Louis, MO), biotinylated peanut agglutinin (PNA) (1250, Vector Laboratories, Burlingame, CA), or biotinylated agglutinin (VVA) (1500, Vector Laboratories, Burlingame, CA) diluted with 1% bovine serum albumin (BSA)/PBS for 16 h at 4C. sections were deparaffinized in xylene and rehydrated in a series of graded alcohols. After quenching the activity of endogenous peroxidase with 1% H2O2 in phosphate-buffered saline (PBS) for 10 min, the sections were rinsed three times with PBS and then incubated with 5% non-fat milk/PBS for 30 min to reduce nonspecific bindings. Sections were incubated with an anti-COSMC polyclonal antibody (130, Sigma, St. Louis, MO), biotinylated peanut agglutinin (PNA) (1250, Vector Laboratories, Burlingame, CA), or biotinylated agglutinin (VVA) (1500, Vector Laboratories, Burlingame, CA) diluted with 1% bovine serum albumin (BSA)/PBS for 16 h at 4C. After rinsing twice with PBS, Super Sensitive? Link-Label immunohistochemistry Detection System (BioGenex, San Ramon, CA) was used and the specific immunostaining was visualized with 3,3-diaminobenzidine liquid substrate system (Sigma, St. Louis, MO). All sections were counterstained with hematoxylin for 1 min and mounted with UltraKitt (Mallinckrodt Baker, Inc., Phillipsburg, NJ). Negative controls were performed by replacing the primary antibody with a control IgG at the same concentration. Real-time RT-PCR Total cellular RNA was isolated from hemangioma tissues or cells grown to 70% confluence by use of the Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer protocols as previously described [14]. For cDNA synthesis, 2 g PRSS10 of total RNA were used as templates in a 25 l reverse transcription reaction. For detection, sense and anti-sense primers were and and was analyzed with MxPro Software (Stratagene, La Jolla, CA). Human umbilical vein endothelial cells (HUVECs) (Lonza, Walkersville, MD) were cultured in Clonetics? EGM-2 BulletKit (Lonza, Walkersville, MD) according S3QEL 2 to the manufacturer’s instruction. Human endothelial cell line EA.hy926 was a gift from Drs. Shu-Huei Wang and Hsiu-Ni Kung (Graduate Institute of Anatomy and Cell Biology, National Taiwan University College of Medicine, Taiwan) and was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo scientific, Barrington IL) containing 10% FBS, 100 IU/mL penicillin, and 100 g/mL streptomycin (Invitrogen, Carlsbad, CA) in a humidified tissue culture incubator at 37C and 5% CO2 atmosphere. COSMC/pcDNA3.1 [15] (a gift from Dr. Richard D. Cummings at the Emory University School of Medicine, Atlanta, USA) and control pcDNA3.1 (Mock) (Invitrogen, Carlsbad, CA) were transfected S3QEL 2 into HUVECs (3-5 passages) using an Amaxa Nucleofector? (Lonza, Walkersville, MD) and the HUVEC Nucleofector kit (Lonza, Walkersville, MD) according to the manufacturer’s instructions. For COSMC knockdown, duplex siRNA against and non-targeting control siRNA were purchased from Invitrogen. Cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) at a final concentration of 10 nM siRNA according to the manufacturer’s instruction. After 48 h of transfection, cells were used for experiments. For analysis of cell signaling, cells were starved for 4 h and then treated with 10% FBS or 20 ng/mL S3QEL 2 of VEGF (Sigma, St. Louis, MO). Western blot analysis Equal amounts of cell or tissue lysates were electrophoresed on an SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were incubated with anti-COSMC polyclonal antibody, anti–actin monoclonal antibody (Sigma, St. Louis, MO), anti-phospho-ERK monoclonal antibody, anti-ERK1/2 polyclonal antibody, anti-phospho-AKT, anti-VEGFR2 monoclonal antibody, anti-phospho-Tyr1175 VEGFR2 monoclonal antibody (Cell Signaling Technology, Beverly, MA), or biotinylated PNA (Vector Laboratories, Burlingame, CA). The membrane was incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were visualized with ECL reagents (Amersham Biosciences, Buckinghamshire, UK) and quantified with ImageQuant 5.1 (Amersham Biosciences, Buckinghamshire, UK). Trypan blue exclusion assay Cells (4104) were seeded in 6-well plates with RPMI 1640 containing 10% FBS (PAA Laboratories, New Bedford, MA). Viable cells in triplicate wells were determined at 24 h intervals for 72 h using hemocytometer with trypan blue exclusion staining (Sigma, St. Louis, MO). MTT cell proliferation assay For viability and proliferation analysis, cells were seeded at 3103 per well in 96-well plates. Triplicates of each cell group were plated. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagents (Sigma, St. Louis, MO) were added to each well, and absorbance was measured according to the manufacturer’s instructions. Lectin pull down assay For lectin pull down assays, 200 g of cell or tissue lysates were incubated with PNA or VVA agarose beads (Vector Laboratories, Burlingame, CA) overnight at 4C. The pulled down proteins were then subjected to Western blotting. S3QEL 2 Chemical inhibition The MEK inhibitor PD98059 and PI3K inhibitor LY294002 (Calbiochem, San Diego, CA), and VEGF receptor tyrosine kinase.