Fig. 3.

Effect of Rhipicephalus microplus saliva on the deposition and cleavage of components of the alternative pathway. a-e Deposition of C3b, Bb, properdin (P), C5b and C9, respectively, onto the activation surfaces. Bars represent the arithmetic mean ± standard deviation (SD) of three biological replicates. Asterisks indicate statistically significant differences between experimental samples and controls (*P < 0.05; **P < 0.01; ***P < 0.001). f, g Inhibition of C3 and factor B (fB) cleavage by the alternative pathway, respectively. The supernatant from the haemolysis assay reactions via the alternative pathway or with salivary proteins (15 μg) or without saliva was analysed by Western blot, which was probed with anti-C3 (f) or anti-fB (g) polyclonal antibodies. h Action of saliva on factor D enzymatic activity. Assays were carried out by combining purified fB, C3b and factor D in the absence or presence of salivary proteins (15 μg) followed by Western blot analysis probed with anti-fB polyclonal antibodies. In f, the α (110 kDa) and β (76 kDa) subunits of C3 as well as the 46 kDa fragment of the α chain are indicated by arrows. In g and h, fB (93 kDa) and its cleavage product Bb (60 kDa) are indicated by arrows. Results of one Western blot out of three are shown in f-h