Anti rabbit or mouse igg antibodies conjugated to horseradish peroxidase

Cells were washed 2 times with phosphate-buffered saline (PBS), harvested with the lysis buffer previously described and centrifuged at 10,000 xg at 4°C, 5 min. For some experiments, cell culture supernatants were concentrated on Microcon concentration units (Amicon, Merck). Protein content was determined by the Biorad Protein assay (Biorad). Proteins (20 μg-40 μg) were then subjected to SDS-PAGE under reducing and non- reducing conditions and then transferred onto a nitrocellulose membrane. The rabbit anti-human MASP-2 H-60 antibody (1/200, sc-50420, Santa Cruz Biotechnology), rabbit anti-human MBL-2 antibody (1/1,000, orb 31822 Biorbyt), rabbit anti-human macrophage migration inhibitory factor (MIF) antibody [81 (link)] and mouse anti-B actin antibody (1/1,000, sc-8432, Santa Cruz Biotechnology) were used as primary antibodies and were detected using anti-rabbit or mouse IgG antibodies conjugated to horseradish peroxidase (1/10,000, Amersham Biosciences). Detection was done with the ECL chemiluminescence kit (Pierce) according to the manufacturer’s protocol.

Cells were harvested with the lysis buffer previously described and centrifuged at 10,000× g at 4 °C, for 5 min. Protein content was determined by the Biorad Protein assay (Biorad). Proteins (20 μg–40 μg) were then subjected to SDS-PAGE under reducing and then transferred onto a nitrocellulose membrane. The rabbit anti-human WWP2 antibody (1/1000, A302-935, Bethyl Laboratories) and mouse anti-B actin antibody (1/1000, sc-8432, Santa Cruz Biotechnology) were used as primary antibodies and were detected using anti-rabbit or mouse IgG antibodies conjugated to horseradish peroxidase (1/10,000, Amersham Biosciences). Detection was done with the ECL chemiluminescence kit (Pierce) according to the manufacturer’s protocol.