Fig. 3. SDS–PAGE/immunoblotting of L.mexicana total cell lysates. (A–C) Lane 1, WT; lane 2, ΔGDPMP; lane 3 ΔGDPMP + cRIBLmxGDPMP. Each lane was loaded with 1 × 107 promastigotes (∼40 µg of protein). (A) Blot probed with affinity-purified rabbit anti-L.mexicana GDPMP antibodies (upper panel) and an identically loaded blot was probed with affinity-purified rabbit anti-L.mexicana PMM antibodies (lower panel) as a loading control. The same or identically loaded blots were then stripped and probed with mAb LT6 (anti-6-Galβ1-4Manα1-PO4-) (B) and mAb LT17 [anti 6-(Glcβ1-3)Galβ1-4Manα1-PO4-] (C). (D) SDS–PAGE/immunoblotting of total cell lysates of L.mexicana promastigotes (lane 1, 2.5 × 106 parasites, corresponding to ∼10 µg of protein) and lesion-derived amastigotes (lane 2, 2.5 × 106 parasites, corresponding to ∼3.5 µg of protein; and lane 3, 7 × 106 parasites, corresponding to ∼10 µg of protein). The blots were probed with affinity-purified rabbit anti-L.mexicana GDPMP antibodies. (E) SDS–PAGE/immunoblotting of total cell lysates of L.mexicana promastigotes fractionated by ultracentrifugation. Lane 1, total cell lysate of 2.5 × 106 parasites, corresponding to ∼10 µg of protein; lane 2, first ultracentrifugation supernatant; lane 3, first ultracentrifugation pellet; lane 4, second ultracentrifugation supernatant; lane 5, second ultracentrifugation pellet. Equivalent sample volumes were loaded and the blots were probed with affinity-purified rabbit anti-L.mexicana GDPMP antibodies. (F) SDS–PAGE/immunoblotting of promastigote lysates (2 × 107 parasites, corresponding to ∼80 µg of protein) from WT (lane 1), ΔGDPMP (lane 2) and ΔGDPMP + cRIBLmxGDPMP (lane 3) probed with affinity-purified rabbit anti-L.mexicana MBAP antibodies. (G) SDS–PAGE/immunoblotting of promastigote lysates with (lanes 1, 3 and 5) or without (lanes 2, 4 and 6) prior GPI-PLC treatment. Lanes 1 and 2, WT; lanes 3 and 4, ΔGDPMP; lanes 5 and 6, ΔGDPMP + cRIBLmxGDPMP. The blot was probed with rabbit anti-CRD antibodies (upper panel). Equal loading was confirmed by stripping and reprobing with rabbit anti-PMM antibodies (lower panel).