Figure 3.

FIF spectrometry as applied to clusters of puncta extracted from fluorescence images of the basolateral membrane of cells expressing wild-type hSecR fused to mEGFP. The analysis was performed on the same images as in Fig. 2. (a, d, and g). Surface plots of the ε_eff occurrence frequency versus receptor concentration of protomers using 3103 (a), 3677 (d), and 3704 (g) total puncta clusters. (b, e, and h) Stacks of cross sections taken from the surface plots in (a), (d), and (g), respectively. Middle range receptor concentration for each range (in protomers × μm⁻²) is indicated above each curve (see explanation in Fig. 2). The vertical dashed lines indicate the peak positions for the brightness spectra of monomers $\left({\epsilon }_{eff}^{mono}=62.3\right)$, dimers, and so on (see Materials and methods). The monomeric brightness was extracted from the depunctate areas of the monomer and tandem-dimer standard samples (c, f, and i), Relative receptor concentration of protomers in each oligomeric species versus total receptor concentration of protomers, as derived from fitting of the curves in (b), (e), and (h), respectively, with a sum of different Gaussian components representing different oligomeric species. Each data point and its error bar represent the mean ± SD, respectively, of 900 different relative fraction values, obtained from the statistical “bootstrapping” procedure mentioned in Fig. 2. To see this figure in color, go online.