Fig. 8
Exocyst molecule counting at vesicle fusion sites. a Stepwise photobleaching of CD86 fused to one or two sfGFP molecules used to determine intensities of known numbers of GFP molecules. b Standard curve for counting protein molecules, from intensities measured on CD86 fused to one, two, or three sfGFP molecules. Error bars = ±s.d.; pink shaded region denotes 95% confidence band. c Fluorescence landscape of SEC8-GFP shows a typical resolvable distribution of molecules within an ROI from which intensity measurements were taken. d Intensity (y) was measured from the peak and converted to the number of molecules using the regression equation determined in b. Numbers for SEC3 and SEC6 were corrected for heterozygosity. Mean ± SD = 7.6 ± 3.6 (SEC3-GFP), 9.8 ± 3.5 (SEC5-GFP), 9.8 ± 3.4 (SEC6-GFP), 9.35 ± 3.2 (SEC8-GFP), and 9.19 ± 2.8 (EXO70-GFP). Cohen’s d value for the difference between SEC3-GFP and SEC5-GFP is –0.208. Centers indicate median, box limits indicate 25th and 75th percentiles, and whiskers extend 1.5x IQR from 25th to 75th percentiles. n = 96, 50, 51, 50, 58. Coefficient of variation (CV) = 47.9, 35.2, 39.2%, 34.3, and 30.7% in the order indicated in the graph. CV between each subunit was 7.9%. Experiments were repeated at least three times with similar results. P-values were computed using one-way ANOVA test followed by Scheffe’s post hoc test
