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The fractional intensity of the clustered regions was then calculated by dividing the integrated intensity of the thresholded image by that of the non-thresholded image. Analyzing the clusters using the triangle algorithm or the Maximum Entropy algorithm selleck chemicals yielded the same critical concentrations. Similar thresholding results were obtained using an iterative manual procedure to identify pixels with intensity greater than three standard deviations above the mean of the non-clustered regions. Thus, our calculation of fractional intensity in the clustered regions and our consequent determination of critical concentration are not dependent on the method used to identify clusters. Size distribution and spatial distribution analyses For the data in Figure 2B,C, 512 by 512 pixel images were taken at 93 randomly selected areas of a sample with clusters made using p-NephrinA488, 1 ?M Nck, and 1 ?M N-WASP. The images were background corrected as described above, flattened using the rolling-ball method in ImageJ, and thresholded using the triangle method. The clusters were binned according to size (excluding those at the image edges) and the distribution was fit to a single-exponential using Graph-pad Prism. The size distributions in Figure 3B were determined similarly from single images Ceftiofur obtained at each time point. To analyze the spatial distribution of puncta, each thresholded image was divided into 25 boxes. In each box, the number of clusters was counted twice��excluding and including clusters at the edges. The average number of edge clusters was obtained from the difference in these values, averaged across all boxes in all images. To eliminate overcounting, for each box half of this value was subtracted from the number of clusters counted including edges. These data were plotted to obtain a frequency histogram using this website Graph-pad Prism and fit to a Gaussian distribution. Fluorescence recovery after photobleaching (FRAP) FRAP was performed using a Nikon Eclipse Ti microscope equipped with an Andor iXon Ultra EM-CCD camera. A circle of 1-?m diameter was initially photobleached and recovery followed for up to 1000 s. The images were corrected for drift using the Sift-Align plugin in ImageJ (Schneider et al., 2012). Background photobleaching was obtained by imaging under the same conditions, excluding the laser illumination used for photobleaching. Background corrected images were normalized to the intensities of the pre-bleached images and fit to either a single or a double-exponential using Graph-pad Prism. F-tests performed in Prism demonstrated that the double-exponential fits are most appropriate (p-values for all experiments were
