Anti vinculin

Protein extraction and western blotting experiments were performed as already described^{61 (link),62} . The antibodies used were: anti-β-actin (sc-1616, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-γ-tubulin (sc-17787, Santa Cruz), anti-GAPDH (sc-32233, Santa Cruz), anti-vinculin (sc-7649, Santa Cruz), anti-PAX8^{63 (link)}, anti-NKX2.1^{33 (link)}, anti-FOXE1^{64 (link)}, anti-SP-B (ab3430, Millipore), anti-SP-C (90716, Abcam), anti-HIPK2 (Novus Biologicals), anti-HMGA1^{65 (link)}. Densitometric analyses of western blot were performed with Image J software.

Anti‐p130cas, anti‐myosin light chain kinase (MLC), anti‐phospho p130Cas (Y165), and anti‐phosphopaxillin (Y118) antibodies were from Cell Signaling Tech., Danvers, MA, USA. Anti‐paxillin antibody was from ECM Biosciences LLC, Versailles, KY, USA, Anti‐FAK (focal adhesion kinase), anti‐vinculin, anti‐phosphotyrosine, and anti‐phospho MLC (Thr18/Ser19 serine) antibodies were from Santa Cruz Biotechnology, Dallas, TX, USA. Anti‐phospho‐vinculin (Y1065) was from abcam, Cambridge, MA, USA. Alexa Fluor 546‐conjugated secondary antibody, Alexa Fluor 488‐conjugated phalloıdin, Fluorescein isothiocyante (FITC)‐labeled dextran and ROCK inhibitor y27632 were purchased from Thermo Fisher Scientific, Waltham, MA, USA. RhoA inhibitor I, (purified C3 Transferase covalently linked to a proprietary cell penetrating moiety), was purchased from Cytoskeleton Inc., Denver, CO, USA. HMEC‐1 (human microvascular endothelial cell‐1) 10 was obtained from the Centers for Disease Control and Prevention (Atlanta, GA) and cultured in vascular cell basal medium supplemented with endothelial cell growth factor kit (Invitrogen, Waltham, MA, USA). Dasatinib was obtained from Selleck Chem., Houston, TX, USA.

40 μg of total protein was analyzed by electrophoresis in an SDS 12%-polyacrylamide gel, transferred onto PVDF membrane and probed with anti-MRM2 (1: 2000, ab60068, Abcam) and anti-vinculin (1: 5000, Sigma) antibodies. Peroxidase-conjugated anti-mouse IgG secondary antibodies (Santa Cruz Biotechnology) were used at a dilution of 1: 2000 and 1: 5000, for anti-MRM2 and anti-vinculin antibody, respectively. The protein bands were visualized by chemiluminescence (ECL, GE Healthcare).

For FACS analysis and immunohistochemistry, anti-PLAP-1 antibody was purchased from Thermo Fisher Scientific. For immunoblot analysis, anti-GOLGA5, anti-Integrin-β5, anti-collagen type-1 α1, anti-collagen type-1 α2, anti-fibronectin, and anti-actin antibodies were purchased from Santa Cruz Biotechnology. For immunocytochemistry, anti-GM130, anti-integrin-β5, anti-calnexin, and anti-vinculin antibodies were purchased from Santa Cruz Biotechnology.

Cell lysate preparation and immunoblot analyses were performed as previously reported [40 (link)]. Filters were probed with the indicated primary antibodies: anti-TIMP-1 and anti-CD99 (R&D Systems, Minneapolis, MN, USA); anti-integrin β1, anti-CD63 and anti-vinculin (Santa Cruz Biotechnology, Dallas, TX, USA); anti-vimentin, anti-integrin αv, anti-PDGFRβ, anti-pAKT, anti-pErk 1/2 and anti-ZO-1 (Cell Signaling Technology Inc., Danvers, MA, USA); anti-ITPRIPL1 (OriGene Technologies, Rockville, MD, USA); anti-NF-kB-p65 (Elabscience, Houston, TX, USA); anti-actin (Sigma-Aldrich); and anti-CD44 (Abcam, Cambridge, UK). Densitometric analysis was performed on at least two different exposures to assure the linearity of each acquisition using ImageJ software (v1.46r).

4–15 or 7.5% SDS–PAGE Mini-PROTEAN TGX gels (BioRad) were blotted using the semidry Trans-Blot Turbo Transfer system (BioRad).The following antibodies were prepared in 5% milk in TBS-T and incubated overnight at 4 °C: anti-Myc (1:1000, clone 4A6, Millipore), anti-p63 (1:2500, ab124762, Abcam), anti-p63α XP (1:1000, D2K8X, Cell Signaling), anti-GAPDH (1:20,000, clone 6C5, Merck), anti-p53 (1:500, DO-1, Santa Cruz), anti-PARP (1:1000, 9542, Cell Signaling), anti-cleaved caspase3 (1:1000, 5A1E,Cell Signaling), and anti-vinculin (1:1000, clone 7F9, Santa Cruz). Secondary antibodies, used for detection, were also prepared in 5% milk in TBS-T and incubated for 1 h at RT (goat anti-mouse HRP, 1:5000, A9917, Sigma–Aldrich; goat anti-rabbit HRP, 1:2000, Jackson Immuno Research Europe Ltd., 111–035–144). Quantification of western blot signals was performed by using ImageJ.

The following antibodies and reagents were used: anti–phospho-Ser1981-ATM, anti-phospho-Chk2(T68), anti-Chk2, anti-phopsho-S15-p53, anti-PARP1 (all from Cell Signaling, Beverly, MA, USA); anti-ATM (2C1), anti-p53 (DO-1), anti-HSP90 (F8), anti-actin B, anti-vinculin, anti-GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-PAR (Ab-1; Sigma Aldrich, St. Louis, MO, USA, cat no AM80); anti-phospho-S824 Kap1 (Abcam, Cambridge, UK, ab70369); anti-SAM68 (purified); neocarzinostatin (NCS) (Sigma-Aldrich, St. Louis, MO, USA, cat no N9162), a radiomimetic chemotherapy drug, was used at 500 ng/mL concentration, and KU55933 (Sigma Aldrich, St. Louis, MO, USA, cat no SML11109), a specific inhibitor of ATM kinase, was used at 10 μM concentration.