FIGURE 4.

Labeling of alternate terminal septin subunit (Shs1) does not establish a FRET system without Cdc11. A, Coomassie gels and typhoon scans of labeled septin octamers with AF555 and AF647 using end subunits of Shs1 and Cdc11. B, FRET emission spectra with 25 nm Shs1^AF555-labeled octamers recorded at increasing concentrations of Shs1^AF647-labeled octamers: 0.78 nm (dashed green), 1.56 nm (dashed cyan), 3.13 nm (dashed red), 6.25 nm (dashed black), 12.5 nm (solid green), 25 nm (solid cyan), 50 nm (solid red), and 100 nm (solid black). The inset illustrates the result of the PCA with at least two principal components. C, titration curves of Cdc11^AF647 capped octamers with Shs1^AF555 capped octamers at varying concentration of Shs1^AF555 (25 nm Shs1^AF555 (red), 35 nm Shs1^AF555 (cyan), and 50 nm Shs1^AF555 (magenta)). D, using PCA, Hill coefficients and IC₅₀ can be determined as a function of ionic strength. The light red line illustrates the location of the IC₅₀ value. The Cdc11^AF555-Cdc11^AF647 (black line) is included for reference.