Ethylene diamine tetraacetic acid (edta)

Cells detached using 200 mg/L EDTA, 500 mg/L trypsin (Cambrex), were lysed in RIPA buffer (10 mM Tris pH 7.5, 140 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% NP40) containing protease inhibitor mixture. Protein content of cell lysates was measured by a colorimetric assay (BioRad). Twenty and forty µicrograms of proteins from each cell lysate were separated on 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% non-fat dry milk and probed with 1 μg/mL R4 anti-uPAR monoclonal antibody recognizing uPAR D3 domain, 1 μg/mL anti-FPR1 polyclonal antibody (Abcam), or 0.2 μg/mL GAPDH Ab (Santa Cruz Biotechnology). Washed filters were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody and detected by ECL (Amersham-GE Healthcare). Densitometry was performed using the NIH Image 1.62 software (Bethesda, MD). Each experiment was performed three times.

Cells detached using 200 mg/L EDTA, 500 mg/L trypsin (Cambrex), were lysed in RIPA buffer (10 mM Tris pH 7.5, 140 mM NaCl, 0.1%SDS, 1% Triton X-100, 0.5% NP40) containing protease inhibitor mixture. Protein content of cell lysates was measured by a colorimetric assay (BioRad). 40 μg proteins or 50 μl concentrated conditioned medium from A375 or A375M6 cells were separated on 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. In all cases, the membranes were blocked with 5% non-fat dry milk and probed with 1 μg/mL R4 anti-uPAR monoclonal antibody recognizing uPAR D3 domain, 1 μg/mL anti-FPR1 polyclonal antibody (#128296 Ab, Abcam), 0.2 μg/mL GAPDH Ab (Santa Cruz Biotechnology), or 1 μg/mL 389 anti-uPA polyclonal antibody (American Diagnostica). Washed filters were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody and detected by ECL (Amersham- GE Healthcare). Densitometry was performed using the NIH Image 1.62 software (Bethesda,MD). Each experiment was performed three times.

Cells detached using 200 mg/L EDTA, 500 mg/L trypsin (Cambrex), were lysed in RIPA buffer (10 mM Tris pH 7.5, 140 mM NaCl, 0.1%SDS, 1% Triton X-100, 0.5% NP40) containing protease inhibitor mixture. Protein content of cell lysates was measured by a colorimetric assay (BioRad). 30 and 60 μg proteins were separated on 10% SDS-PAGE under unreducing (to detect uPAR) or reducing conditions (to detect FPR1) and transferred onto a polyvinylidene fluoride membrane. Membranes were blocked with 5% non-fat dry milk and probed with 1 μg/mL R4 anti-uPAR mAb, recognizing the DII-DIII uPAR domains, 1 μg/mL anti-FPR1 polyclonal antibody (Ab) (#128296 Ab, Abcam), or 0.2 μg/mL GAPDH Ab (Santa Cruz Biotechnology). Washed filters were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody and detected by ECL (Amersham- GE Healthcare). Densitometry was performed using the NIH Image 1.62 software (Bethesda, MD.