Figure 7. Paracoccidioides Rbt5 binds heme-containing molecules.

A. Recombinant protein Rbt5 was pre-incubated with (+) or without (−) hemoglobin (Hb) for 1 h. Subsequently, the samples were incubated with hemin-agarose resin for 1 h. After, the samples were centrifuged, the supernatants (S) were collected and the resin (R) was washed twice. After adding the buffer, the samples were boiled for 5 min and submitted to SDS-PAGE and Western blot analysis. A single 22 kDa immunoreactive species, which corresponds to the Rbt5 recombinant protein, was detected bound to the resin in the absence of hemoglobin or in the supernatant in the presence of hemoglobin. B. Upper and lower panels represent systems where protoporphyrin or hemoglobin Rbt5 recognition was assessed, respectively. The sequential steps of incubation in each system are indicated on the bottom of each panel. Pb01 denotes the background fluorescence of fungal cells alone; Pb01 + anti-Rbt5 represents systems where fungal cells were sequentially incubated with primary and secondary antibodies; Pb01 + protoporphyrin/hemoglobin + anti-Rbt5 is representative of systems that included the blocking of yeast cells with heme-containing molecules before exposure to antibodies; and Pb01 + anti-Rbt5 + protoporphyrin/hemoglobin represents systems that included the incubation of yeast cells with heme-containing molecules after exposure to antibodies.