FIG. 2.

Antibodies to LdMT and LdRos3 cross-react with their L. braziliensis orthologues. (A) Specificity of the anti-LdMT and anti-LdRos3 antibodies. Crude membrane fractions from promastigotes of wild-type (WT) L. donovani and the LdMT^−/− and LdRos3^−/− lines were subjected to SDS-PAGE and immunoblotted with rabbit polyclonal antibodies against recombinant polypeptides of LdMT and LdRos3. An anti-α-tubulin monoclonal antibody was used as a probe for a protein loading control. The positions of molecular mass markers (kilodaltons) are indicated. (B) Level of recognition of LbMT by anti-LdMT antibodies. Aliquots of crude membrane fractions of L. donovani LdMT^−/− promastigotes transfected with LbMT-GFP or LdMT-GFP were subjected to SDS-PAGE and immunoblotted with anti-LdMT and anti-GFP as a normalization control. A Western blot representative of at least three independent experiments is shown. The positions of molecular mass markers (kilodaltons) are indicated on the left. (C) Level of recognition of LbRos3 by anti-LdRos3 antibodies. Aliquots of six-His-tagged LdRos3 and LbRos3 recombinant polypeptides (LdRos3-His, LbRos3-His) were subjected to SDS-PAGE and immunoblotted for anti-LdRos3. HisProbe-HRP was used as a normalization control. A Western blot representative of at least three independent experiments is shown. The positions of molecular mass markers (kilodaltons) are indicated on the left. (D) LdRos3 and LbRos3 are glycosylated proteins. Crude membrane fractions of L. donovani and L. braziliensis LH-2419 wild-type strain promastigotes were treated with PNGase F at 37°C for 1 h and analyzed by Western blotting with anti-LdRos3 antibodies. Lanes 1 and 3, untreated protein samples; lanes 2 and 5, PNGase F-treated samples; lane 4, sample incubated with the N-glycosidase buffer. The positions of molecular mass markers (kilodaltons) are indicated on the left. The L. braziliensis 38- to 70-kDa proteins recognized by anti-LdRos3 are indicated on the right.