Fig 6.

Released proteins from sterol-depleted promastigotes. (A) Stationary-phase promastigotes were treated with the indicated concentration of MβCD (0 to 10 mM) for 48 h. Filtered supernatants (S) and cells (C) were subjected to SDS-PAGE. MSP was detected by Western blotting (top panel). The same filter was reprobed with monoclonal antibody to α-tubulin (bottom panel) for a loading control. A total of 1 × 10⁷ cell equivalents were loaded. The percentage of MSP in the supernatants of each treatment is shown. Results from a representative of two experiments are presented. (B) Released proteins were directly collected from the extracellular medium of metacyclic promastigotes that were untreated (lane 2) or subjected to 25 mM MβCD treatment (lane 1) for 1 h. A silver-stained SDS-polyacrylamide gel is shown. A total of 8 × 10⁸ cell equivalents were loaded. There is an empty lane between lanes 1 and 2. Gel slices were excised for LC-MS/MS analysis (arrowhead), and the results are summarized in Table 2.