Anti masp 2

Cell lysates were prepared in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% Nonidet P-40) containing 1× protease inhibitors (cOmplete™ EDTA-free Protease Inhibitor Cocktail, Roche) with 2 mM CaCl₂ or 1 mM EDTA. Soluble proteins were subjected to immunoprecipitation with anti-Flag M2 agarose (Sigma). Then, the adsorbates were separated by SDS-PAGE and transferred onto an Immobilon-P transfer membrane (Millipore) by semi-dry transblot (Bio-Rad). The membrane was blocked with 5% Western-Blocker (Bio-Rad). Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma), anti-β-actin (Sigma), anti-green fluorescent protein (GFP) (Clontech), anti-MASP-2 (Santa Cruz), anti-C4α (Santa Cruz), HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies. The antigen-antibody complexes were visualized by chemiluminescence (GE Healthcare).

Purified MASP-2 (8 nM) was incubated at 37 °C for 3 h in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 2 mM CaCl₂ with purified C4 (Calbiochem), recombinant MBL (Calbiochem), mannan (Sigma), and SARS-CoV N protein with/without C1INH (Calbiochem), anti-MASP-2 (Santa Cruz), and anti-SARS-CoV N antibody (Sino Biological) at concentrations of 50 nM, 30 nM, 15 ng/ml, 10 nM, 40 μg/ml, 2.4 μg/ml and 12 μg/ml, respectively. The cleavage was followed by SDS-PAGE under reducing conditions, and the C4alpha’ fragments, which are left by C4a release after C4 cleavage and can indicate C4 activation, and MASP-2 were detected by an immunoblot analysis with an anti-C4alpha antibody (Santa Cruz) or anti-Flag antibody (Sigma). Purified C4b (Calbiochem) containing C4alpha’, but not C4a, was used to indicate the C4alpha’ band position.