Anti upar

Endothelial cells were cultured with only endothelial growth media, microvascular endothelial growth media, or each CM and seeded at a density of 2000 cells per well in 96-well tissue culture plates and incubated for 48 h at 37 °C. Anti-uPAR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-HGF antibodies (Santa Cruz Biotechnology) were added at a concentration of 2 μL/mL for 2 h to endothelial cells cultured in CM and treated with negative siRNA. A cell growth assay was performed using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) [37 (link)]. The interactions of HSC3 cells and endothelial cells were tested using a CytoSelect Tumor-Endothelium Adhesion Assay (Cell Biolabs, San Diego, CA, USA) and CytoSelect Tumor Transendothelial Migration Assay systems (Cell Biolabs), as described in our previous reports [3 (link),10 (link)]. Briefly, a suspension of fluorescently labeled OSCC cells was cultured with a monolayer of endothelial cells and lysed in lysis buffer. Absorbance at 450 nm (to measure cell growth) and 480/520 nm (to measure adherent or migrating cells) was determined using a Multiskan GO Microplate Spectrophotometer (Thermo Fischer Scientific).

Human samples and ISK cells were lysated in a lysis buffer (RAPI buffer: 50 mM Tris-HCl, pH8.0, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS). Western blot analysis was performed as described in our previous study [56 (link)]. Equal amounts of protein were subjected to 8% SDS-PAGE and were transferred onto nitrocellulose membranes. The transferred membrane was blocked with 5% skim milk in TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20, pH 8.0) for 1 h. The membrane was then incubated with the primary antibody in TBST plus 2% fat-free milk at 4°C for overnight. Antibodies used in this study were: Anti-CFTR (Almone Lab, catalog No.: ACL-006, 1:500), anti-uPAR (Santa Cruz, catalog No.: sc-10815, 1:500), anti-NFκB p65 (Cell signaling, catalog No.: 9496, 1:500), anti-GAPDH (Santa Cruz, catalog No.: sc-47724, 1:5000), anti-β-tubulin (Santa Cruz, catalog No.: sc-9104, 1:2000), anti-β-actin (Sigma, catalog No.: A2066, 1:5000). The membrane was subsequently washed with TBST and incubated for 1 hour with peroxidase-conjugated secondary antibody. The membrane was washed 3 times with TBST and then detected by chemiluminescence (Amersham, Piscataway, NJ).

Cultured podocytes were lysed in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitors (Roche). The lysates were fractionated in SDS-PAGE, followed by protein transfer to blots and incubation with primary antibodies, including polyclonal anti-uPAR (1:200, SC-10815, Santa Cruz, Texas, USA), polyclonal anti-ITGB3 (1:200, SC-14009, Santa Cruz, Texas, USA), polyclonal anti-synaptopodin (1:200, SC-21536, Santa Cruz, Texas, USA), and polyclonal anti-GAPDH (1:5000, AP0063, Bioworld, MN, USA), respectively.