Figure 1

AdcA is phosphorylated in response to various stresses. Parental KAx-3 cells in suspension in nutritive medium were subjected to different stress conditions. Protein samples were prepared as described in Materials and Methods and AdcA electrophoretic profile was followed by Western blot with anti-AdcA antibodies. A. Effect of sorbitol on AdcA electrophoretic mobility. Cells were treated with 200 mM sorbitol. The bottom panel represents the pixel intensities along the scanning lines a, b, c and d on the top panel. Bands 1, 2 and 3 were quantified using ImageJ software and the signal intensity of bands 2 + 3 was expressed as a percentage of the total and plotted as a function of time. The data shown correspond to the mean ± s.d of 4 experiments. B. Effect of increasing concentration of sorbitol on AdcA. Cells were subjected to concentrations of sorbitol ranging from 50 to 200 mM and the consequences on AdcA migration were analysed by Western blot. C. AdcA responds to successive sorbitol shocks. Cells were stimulated with 200 mM sorbitol at t = 0 and again at t = 180 min. Samples were collected at indicated times to analyse AdcA response. D. Treatment of AdcA with the CIP phosphatase abolishes its mobility shift. Protein samples obtained from cells subjected to 200 mM sorbitol were treated with CIP for 30 min at 37°C (+). Controls (−) were subjected to the exact same procedure but received no enzyme. E. AdcA responds to other stresses. Cells were faced with different stress conditions. The amount of AdcA as bands 2 + 3 at t = 30 min was represented as a percentage of the total amount of AdcA. The data are shown as the mean ± s.e.m of 3 independent experiments for each stress and 8 for the control (−) experiments.