FIGURE 1: Conditional expression of YFP-ATG8 proteins in procyclic form T. brucei.
(A) Expression of individual YFP-ATG8 fusion proteins was induced by growing cells for 24 h in media containing tetracycline (tet). 1 x 106 cells were loaded per lane and immuno-blotted with anti-ATG8.1 or anti-GFP antibody. Anti-EF1α was used to demonstrate equal loading.
(B) Endogenous ATG8 expression. 5 x106 wild type BSF and PCF cells loaded per lane, were probed with anti-ATG8.1. Anti-EF1α was used to demonstrate equal loading.
(C) Fluorescent microscopy was used to monitor the presence of autophagosomes in PCF cells conditionally expressing YFP fusion proteins following growth in SDM79 media, starvation in PBS for 2.5 h or 2.5 h nutrient starvation in PBS supplemented with 10 µM wortmannin. The mean number of autophagosomes per cell was determined by counting >200 cells with data displayed as a mean of three replicate experiments from three inde-pendent clones. Error bars represent standard deviation and asterisks indicate where data differed significantly from the mean of the non-starved controls *p<0.05, ***p<0.001.
(D) Representative images of cells assessed as for C. Left hand images FITC filter set, right hand images DIC FITC merge. Scale bar 5 µm.