The following mutations analyzed in this study have been previously reported in aHUS patients: C25F, P32A, N133S, H165R 32, W127x, L289x (c.893delC) 8, A222G, R299W, W468x (c.1446-1450 delTTCAC), D501N 4, R456x, W528x 7 and T520x (c.1610insAT) 31. The M120V mutation was identified in a Caucasian patient from Saudi Arabia. The sequencing of CFI was performed by Dr. Fremeaux-Bacchi in Paris. The numbering excludes selleck inhibitor the signal

peptide and +1 corresponds to the first amino acid in the mature protein. In order to convert the numbering to that for the full-length protein starting with Met1, 18 amino acids must be added. Human C4BP 38 and FH 39 were purified as described previously. C1, C4, C2, C3, C3b, C4b, FB, factor D (FD) and properdin were purchased from Complement

Technology (San Diego, CA, USA). C3b and C4b were labeled with Opaganib order 125I using the chloramine T method 40. Full-length cDNA encoding the human CFI gene was cloned into the eukaryotic expression vector pcDNA3 (Invitrogen, Carlsbad, CA, USA) with addition of a N-terminal His-tag as described earlier 10. The mutations reported in aHUS patients were introduced in the CFI gene using the primers listed in Table 3 and a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The mutations were confirmed by automated DNA sequencing using a Big dye terminator kit (Applied Biosystems, Foster City, CA, USA). The transient transfection Florfenicol and ELISA were performed as described before 34. The experiment was

conducted in triplicate. HEK 293 cells stably transfected with WT FI or mutants C25F, N133S, A222G and D501N, and were detached using trypsin, washed and suspended at 1.0×106 cells/mL in DMEM. The cells were then permeabilized using PBS containing 0.5% Tween 20. Permeabilized cells were incubated with monoclonal Ab against FI (Quidel, San Diego, CA, USA) diluted in PBS, 0.05% Tween 20, 1% BSA, 30 mM NaN3 and washed twice before incubation with the secondary, FITC-conjugated Ab against mouse immunoglobulins (Dako, Denmark). As a negative control HEK 293 cells stably transfected with C4BP were used. HEK 293 cells stably expressing FI WT and mutants C25F and N133S or human C4BP as a negative control were lysed and subjected to immunoprecipitation with polyclonal goat anti-human FI Ab (Quidel). The immunoprecipitates were treated with EndoH (Roche Applied Science, Mannheim, Germany) for 18 h at 37°C. Treated samples were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and visualized using a polyclonal goat anti-human FI Ab (Quidel), followed by rabbit anti-goat Ab conjugated to HRP (Dako). The expression and purification of FI WT and mutants were done as described previously 34. Briefly, 3 L of conditioned serum-free Optimem Glutamax was applied to a Ni-NTA Superflow column (Qiagen, Hilden, Germany).