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Blood, Vol. 112, Issue 13, 4948-4952, December 15, 2008
Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome
Blood Frémeaux-Bacchi et al.
112: 4948
Supplemental materials for: Fremeaux-Bacchi et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 71.9 KB)
- Table S1. C3 primer sequences and PCR conditions (PDF, 61.1 KB)
- Table S2. Clinical and laboratory data of aHUS patients carrying C3 mutations (PDF, 34.2 KB)
- Figure S1. Large aHUS pedigree. IV.1, IV.8, IV.9, and V.9 all carry the R570W mutation (JPG, 61.6 KB)
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- Figure S2. Western blot showing expression of C3 in 293T cells (JPG, 40.9 KB)
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(A) Supernatants from 293T cells transfected with wild-type or mutant C3 were concentrated and evaluated by 10% reducing SDS-PAGE. (B) Nonreduced samples were electrophoresed on a 4-12% SDS-PAG. Blots were probed using goat anti-human C3 (1:5000), a horseradish peroxidase secondary antibody (1:5000, Sigma-Aldrich), and developed using a chemiluminescent substrate. C3 MA is a commercially available C3 preparation (Complement Technologies) that was treated with methylamine (0.2M). Approximately 10 ng/lane. Representative experiment of over 20 independent transfections.
- Figure S3. Kinetic analysis of factor H cofactor activity of wild-type C3 versus C3 mutant Q1139K (JPG, 24.7 KB)
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C3 preparations were incubated for 0 to 30 min at 37°C with factor I and cofactor, factor H. Samples were reduced and analyzed by Western blotting using chicken anti-human C3 and HRP-donkey anti chicken IgY (Jackson Immunoresearch). A decrease in factor H cofactor activity was reproducibly observed for Q1139K which also had lower C3 binding to factor H (see Fig. 2A). A representative experiment from five is shown.
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