Fig. 1
a Akt and b Aktp473 antibody validation for reverse phase protein array (RPPA). MDAMB468 (red), ZR75-1 (black) and T47D (blue) cells were left untreated followed by no stimulation (control) or by stimulation with epidermal growth factor (EGF) or were treated with LY294002 (phosphatidylinositol-3-kinase (PI3K) inhibitor), perifosine (Akt inhibitor), rapamycin (mTOR inhibitor), or ultraviolet (UV) irradiation and then stimulated with epidermal growth factor (EGF) in the case of treatment with the three inhibitors. Lysates were then probed with antibody to total Akt (a) or to phosphorylated Akt at serine 473 (Aktp473, b) by RPPA in triplicate (panels A–C) and by western blotting (panel D) and the derived signals for total Akt and for Aktp473 were quantified and correlated (panel E in a and b). For RPPA, each lysate was arrayed in five serial 2-fold dilutions on nitrocellulose slides (with increasing dilution from left to right on each slide for each lysate as shown in panel B). A control spot (a mixed cell line lysate) was placed at the end of each sample lysate’s five serial 2-fold dilution series to give six spots. Four samples are arrayed in this fashion in each grid of 24 spots on the nitrocellulose slides shown. The correlation coefficients between signals derived using RPPA and western blotting for Akt and Aktp473 were 0.897 and 0.93, respectively (panel E in a and b). These correlation coefficients were based on 18 data points as shown and indicate valid antibodies for RPPA. Panel A in a and b demonstrates the process of curve fitting for RPPA that is applied by the R package SuperCurve (version 1.01)18. In the upper left of panel A, estimated protein concentration (x-axis) is plotted against signal intensity (y-axis). In the upper right of panel A, residuals from model fitting (y-axis) are plotted against estimated protein concentration (x-axis). Ideally, the residuals should be symmetrical about the horizontal 0 line and should not increase with increasing concentration. In the lower left of panel A is an image plot of squared residuals from model fitting. This plot shows that the squared residuals are largely homogeneous. In the lower right of panel A, the intensity differences of adjacent dilution steps are plotted (y-axis) against the averaged intensities of adjacent dilution steps (x-axis). If this curve is flat and close to the horizontal line, the dilutions were unsuccessful and the data are not reliable