The localization and internalization of the CysLT1R in epithelial cells.
Immunofluorescent staining of endogenous CysLT1R (A, D) and Flag-tagged CysLT1R (C, F) in Int 407 (A, C) and Caco-2 cells (D, F). Cells were grown on cover slips to 50–60% confluency pre-treated, or not, with ZM198,615 (40 µM, 15 minutes) and treated with or without 80 nM LTD4 for 5 minutes or as indicated. The cells were fixed and stained with CysLT1R antibody (1∶250) or Flag antibody (1∶2500) and mounted as described in Methods. The mounted slides were examined using a Nikon TE300 microscope (100×1.4 plan-apochromat oil immersion objective). The scale bar represents 10 µm. Int 407 cells (B) and Caco-2 cells (E) were grown to 80% confluency, serum-starved for 2 hours and treated with or without 80 nM LTD4 for indicated time points. Plasma membrane fractions were prepared as described in Methods and samples were subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis. The PDVF membranes were stained with the CysLT1R (1∶1000) and re-probed with actin (1∶2000) antibodies. The data are given as percent of control and represent means ± S.E.M. of at least three separate experiments. The statistical analysis was performed with a Student's t test. *P<0.05 and ** P<0.01. (G) A representative FACS analysis histogram overlay displaying the relative fluorescence intensity of CysLT1R surface expression is shown for Caco-2 cells.