Figure 1.

ARF localizes to the plasma membrane during adhesion/spreading process. HeLa cells were detached by gentle trypsin treatment, re-suspended in growth medium and allowed to adhere onto fibronectin-coated coverslip, and at various times following plating, rinsed in PBS (4 °C) and fixed with 3.75% PFA. Cells were fixed at different time points after plating (30’, 1 h, 3 h and 5 h), permeabilized and subjected to IF with anti-ARF antibody and both tetramethylrhodamine phalloidin and DAPI staining to visualize actin cytoskeleton and nuclei. Representative images of ARF subcellular localization are shown for each time point. Images were taken with a Zeiss confocal laser-scanning microscope LSM 510 (Oberkochen, Germany) (scale bar, 7μm). A 40 × objective was used and image analysis was performed using ImageJ. Samples that were to be directly compared were imaged at the same sitting, and the same gain and exposure time were used. Histograms, representing the mean of three independent experiments, reports the percentage of cells in which ARF localize with f-actin. For each time point, 50 cells have been analyzed. S.d. are also shown. Asterisks (*) indicate statistically significant differences by unpaired two-tailed t-test with Welch correction: *P<0.001 between each point and 1-h time point.