Wide distributions are observed in endogenous protein levels for cultured cell lines. Antigen intensity was determined by high content screening following fixation and staining with antibodies that were specific for the indicated protein. Quantitation was achieved by a whole cell mask, which was dilated out from the nuclear region identified by DAPI staining. A. Endogenous expression levels of proteins are shown for two breast cell lines, the immortalized line 184B5 and the estrogen-sensitive breast cancer cell line T47D, shown as well or sample mean values of all of the cells. Proteins examined in this study, as indicated in the figure, were quantitated by indirect immunofluorescence and image analysis, and values for each cell are plotted as individual points. All graphs are to the same scale, indicated in the lower left. Approximately 7000 cells were quantitated per sample (antigen/cell line). B. Display of sample distributions for the proteins indicated in (A). Data is presented as histograms of cells with increasing levels (total fluorescence intensity, or the sum of all pixel intensities, per cell) of the indicated proteins. C. Contribution of cells stratified by antigen intensities on overall abundance measurements. Data is as in panel B, but calculated as the product of the number of cells per bin times the average antigen intensity for that bin. As such, the contribution of each bin to the total well mean response is represented. D-F. Correlations between two proteins in cell populations. Protein levels per cell are shown for T47D cells, as indicated in the graphs. Protein levels are mean average fluorescence intensities per cell, as indicated in the axes labels.