FIGURE 1:

The cellular distribution of endogenous and FP-tagged EB1 is similar. (A) Schematic presentation of the EB1-FP expression vector. The sequence for either GFP or NG were integrated into the genomic DNA encompassing the EB1 gene, including its endogenous promoter (pro) and terminator (term). The selectable marker gene aphVIII was present on the same plasmid. The arrows indicate the orientation of the genes. (B) Western blot analysis of whole cells and isolated flagella of wild type (control) and strains expressing EB1-GFP or EB1-NG probed with antibodies to EB1, and as loading controls, to IC78 and α-tubulin. The flagellar samples were 70 times more concentrated than the whole-cell samples (i.e., ∼140 flagella/cell). (C) Flagellar extracts from a EB1-GFP–expressing strain and a wild-type control were incubated with anti-GFP beads and the depleted extract (Unbound), the bound fraction (Eluate), and the original extract (Input), were analyzed by SDS–PAGE and Western blotting with anti-EB1. Note that endogenous EB1 copurifies with EB1-GFP. (D) Silver staining of the eluate obtained from a strain expressing EB1-GFP by GFP affinity purification. (E) DIC (a), TIRF (b), and the corresponding merged image (c) of live EB1-GFP cells. Scale bar: 3 μm. (F) Schematic representation (left) and live images of a focal series through a EB1-NG cell. Arrowheads in a, flagellar tips; arrows in d, punctae of EB1-NG in a posterior region of the cell. Scale bar: 3 μm.