Spin x uf concentrator columns

To detect MMP-9 and -2 activity in conditioned media, CRC-SC transiently transfected with si-β-arr1 or with negative control, were stimulated with ET-1 or MAC, alone or in combination for 24 h. Conditioned media was collected and concentrated by using Spin-X UF concentrator columns (Corning, New York, NY, USA). Conditioned medium was separated by 9% SDS-PAGE gels containing 1 mg/ml gelatin. The gels were washed for 30 min at 22 °C in 2.5% Triton X-100 and then incubated in 50 mM Tris (pH 7.6), 1 mM ZnCl2, and 5 mM CaCl2 for 18 h at 37 °C. After incubation the gels were stained with 0.2% Coomassie Blue. Enzyme-digested regions were identified as white bands on a blue background and quantified by computerized image analysis of the band. Molecular sizes were determined from the mobility, using gelatin zymography standards (Bio-Rad Laboratories, Richmond, CA, USA).