Fig. 4

Metacyclogenesis and Western blot analysis of LPG from different L. major lines. Proportion of metacyclic promastigotes of different L. major lines including: control (WT, black histograms, diamonds), ΔLABCG1-2 (grey histograms, squares) and add-back parasites (ΔLABCG1-2 + LABCG1-2, white histograms, triangles), in stationary phase of culture (day 5), assessed using the PNA assay (a). b Promastigotes (1 × 10⁷/ml) in the logarithmic (b1) or stationary phase (b2) of the growth curve were exposed to increasing concentrations of fresh human serum for 30 min at 37 °C. Resazurin was then added and the promastigotes incubated at 28 °C for 24 h in order to determine cell viability. Data are the mean ± SD of three independent experiments. Significant differences versus the respective control line were determined using the Student’s t-test (*P < 0.05). c Promastigotes (1 × 10⁷) of WT, ΔLABCG1-2 and ΔLABCG1-2 + LABCG1-2 were lysed and collected in the stationary growth phase and Western blot analysis with the antibodies HASPB and HMGS was performed. A Western blot assay representative of at least three independent experiments is shown. The positions of molecular markers (kDa) are indicated on the left. d Promastigotes (1 × 10⁷) of WT, ΔLABCG1-2 and ΔLABCG1-2 + LABCG1-2 were collected at stationary phase of growth (8 days) and lysed as described in Materials and Methods. Western blot analysis of total proteins from parasites incubated with the antibodies WIC79.3 (d1) or 3 F12 (d2) were performed at 1:250 and 1:500 dilutions, respectively. A Western blot assay representative of at least three independent experiments is shown. The positions of molecular markers (kDa) are indicated on the left