Figure 1

NHERF1 translocates from cytosol to plasma membrane upon LPA stimulation, and then to surface protrusions. (a) Time-lapse imaging of LPA-induced NHERF1 translocation. OVCAR-3 cells were plated onto type I-coated glass-bottom dishes and transfected with GFP-tagged NHERF1 construct. After serum deprivation for 24 h, the cells were incubated in Phenol Red-free DMEM and stabilized for 20 min. After adjusting the plane of focus near the bottom region close to the glass surface, time-lapse scanning was performed at 2 min intervals after 1 μM LPA stimulation. Scale bar, 20 μm. Relative intensities of GFP fluorescence are shown along the broken line of the long arrow (yellow) overlaid on each image, using the line scan tool of Image J software. The relative distribution of GFP-NHERF1 at each time point is plotted in the graph in the right lower corner. (b) High-resolution image of NHERF1 translocation from cytosol to surface protrusions in response to LPA stimulation. OVCAR-3 cells expressing GFP-NHERF1 were stimulated with 1 μM LPA, fixed, and observed by confocal microscopy. The focuses of the confocal imaging are adjusted to the top and the bottom region sequentially, as indicated. Scale bar,20 μm. Relative intensities of GFP fluorescence intensities are shown along the broken line of the long arrow (yellow) overlaid on each image. The relative distribution of GFP-NHERF1 is plotted in the graph immediately below each image.