Ab180139

Samples were subjected to SDS-PAGE using 4 to 15% Criterion TGX Precast Midi Protein Gels and transferred to nitrocellulose membranes using the semidry Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked by incubation in TBS and 0.1% (v/v) Tween 20 for 1 hour before addition of the primary antibodies, which were diluted in 25 mM tris, 0.05% (v/v) Tween 20, 1 mM EDTA, HSA (1 mg/ml; CSL Behring), and human IgG (100 μg/ml; Beriglobin, CSL Behring) (pH 7.4). Detection of human ITIH4 was achieved using monoclonal rabbit anti-ITIH4 (0.05 μg/ml; ab180139, Abcam) mixed with polyclonal rabbit anti-ITIH4 (0.05 μg/ml; HPA001835, Sigma-Aldrich), which ensures that both N- and C-terminal fragments of ITIH4 are detected. Murine ITIH4 was detected using monoclonal rabbit anti-ITIH4 (0.1 μg/ml; ab180139, Abcam). MASP-1 was detected using mouse anti–MASP-1 antibody (1 μg/ml); MASP-3 was detected using a monoclonal rat anti–MASP-3 antibody (1 μg/ml), while MASP-2 was detected using polyclonal antiserum (1:1000 dilution) from a rat that was immunized with a MASP-2 fragment. In all cases, the secondary antibody was the appropriate species-specific anti-IgG antibody conjugated with horseradish peroxidase (Dako).