Figure 7
From: Dynamic microtubule association of Doublecortin X (DCX) is regulated by its C-terminus
FRAP analysis reveals that hyperosmotic stress conditions affect GFP-α-tubulin exchange dynamics. COS-1 cells were transfected to express (A) GFP-α-tubulin, GFP-α-tubulin together with myc-DCX WT or GFP-α-tubulin together with myc-DCX ΔC and then exposed to sorbitol (0.5 M, 1 h) as indicated to induce hyperosmotic stress. A FRAP protocol with a small bleach area and measuring recovery into the bleached area was utilized (A,B). A small ROI (indicated by the white rectangle in each cell image) was photobleached in (A) and the fluorescence recovery subsequently monitored at 3 s intervals for 60 s. (B) Plots of the recovery of fluorescence in small area of bleach in GFP-α-tubulin with or without myc-DCX constructs in the presence and absence of sorbitol treatments are shown. Regression values for the accuracy of the respective curve fits are shown. Insets represent the recovery of fluorescence in bleach area for first three time-points (0–6 s of post-bleach) with the line of best fit used for the calculation of initial rate. (C) Equivalent expression of the GFP-α-tubulin and myc-DCX constructs was detected in the presence and absence of sorbitol by immunoblotting for c-myc (upper panel) and GFP (lower panel). Scale bars represent 10 µm. See Fig. 8 for quantitative pooled data.
