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. 2014 Jan 29;9(1):e87316. doi: 10.1371/journal.pone.0087316

Figure 4. Western blot analyses of C3b, C4b and properdin complexes of RBCs from five PNH patients (lanes 4–8) compared with normal RBCs (lanes 1–3).

Figure 4

Normal human serum was utilized in lanes 1–3, with zymosan activated serum being added in lane 2. See methods for details. Three independent experiments were performed and these are representative blots. Normal serum (lane 1) in the presence of normal RBCs contained detectable levels of full length C3, C4, and properdin. When serum was activated with zymogen (lane 2) normal and PNH RBCs showed bands corresponding to C2aC3bC4b and PC3Bb when developed with a C3 antibody, C2aC3bC4b and C2a4b when developed with a C4 antibody, and PC3Bb when developed with a properdin antibody. These data show that both the classical and alternative pathways have been activated on the surface of RBCs by zymogen treatment. Note that there were C3 convertases (C2aC4b and PC3bBb) and C5 convertases (C2aC3bC4b and PC3bBbC3b) on the surface of PNH RBCs despite eculizumab treatment. Normal RBCs were first solubilized with sample loading buffer. PNH RBCs were directly treated with sample loading buffer.