Fig. 3. SDS–PAGE/immunoblot analysis of L.mexicana and L.donovani LPG-deficient mutants. (A–D) Total promastigote lysates (100 µg protein). Lane 1, L.mexicana Δlmexlpg1, clone I/8D; lane 2, L.mexicana Δlmexlpg1, clone II/5C; lane 3, L.mexicana Δlmexlpg1, clone IV/2B; lane 4, L.mexicana wild type. The blots were probed with the mAbs LT6 (A), L7.25 (B) and LT17 (C). Equal loading of lanes 1–4 was confirmed by probing a blot with the anti-leishmanolysin/gp63 mAb L3.8 (D). (E–F) LPG from 10⁸ promastigotes enriched by solvent extraction (McConville et al., 1990). Lane 5, L.mexicana wild type; lane 6, L.mexicana Δlmexlpg1, clone I/8D. The blots were probed with LT6 (E) and L7.25 (F). (G) Total promastigote lysates (100 µg protein) digested with proteinase K in SDS–PAGE sample buffer (200 µg/ml final concentration, 15 min, 37°C). Lane 7, L.mexicana wild type; lane 8, L.mexicana Δlmexlpg1, clone I/8D; lane 9, L.mexicana Δlmexlpg1, clone I/8D + pXlmexlpg1. The blot was probed with mAb LT6. (H and I) Total promastigote lysates (100 µg of protein). Lane 10, L.donovani wild type; lane 11, L.donovani R2D2; lane 12, purified L.donovani LPG (2.5 µg), for reference. The blots were probed with mAb LT6 (H) or L7.25 (I). Arrows mark the border between the stacking and the separating gel. The molecular masses of standard proteins are indicated in kDa.