Fig 6. Flagellum purification demonstrates an enrichment of LiAlba3 in the Leishmania flagellum during heat stress.

Phase contrast images of intact L. infantum promastigotes (A) and of purified flagella after sucrose gradient isolation (B) as described in Materials and Methods. (C) Summary of MS/MS identified proteins from four independent experiments of flagellum purification (two from promastigote cell extracts and two from 8 h-differentiating amastigotes). Identified genes were classified according to their gene ontology and to characterized orthologs in Trypanosoma spp. based on GeneDB and TriTrypDB gene annotations. Only genes identified at least twice with a minimum of 2 peptides are shown here (see Tables 2, S3 and S4 for the complete list of the identified L. infantum flagellum proteins). (D) Confirmation of flagellar localization of PFR2C-HA in recombinant L. infantum expressing pSP-alphaIRNEOalphaIR-PFR2C-HA. (E) Western blot analysis and quantification of endogenous LiAlba3 in purified flagellum fractions upon promastigote conditions of growth (Pro) or following 8 h of temperature stress (37°C). After flagellum purification, flagella from promastigotes and heat-stressed parasites were counted on a Malassey (hemocytometer) to load an equivalent number of flagella on the gel. Total proteins of promastigote cells were loaded as a control. Relative quantification was performed using ImageJ blot.