Figure 2

Absence of cortactin causes increased ROCK1 protein levels.

Cortactin-depleted HMEC-1 and scrambled control cells were grown to confluency and lysed for either protein or RNA extraction. (A) Proteins were analysed by western blot for cortactin, MLCK and ROCK1 expression. γ-tubulin served as loading control. Left graph shows quantification of cortactin expression and right graph shows quantification of ROCK1 expression of three independent experiments each. **p < 0.01; ***p < 0.001 (B) cDNAs were analysed by real-time RT-PCR for expression of ROCK1 and MLCK (transcript variants (Tv) 1 and 2). Data were normalized to β-actin as house-keeping gene and WT controls were set to 1 for each amplicon. Cryotissue sections from lungs (C) and brains (D) were analysed by immunofluorescence using anti-PECAM-1 and anti-ROCK1 antibodies. White squares indicate zoomed areas of interest in brain vessels (D) where colocalization of PECAM-1 and ROCK-1 can be observed (arrowheads). Bars, 20 μm (brains) and 10 μm (lungs). Pixel intensities for ROCK1 (graphs on the right) were quantified using ImageJ software. Graphs show means +/−SDM of three (lungs) and four (brains) independent tissue preparations. ***p < 0.001.