Cxcl8 mab208

Western blotting using 12% SDS‒PAGE was performed, and 30 μg of cell lysates were analyzed as previously described 12 (link). Antibodies against human NOX4 (GTX121929) and pCHOP Ser30 (GTX55401) (GeneTex, Hsinchu, TWN); ANGPTL4 (#409800) and pFAK Tyr397 (44-624G) (Invitrogen) (BD Transduction Laboratory, Los Angeles, CA, USA); p41/42 MAPK (#9102), pAKT Ser473 (4051S) and pEGFR Y1068 (3777S) (Cell Signaling Technology, Inc., Danvers, MA, USA); GAPDH (sc-32233) and EGFR (A-10) (sc-373746) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA); CXCL8 (MAB208) (R&D); CHOP (A20987) (Abclonal); and NOX4 (ab109225) (Abcam) were used as the primary antibodies.

Two hundred and fifty microliters of polymerized Cultrex BME (12–18 mg/ml, Trevigen) were dropped onto each well of a 24-well plate and were allowed to solidify for 1 h at 37 °C in humidified 5% CO₂ incubator. Transfected 2 × 10⁵ melanoma cells were seeded in serum-free medium onto the gelled BME and incubated at 37 °C for 18 h. Cells were incubated alone or in the presence of specific human IL-8- or VEGF-neutralizing antibodies (0.2 μg/mL, CXCL8-MAB208 and VEGF-MAB293, R&D Systems) and capillary-like structures (CLS) evaluated. CLS formation was photographed using light microscopy and quantified by evaluating the tube length and counting the number of cell junctions in 10 sets of images for condition. Each condition was analyzed in duplicate in three different experiments using image analysis program (Image J v.1.34 s; http://rsb.info.nih.gov/ij/).