Depletion of Alba proteins does not affect GPEET and EP procyclins, but affects translation of a reporter.

(A) Western blot analysis was performed on total lysates (1.5×10⁶ cell equivalents per lane). Alba 3&4 RNAi cultures were incubated for 4 days in the presence or absence of tetracycline (+/− Tet). Western blot analysis was performed with antibodies against GPEET and EP. Mitochondrial HSP60 was used as a loading control. Early procyclic forms of AnTat 1.1 (early PCF) possess two copies of GPEET. In Alba3&4 RNAi the coding region of one copy of GPEET is replaced by GFP and the adjacent copy of EP3 is replaced by a puromycin-resistance cassette. (B–E). 2D-DIGE detects a limited number of differences in following knockdown of Alba proteins. Alba3&4 RNAi cells were cultured for 4 days in the presence (+Tet) or absence (−Tet) of tetracycline, which is before the onset of the slow growth phenotype. (B) Merge of a representative pair of gels (one of 4 biological replicates) showing significantly regulated proteins. (C) Enlarged regions of 2D-DIGE gels for Cy3-labeled proteins from induced (+Tet; green) and Cy5-labeled proteins from uninduced (−Tet, red) cultures, and the corresponding 3D views. The lower panel shows a graphic representation of differences in abundance of these proteins across the 4 independent experiments. (D) Protein identities, fold difference (+Tet/−Tet) and statistical significance. (E) Northern blot analysis and quantification of GFP mRNA in Alba3&4 RNAi cells. The entire ORF of GFP was used as a hybridisation probe.