Polyclonal anti αv

Whole cells were lysed as described [42 (link)], and resolved by SDS-PAGE, followed by Western blotting with PVDF membranes (Millipore, Burlington, MA, USA). The filters were probed with the following antibodies: polyclonal anti-VE Cadherin (Bioss Antibodies, Woburn, MA, USA), polyclonal anti-αV (Millipore), polyclonal anti-GAPDH (Elabscience, Houston, TX, USA), and polyclonal anti-actin (Sigma-Aldrich, St. Louis, MO, USA). Then, goat anti-rabbit IgG-HRP (Sigma) or goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA) were employed as secondary antibodies. The peroxidase activity was revealed with Immobilon^TM Western HPR Substrate (Millipore). Films were imaged using the CanoScan 4400F (Canon, Tokyo, Japan) at 300 dpi, with the Adobe Photoshop Creative Suite 2 or CS2, and bands were quantified with the ImageJ 1.52a software (NIH, Bethesda, MD, USA).

The U87-MG, U251-MG, and U138-MG cell lines were harvested and acid-treated, as described [41 (link)]. Then, 2 × 10⁶ cells/sample were exposed for 30 min at 4 °C to uPAcyclin or S-uPAcyclin at the indicated concentrations, or with the following antibodies: VNR147 monoclonal anti-αV integrin (Chemicon Int. Inc., Temecula, CA, USA), polyclonal anti-αV (Millipore, Burlington, MA, USA), monoclonal anti-α2 (Chemicon Int. Inc., Temecula, CA, USA), polyclonal anti-actin (Sigma-Aldrich, St. Louis, MO, USA), monoclonal anti-GAPDH (Abcam, Cambridge, UK), and purified vitronectin (VN, Corning, Glendale, AZ, USA). The cells were subsequently incubated with 50 nM FITC-uPAcyclin for 2 h at 4 °C, as described [25 (link)]. At the end of incubation, the cells were washed and the cell surface-associated fluorescence was determined using a fluorimeter (VICTOR^TM X3-PerkinElmer, Waltham, MA, USA). All binding assays were performed three times, with duplicate samples, and analyzed as described in Section 2.11.