Figure 2.

Differential protein expression and phosphorylation in the context of E-cadherin expression. (a) Experimental workflow for the RPPA analysis. After collection, dilution and spotting of the cell lysates, each of 16 sub-arrays (pads) per nitrocellulose slide were probed with a different validated primary antibody (Ab). A fluorescent secondary antibody was used for signal detection and quantification (quant.). Mean intensities of the biological replicates were used to perform cluster analysis. E+, E-cadherin-expressing cells; E−, E-cadherin-negative cells. (b) Hierarchically clustered heat map showing the relative levels of differentially regulated proteins and phosphoproteins (Q = 0.05) in whole cell lysates from mouse (Trp53^Δ/Δ-3, Trp53^Δ/Δ-7, mILC-1, mILC-2) and human (MCF7, IPH-926) cell lines as determined by RPPA. (c) Hierarchically clustered heat map showing the relative levels of phosphoproteins related to the Akt signalling pathway. Heat maps display the relative expression (Z-scores) of proteins or phosphoproteins (red, up-regulated; blue, down-regulated). (d) Western blot analysis of differentially regulated proteins and phosphoproteins identified by RPPA. Phosphorylation levels of Akt (p-Akt; Thr308 and Ser473) were assessed and normalised over the corresponding total protein levels, while PTEN expression levels were normalised over GAPDH levels. For mouse cells, normalised phosphoprotein levels in Trp53^Δ/Δ-3 cells were set to 1; for human cells, normalised phosphoprotein levels in MCF7 cells were set to 1. For analysis of phospho-Akt (Ser473), blot lanes for additional mILC sub-clones were removed, as denoted by the dashed lines. (e) Representative immunohistochemistry image of expression of phospho-Akt (Ser473) in ILC patients (see Table 2). Scale bars, 50 µm.