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Resistin regulates human choriocarcinoma cell invasive behaviour and endothelial cell angiogenic processes.

Western blot analysis

Monolayer HUVEC cultures were treated with 5 mM EDTA (ethylenediaminetetraacetic acid) in PBS for 3 minutes at room temperature; cells were scraped and collected by centrifugation. Collagen gels with embedded HUVECs were incubated with 0.5 U/mL collagenase for 3 minutes at 37°C. The collagen matrix was dispersed by shaking vigorously, and the gel homogenates were diluted 10-fold with PBS. Cells were recovered by centrifugation, washed 3 times with PBS, and counted. Cell pellets were lysed in 0.5 mL lysis buffer (10 mM HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], pH 7.4, 135 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 2 mM NaF) and the lysates were clarified by centrifugation at 17 000g for 10 minutes. The protein concentration of the supernatants was determined by use of the BCA protein assay reagent. Samples were resolved by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred onto nitrocellulose Hybond C Extra membranes. Blots were probed with the first antibody (anti–c-met C-28, 1:800, or anti–VE-cadherin, 1:1000) overnight at 4°C, followed by the peroxidase-conjugated antirabbit, or antimouse secondary antibody for 1 hour at room temperature. Membranes were developed with the ECL plus Western blotting detection reagent and scanned using a Hewlett Packard ScanJet ADF laser densitometer.