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. 2015 Apr 24;11(4):e1005140. doi: 10.1371/journal.pgen.1005140

Fig 4. Oligomerization properties of FloA and FloT.

Fig 4

(A) BTH analysis to study the interactions between FloA and FloT. Interaction activates lacZ and this degrades X-Gal (blue). The two cytoplasmic domains of a leucine-zipper represent a positive control (pKT25-zip + pUT18C-zip). The negative control is represented by the E. coli strains harboring empty plasmids (pKNT25 + pUT18). Plasmids containing FloA, FloT and EA variants are pKNT25 (T25) or pUT18 (T18). Dashed line indicates the threshold limit of 700 Miller Units that defines a positive (≥ 700 Miller Units) and a negative interaction signal (≤ 700 Miller Units) according to the instructions of the manufacturer. (B) BTH analysis between the EA1 to EA4 variants of FloA and FloT. Positive controls are wild type FloA and FloT. Dashed line indicates the threshold limit of 700 Miller Units that defines a positive and a negative interaction signal. (C) Fluorescence microscopy of cells expressing GFP-tagged versions of EA variants of FloA (Upper row). Fluorescence microscopy of cells expressing GFP-tagged versions of EA variants of FloT (Bottom row). Scale bar is 2 μm. (D) Quantification of the number of foci per cell (n = 400) of the distinct GFP-tagged versions of EA variants of FloA (left panel) and FloT (right panel). (E and F) PALM images of cells expressing the EA4 variant of FloA-mEoS2 (E) and EA2 variant of FloT-mEoS2 (F). Scale bars are 500 nm. Detail of the right bottom of each panel shows a dashed-line decorated PALM picture as a general indicator of the cell outline. Scale bar is 500 nm.