Reverse phase protein (RPP) microarray profiling of Notch signals in T-ALL cell lines. (A) Six γ-secretase inhibitor (GSI)–sensitive T-ALL cell lines were treated with 1 μM compound E, a potent GSI, or vehicle (DMSO) for 7 days. Cell cycle distribution was determined based on DNA content of propidium iodide–stained populations. Green bars highlight the difference between mock- and GSI-treated cells in the G₀/G₁ fraction. (B) Cell cycle analysis of 7 GSI-resistant cell lines. (C) Lysates derived from GSI- and mock-treated cells were diluted (1:1, 1:3, 1:9) and printed on nitrocellulose-coated slides in 6 replicates. Fluorescent image of a representative RPP microarray probed with an antibody specific for the CDK inhibitor, p27^Kip1, and stained with Alexa Fluor 647 is shown. Each subarray contains lysates derived from a single cell line. For the sole purpose of presentation, brightness and contrast were adjusted equivalently across the entire array to highlight differences in feature intensities. (D) Presentation of 133 phospho-protein or total protein fold change measurements (GSI/DMSO) for each cell line as a scatter plot. Each dot represents an individual measurement. Scatter plots of sensitive cell lines are on the left side and resistant cell lines on the right. See “Materials and methods” for details in the calculation of fold change. All data points are logged in base 2 and median-centered for their corresponding cell line. The blue horizontal lines are arbitrarily placed to assist the comparison of scatter plots.