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. Author manuscript; available in PMC: 2016 Mar 1.
Published in final edited form as: Mol Immunol. 2014 Nov 15;64(1):55–62. doi: 10.1016/j.molimm.2014.10.022

Figure 4.

Figure 4

Thermally denatured proteins activate the complement system via the alternative pathway. NHS was heated to 60°C for 1 hour (Δ serum) and then added to equal volume of either fresh NHS (lanes 2), factor B depleted serum (lanes 5), factor D depleted serum (lanes 6) or C3 depleted serum (lanes 7) and incubated for either 15 or 60 minutes at 37°C. The negative control was untreated NHS (lanes 1) or NHS that received equal amount of Δ serum in the presence of 10 mM EDTA (lanes 3) to prevent complement activation. The positive control was a mixture of Δ serum plus fresh serum treated with CVF (lanes 4) to activate the alternative pathway. Samples were separated using an 8% SDS-PAGE gel and blotted for factor B (alternative pathway activation and generation of the 33 kDa Ba fragment), C3 (110 kDa α-chain cleavage and 67 kDa iC3b generation) and α1PI (iC3b:α1PI complex formation).