Analysis of CFHL1 and CFH binding to BbCRASP-2 mutants. Purified
GST-BbCRASP-2 (positive control), GST-BbCRASP-2 mutants as well as purified
GST protein (negative control) were immobilized onto microtiter plates and
incubated with purified CFH or CFHL1 as described under “Experimental
Procedures.” Binding of CFH or CFHL1 was then assessed by an ELISA
together with a polyclonal anti-CFH antibody. A, BbCRASP-2 mutants
representing binding region 2, B, BbCRASP-2 mutants representing
binding region 3; C, BbCRASP-2 mutants representing binding region 4.
Experiments were performed at least three times, and one representative
experiment is shown. Bars represent the mean of triplicates within
one experiment ± S.D. One-way analysis of variance was performed to
analyze statistical significance in CFH/CFHL1 binding between BbCRASP-2 and
mutant proteins, and significant differences from the wild-type (p
< 0.001) are indicated by asterisks.