Figure 3
From: The arrestin-domain containing protein AdcA is a response element to stress
Responses of vegetative KAx-3 cells to hyperosmolarity. Cells maintained in culture medium were treated with 200 mM sorbitol and the consequences of hyperosmolarity were followed on different aspects of the cell biology. A. The effect on cellular morphology was monitored by time-lapse video microscopy using DIC optics and a 63x objective. The images shown illustrate the morphology of the cells after 5 min of stress. Scale bars: 10 μm. B. The distribution of F-actin was analysed using Alexa 594-phalloidin after cell fixation/permeabilisation in 4% PFA, 0.1% Triton X-100. To enhance resolution, optical sections were taken throughout the cell and digitally deconvolved using Axiovision software. One optical section and a z-stack maximal intensity projection are shown. The phosphorylation of actin in response to sorbitol treatment was examined by Western blot on whole cell extracts using the anti-phospho-Tyr antibody (PY20). Scale bars: 5 μm. C. Endogenous STATc and mycPTP3ΔC649S phosphorylation was analysed using the STATc-specific antibody CP22 and 7H3 or the anti-myc antibody 9E10. D. STATc nuclear translocation in response to stress was detected by immunofluorescence using the 7H3 antibody on cells kept in nutritive medium or starved for 4 h in 12 mM Na, K-phosphate buffer, pH 6.2, subjected or not to 200 mM sorbitol for 5 min. Scale bar: 10 μm.