Heterodimerization of CysLT1 and CysLT2 receptors. FLIM images (A,B) showing interactions between the indicated receptors on LAD2 cells stained with directly labeled Alexa Fluor 488–conjugated anti-CysLT1 EC3 Abs (RB34) without or with Alexa Fluor 546–conjugated anti-CysLT2 N-terminal polyclonal Abs (A). Pseudocolor images are from 1 experiment representative of 4 performed and are similar to experiments performed with anti-CysLT2 N-terminal monoclonal directly or indirectly labeled with Cy3 (not shown). (B) Noninteracting (BLT1/CysLT2) and interacting (α4/β1) controls for FRET. Combinations of Abs against BLT1 (Alexa Fluor 488) plus CysLT2 (Cy3) (B left), or α4 (Alexa Fluor 488) and β1 (Cy3) integrins (B right). Data are from 1 experiment representative of 3. Orange color (see scale) indicates the areas of strongest FRET. See “FLIM” for complete image acquisition information. (C) Biexponential analysis of FRET comparing cells stained with the directly fluorophore-conjugated Abs (Alexa Fluor 488–conjugated RB34, plus Cy3- or Alexa Fluor 546–conjugated rabbit antihuman CysLT2 N-terminal Ab). Similar results were obtained with indirect labeling using RB34 and monoclonal antihuman CysLT2 Abs. Results are the mean of 3 to 4 experiments using each Ab combination. *P < .001 relative to the negative control (CysLT1-Alexa-488 alone). (D) Coimmunoprecipitation of CysLT1 and CysLT2 from lysates of hMCs. CysLT1 was precipitated and detected with rabbit antihuman EC3 Ab (RB34), whereas CysLT2 was precipitated with rabbit anti–N-terminal CysLT2 Ab and detected with mouse anti-CysLT2 monoclonal Ab 1B3. Note the presence of the likely rabbit IgG heavy chain (top). Protein equivalents of 2.5 × 107 cells were used to generate the precipitates, whereas 1 × 106 cell equivalents of whole-cell lysates were used as controls. Data are from a single experiment representative of 4 performed with cells from different donors. Vertical lines have been inserted to indicate repositioned gel lanes.
