor expression levels. (A) Cell surface xpression of in HEK293T cells transiently cotransfected with X1- /X1-Y and pires2-acgfp1-nuc or empty vector, as determined in low cytometry. () Cell surface expression of in HEK293T cells traniently cotransfected with X2-C/X2-Y and pires2-acgfp1-nuc or mpty vector, as shown using flow cytometry.

7 CFP-Pre YFP-Pre CFP-Post YFP-Post FRET on bleached areas X1-C/X1-Y piresgfpnuc pires-x2gfpnuc %FRET efficiency %FRET efficiency X2-C/X2-Y piresgfpnuc pires-x1gfpnuc CFP-Pre YFP-Pre CFP-Post YFP-Post FRET on bleached areas %FRET efficiency %FRET efficiency Supplemental Data 7. FRET analysis by photobleaching of X1/X1 and X2/X2 homodimers in the presence of X2 or X1. (A) FRET was measured in unstimulated HEK293T cells transiently cotransfected with X1-C, X1 Y, and pires2-acgfp1-nuc or pires2-x2- AcGFP1-Nuc. One representative image (of >) shows CFP and YFP staining before (CFP-pre, YFP-pre) and after photobleaching (CFP-post, YFP-post), with a zoom image of FRET at the photobleached area (inset). FRET efficiency ± SEM is shown (right). The DIC image of both cell types is shown (left panels). Areas showing a ~1:1 YFP/CFP ratio were selected for bleaching and analysis (white outline). () FRET was measured in unstimulated HEK293T cells transiently cotransfected with X2-C, X2 Y, and pires2-acgfp1-nuc or pires2-x1-acgfp1-nuc and analyzed as in (A).