Anti vegf

To better understand and evaluate the adhesion on G, the hPDLSCs on granules were processed for immunofluorescence labeling. Samples were fixed for 10 min at room temperature (RT) with 4% paraformaldehyde in 0.1M sodium phosphate buffer (PBS), pH 7.4. Then, samples were permeabilized with 0.5% Triton X-100 in PBS, followed by blocking with 5% skimmed milk in PBS. Primary monoclonal antibodies to antivinculin (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-VEGF (1:250; Santa Cruz Biotechnology) were used, followed by Alexa Fluor 568 green fluorescence conjugated goat antimouse as secondary antibodies (Molecular Probes, Invitrogen, Eugene, OR, USA). Subsequently, samples were incubated with Alexa Fluor 488 phalloidin green-fluorescence conjugate (Molecular Probe) as cytoskeleton actin marker. Samples were placed face-down on glass slides and mounted with Prolong antifade (Molecular Probes) [49 (link)]. Samples were observed with CLSM (Zeiss LSM510META) and connected to an inverted Zeiss Axiovert 200 microscope equipped with a Plan Neofluar oil-immersion objective (40×/1.3 NA).

The infarct border zone tissue was homogenized in RIPA buffer with proteinase inhibitor for 1 h at 4 °C. Proteins were collected by centrifugation, and the supernatant boiled with 4× Laemmli sample buffer at 96 °C for 10 min. The denatured proteins were separated on a 10% SDS-PAGE gel and transferred to a membrane. The membrane was incubated with anti-VEGF (Santa Cruz, 1:1000), anti-angiogenin (Abcam, 1:1000), anti-angiopoetin 2 (Abcam, 1:1000), and b-actin (Abcam, 1: 4000) antibodies overnight at 4 °C. After washing, anti-rabbit HRP secondary antibody was added and target proteins were visualized using Clarity ECL Western Blotting Substrate (Bio-Rad, Hercules, CA, USA). Quantification of protein bands was performed using ImageJ software.

Total cytosolic and nuclear extracts were prepared, as previously described [26 (link)], on saphene veins. The following primary antibodies were used: anti- IL-1β (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500 #sc12742, D.B.A, Milan, Italy), anti-TNF-α (Santa Cruz Biotechnology; 1:500 #sc52746), anti-TGFβ1 (Santa Cruz Biotechnology, 1:500 #sc130348, D.B.A, Milan, Italy), anti-VEGF (Santa Cruz Biotechnology; 1:1000 #sc7269), anti- αSMA (Santa Cruz Biotechnology; 1:500 #sc53015) and anti-prolyl endopeptidase (Abcam; 1:1000,#ab58988) in 1× phosphate-buffer saline (Biogenerica srl, Catania, Italy), 5% w/v non-fat dried milk, 0.1% Tween-20 at 4 °C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:2000) for 1 h at room temperature. Anti-β-actin (Santa Cruz Biotechnology; 1:1000 #sc47778) and anti-βTubulin (Santa Cruz Biotechnology; 1:1000 #sc5274) antibodies were used as controls. Protein expression was detected by chemiluminescence (ECL) system (Thermo, Waltham, MA, USA), visualized with the ChemiDoc XRS (Bio-Rad, USA), and analyzed by using Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA) as previously reported [27 (link)].

Cells were lysed in 200 μl RIPA buffer (150 mM sodium chloride, 1% NP 40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl [pH 8.0], 100 mM PMSF) and centrifuged at 14,000 g for 10 min at 4°C. The supernatant was mixed with denaturing sample buffer (1:1) and boiled for 5 min at 94°C. Equal amounts of protein (50 μg) were loaded and separated by 10% SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes (BioRad, Hercules, CA, USA). The membranes were blocked in Tris-buffered saline with Tween 20 (TBST) containing 5% non-fat dry milk for 1 h at 4°C and incubated with anti-4E-BP1 (1:2000), anti-phospho-4E-BP1 (1:1000), anti-mTOR (1:2000), anti-E-cadherin (1:1000) (all from Cell Signalling Technologies, Danvers, MA, USA), or anti-VEGF (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies overnight at 4°C. Anti-β-actin antibody (1:5000; Sigma-Aldrich) was used as a control. Membranes were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) for 1 h at room temperature and washed again in TBST. The signal was detected using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA), and intensity was quantified using ImageJ software.

The protein extracts of lung were incubated with the primary antibody [anti-phosphoinositide 3-kinases (PI3K) (1:1000; Cell Signaling Technology); anti-nuclear factor kappa B (NFκB, p65) (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.); anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) (1:10000; Abcam plc); anti-phosphorylated IκBα (1:1000; Cell Signaling Technology); anti-VEGF (1:1000; Santa Cruz Biotechnology); anti-VEGFR-1, -phosphorylated VEGFR-1 (1:1000; Abcam plc); anti-VEGFR-2 (1:500; Millipore Corporation); anti-phosphorylated VEGFR-2 (1:1000; Cell Signaling Technology); anti-Rho-associated kinase (RhoA) (1:1000; Cell Signaling Technology); anti-Akt (1:500, Cell Signaling Technology); anti-phosphorylated Akt (1:2000, Cell Signaling Technology); anti-extracellular signal-regulated kinase (ERK), -phosphorylated ERK (1:3000, Millipore Corporation)]. Then the blots were incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG antibody, Sigma Chemical Co., St. Louis, MO, U.S.A.). With a computer assisted video densitometer and digitalized software (Kodak Digital Science^TM ID Image Analysis Software, Eastman Kodak Co., Rochester, NY, U.S.A.), the blots were scanned, photographed then the signal intensity (integral volume) of the appropriate bands were analyzed.

Western blotting was performed as previously described [51 (link), 52 (link)]. Briefly, cells were collected and lysed in RAPI buffer. Cleared total cell lysate was denatured by boiling and loaded onto a 10% gradient SDS–PAGE. After electrophoretic separation, proteins were transferred to an Immobilon-P membrane. Membrane was blocked with SuperBlock Blocking Buffer, and probed with the primary antibody, anti-HIF-1α, anti-VEGF, anti-p-Akt, anti-β-actin (Santa Cruz, CA) and anti-p-ERK1/2 (Cell Signaling Technology, Vancouver, Canada), followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The proteins of interest were detected by using SuperSignal West Pico Chemiluminescent Substrate kit.