Anti cortactin antibody

Following siRNA transfection, HAECs or HMVECs, were trypsinized and seeded on collagen I-coated coverslips in complete growth medium. Adherent cells were treated with VEGF-A₁₆₅ (25 ng/ml) and select experimental compounds [PKAi cocktail, PP2 (10 µmol/L), ML141 (10 µmol/L) or the appropriate vehicle] for a period of 24 hours, then fixed in 4% paraformaldehyde. To visualize actin and cortactin, the fixed cells were and stained with an anti-cortactin antibody (#05–180, clone 4F11; Millipore) and with TRITC-conjugated phalloidin. DAPI was also used to stain cellular nuclei. For high resolution images of podosomes and podosome rosettes, actin/cortactin-stained cells were imaged with a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems, Concord, ON, CA). For quantification of podosome-positive and podosome rosette-positive HAECs or HMVECs, images were acquired with a Zeiss Axiovert S100 microscope. Individual podosomes were identified by co-localization of F-actin and cortactin staining in a dot-like distribution (0.5–1 µm)^{59 (link)} while podosome rosettes were characterized by individual podosomes clustered into a ring or circular-like structure of dimensions (5–10 µm in diameter)^{59 (link)}. Identification of individual podosomes or podosome rosettes was quantified from at least three separate experiments in which 50–100 cells were examined per experiment.

The following reagents were used: α-minimum essential medium, (α-MEM; 135-15175, WAKO, Osaka, Japan); fetal bovine serum (FBS; 10270, Gibco), anti-Arp3 antibody (sc-48344, 1:100, Santa Cruz Biotechnology); anti-β-actin antibody (ab8226, 1:100, Abcam); anti-non-muscle myosin heavy chain IIA antibody (PRB-440P-100, 1:100, BioLegend, Dedham, MA); anti-cortactin antibody [5-180, 1:100, Millipore (Upstate)]; anti-paxillin antibody (610051, 1:100, BD Biosciences); anti-vinculin antibody (V9264, 1:100, Sigma-Aldrich); anti-zyxin antibody (sc-6438, 1:100, Santa Cruz Biotechnology); rhodamine-phalloidin (R415, Molecular Probes, Thermo Fisher Scientific); fluorescein isothiocyanate-phalloidin (P5282, Sigma-Aldrich); DAPI (D1306, Molecular Probes, Thermo Fisher Scientific, CA); recombinant mouse receptor activator of nuclear factor kappa-Β (sRANK) ligand (315-11, PeproTech, NJ); fluorescein isothiocyanate- and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies (715-095-151, 711-095-152 and 712-096-153, Jackson ImmunoResearch, PA); CK-666 (182515, Calbiochem, Merck Millipore, Darmstadt, Germany) and ML-7 (475880, Calbiochem).