Western Blot Analysis

Proteins in the samples were separated by SDS-PAGE under both reducing and non-reducing conditions. Stacking gels (125 mM Tris, pH 6.8) contained 5%, separating gels (750 mM Tris, pH 8.8) contained 12.5% acrylamide-bisacrylamide (37.5:1) solution, separation was performed at a constant voltage of 180 V in running buffer (25 mM Tris, 192 mM glycine, 0.1% w/v SDS, pH 8.3). Proteins were transferred to nitrocellulose membranes (Bio-Rad) in 25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.4 transfer buffer. Blotting was performed at a constant current of 120 mA for 60 min. After blocking for 1 h in 20 mM Tris, 200 mM NaCl, 0.02% w/v NaN₃, 5% w/v nonfat dry milk, pH 7.2 blotting buffer, membranes were incubated overnight at room temperature with MASP-1-SP-AP antibody or MASP-3 monoclonal antibody in the same buffer at 1 or 2 µg/ml, respectively. Membranes blotted with the MASP-3 antibody were washed (20 mM Tris, 200 mM NaCl, 0.02% w/v NaN₃, 0.3% v/v Tween-20, pH 7.2) for 30 min, then incubated for 1 h at room temperature with alkaline phosphatase conjugated goat anti-mouse antibody at 2,000-fold dilution in blotting buffer. After washing for 45 min, membranes were visualized with NBT/BCIP solution (5 mM MgCl₂, 100 mM Tris, 400 mg/l NBT, 200 mg/l BCIP, pH 9.0). Blots were scanned with an Epson Perfection 4490 scanner in 16-bit grayscale reflective mode. Densitometric analysis of the Western blots was performed using the Quantity One software (Bio-Rad).