Mouse monoclonal anti vinculin hvin 1

Western blot analysis was carried out as described elsewhere [49 (link)]. The following antibodies were used: rabbit polyclonal anti-β-tubulin (H-235) and mouse monoclonal anti-fibronectin (IST-9) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-Integrin α5 (AB1949) and goat polyclonal anti-HGF from R&D System (Minneapolis, MN, USA); rabbit polyclonal anti-RSK1, rabbit monoclonal anti-RSK2 (D21B2) XP, rabbit monoclonal anti-phospho-p90RSK (380), rabbit monoclonal anti-phospho-RSK2 (227) (D5EA11), rabbit polyclonal anti-YB1 (D299), rabbit monoclonal anti-phospho-YB1 (Ser102) (C34A2) and rabbit monoclonal anti-SNAIL (C15D3) obtained from Cell Signaling Technology (Beverly, MA, USA); mouse monoclonal anti-Vinculin (hVIN-1) from Sigma, Saint Louis, MO, USA. It is worth noting that two bands of YB-1 become visible when the level of YB-1 expression is very high as in the case of rescued cells. Indeed, two bands are recognized by several antibodies and are differently interpreted [see e.g. ref. 52 (link)].
Bound antibodies were detected using the appropriate peroxidase-conjugated secondary antibody and revealed by enhanced chemiluminescence (Pierce, Rockford, IL, USA).

STED microscopy was performed on a Leica TCS SP8 STED 3X microscope. HUVEC monolayers were stained using the following antibodies: rabbit polyclonal anti-pacsin2 (Abcam), mouse monoclonal anti-vinculin hVIN-1 (Sigma), mouse monoclonal anti-p120-catenin clone 98/pp120 (BD Bioscience), rabbit anti-α-catenin and anti-β-catenin serum (Sigma) combined with anti-VE-cadherin mouse monoclonal F-8 (Santa Cruz) or rabbit polyclonal (Cayman) antibodies. Secondary antibodies used were STAR 580 and STAR 635P (Abberior). Samples were irradiated with a pulsed white light laser at wavelengths 587 and 633 nm. A pulsed STED laser line at a wavelength of 775 nm was used for the depletion of the 587- and 633-nm fluorophore, reaching a lateral resolution of ∼45 nm to distinguish individual fluorescent signals in these IF stainings. The signal was detected using a gated hybrid detector in counting mode. STED images were acquired using a dedicated 100 × 1.4 NA oil objective. Finally, deconvolution was performed with Huygens Professional (Scientific Volume Imaging).