Fig. 6: Validation of phosphoproteomic data.

a, b Proteome Profiler Human Phospho-Kinase Array and western blot analysis validating pathway regulation downstream of EphA2 kinase activation in PC-3 cells. PC-3 cells were left untreated or incubated with clustered Fc control (Fc) or clustered ephrin-A1-Fc (A1) for 20 min to activate the EphA2 receptor. Representative images of array and western blot membranes are shown. β-actin is the western blot loading control. Increased PARD3 tyrosine phosphorylation was shown by immunoprecipitating PARD3 and subsequent probing with an anti-phospho-tyrosine antibody. Bar graph shows the quantification of the Proteome Profiler array as log2(fold change (efnA1-Fc:Fc control)). Mean ± SE, (n = 4 biological replicates). c Western blot analysis assessing EphA2-signaling in a panel of prostate, breast, colon and brain cancer cell lines. Cell lines were incubated with clustered Fc control (Fc) or clustered ephrin-A1-Fc (A1) for 20 min. SHB phosphorylation on Y246 is consistently upregulated by EphA2 receptor activation across all tested cell lines. Downregulation of Afadin (S1718/S1799) and NDRG1 was observed only in cell lines which responded with a robust downregulation of Akt phosphorylation on S473 (PC-3, MDA-MB-231, BT-519, HCT-116 and MN1).