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- Histogram overlays are another way of assessing the signaling response of a particular protein in each of the conditions measured. In this case, we are interested in seeing the response for each of the phospho proteins (e.g. p-STAT1) in a condition (e.g. IL-4) when compared to the Unstimulated or basal state.
- Let's start with the saved illustration from the previous section. In the experiment summary page, click on Edit next to "U937 contour plots."
- Change the plot type to Histogram overlay and click on Update plots.
- In the dimensions box, you'll see conditions referred to as columns, channels referred to as overlaid and populations referred to as table.
- Make Conditions the overlaid dimension by dragging Conditions past the Channels dimension and clicking on Update dimensions.
- The illustration tab will display a histogram overlay for each of the proteins selected in the channels dimension. Each overlay will contain the conditions selected in the conditions dimension. The table underneath the histogram overlay is reporting a statistic. In this case each value in the table is the median intensity of the corresponding phosphoprotein and condition. A more useful statistic would be how much more a protein responded when compared to its unstimulated state (fold change). Configure this by choosing Fold change equation in the "Plot Statistics" section on the left menu bar and choosing the First Row as the control. Click on Update Plots to see the results. You will notice that p-Stat1 had a large response to IFN-gamma and IL-4+IFN-gamma wheareas p-Stat6 had a high response to IL-4 and IL-4+IFN-gamma.
- Save the illustration as "U937 histogram overlays"
