FIG. 5.
Analysis of L. mexicana wild type, a ΔPMM mutant, and a PMM gene addback mutant by SDS-PAGE and immunoblotting, SDS-PAGE and fluorography, and TLC analysis. (A) SDS-PAGE and 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; lane 3, 7 × 106 parasites, corresponding to ∼10 μg of protein). The blots were probed with affinity-purified rabbit anti-L. mexicana PMM antibodies. (B) SDS-PAGE and 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 PMM antibodies. (C) SDS-PAGE and fluorography of delipidated total promastigote lysates from [3H]myo-inositol-labeled L. mexicana wild type (lane 1), ΔPMM (lane 2), and ΔPMM + cRIBΔPMM (lane 3). Each lane was loaded with 2.5 × 107 delipidated promastigotes labeled overnight with [3H]myo-inositol. The positions of 14C-labeled protein markers, LPG, and the major GPI-anchored surface metalloproteinase gp63 are indicated. (D) SDS-PAGE and immunoblotting of promastigote lysates (2 × 107 lysates, corresponding to ∼80 μg of protein) of wild type (lane 1), ΔPMM (lane 2), and ΔPMM + cRIBPMM (lane 3). The blots were probed with affinity-purified rabbit anti-L. mexicana MBAP antibodies. The molecular masses and relative positions of standard proteins and the positions of PMM and MBAP are indicated (A to D). (E to H) Silica gel 60 HPTLC analysis of the predominant promastigote glycolipids of L. mexicana in wild type and ΔPMM mutant promastigotes. Lanes 1, wild type; lanes 2, ΔPMM; lane 3, ΔPMM + cRIBPMM. (E) Total lipids from 2 × 108 promastigotes visualized by orcinol/H2SO4 spraying. (F) Fluorography of total lipids from 2.5 × 107 [3H]Man-labeled promastigotes (approximately 100,000 cpm). (G) Fluorography of total lipids from 5 × 106 [3H]GlcNH2-labeled promastigotes (approximately 100,000 cpm). (H) Fluorography of total lipids from 5 × 106 [3H]myo-inositol-labeled promastigotes (approximately 100,000 cpm). Bars, positions of abundant L. mexicana GIPLs (30); S, start of TLCs; ∗, new [3H]GlcNH2-labeled compounds accumulating in the ΔPMM mutant; X, two lanes loaded with samples irrelevant to this study.