Figure 4. Analysis of microtubule stability in β3WT, β3HET and β3NULL endothelial cells.

1. Top: β3WT, β3HET and β3NULL endothelial cells were adhered to fibronectin‐coated coverslips for 75 min at 37°C before being moved to ice for 15 min. Soluble tubulin was then washed out using PEM buffer (see Materials and Methods) before fixing with −20°C methanol (Note: this protocol leads to nuclear auto‐fluorescent background in all three channels used). Immunostaining was carried out for α‐tubulin (green) and talin‐1 (Tln1, red). DAPI (blue) was used as a nuclear stain. Images shown are representative of the data shown in the bar graph shown below. Scale bar = 5 μm. Bottom: Dot plots = mean (±SEM) number of cold‐stable microtubules per cell (n ≥ 300 cells per genotype, from three independent experiments). Significant differences between means were evaluated by unpaired two‐tailed Student's t‐test.
2. β3WT, β3HET and β3NULL endothelial cells were adhered to fibronectin for 75 min at 37°C before being moved to ice for 15 min. Cold‐soluble tubulin (left blot) was then washed out using PEM buffer and Western‐blotted for α‐tubulin and Gapdh (as a loading control). Cold‐insoluble tubulin (middle blot) from the same cells was obtained by lysing the remaining cells and Western blotting for α‐tubulin and Gapdh (as a loading control). Right bar chart: Bars = mean (±SEM) relative cold‐soluble and cold‐insoluble α‐tubulin levels for each genotype. Data are representative of four independent experiments. *indicates statistical significance compared to WT (P < 0.05). Significant differences between means were evaluated by unpaired two‐tailed Student's t‐test.