Biological quadruplicates of BT474 cells were treated for 6 or 48 hours with 100 nM TZB, 10 μM ARRY, the combination of 100 nM TZB and 10 μM ARRY (termed A+T), or 100 nM DARPin 6L1G. Cells were washed with ice-cold PBS and scraped in PBS containing Halt phosphatase and Halt protease inhibitor cocktails (Pierce) on ice. Cells were centrifuged at 300 rpm for 2 min, and afterward pellets were lysed by addition of M-PER mammalian extraction buffer (Pierce) containing both inhibitor cocktails for 30 min at 4°C on a rocker. Cell extracts were cleared by centrifugation for 10 min at 12,000 rpm at 4°C, and protein concentrations were determined by BCA assays (Pierce). Aliquots of 1 mg/ml were prepared and snap-frozen in liquid nitrogen. Five and 1.5 μg of protein extract were used for the PTK array protocol (V1.9) and the STK array protocol (V4.1), respectively. Measurements were performed on a PamStation12 from PamGene (Wolvenhoek 10, ‘s-Hertogenbosch, Netherlands). Briefly, the PTK array was processed in a single-step reaction. Cell extracts, ATP, and fluorescein isothiocyanate (FITC)–labeled pY20 antibody were incubated on the chip, and the phosphorylation of the individual Tyr peptides was followed by fluorescence detection in real time. The STK array was processed in a two-step reaction. First, the cell extracts, ATP, and the primary antibody mixture were incubated with the chip for 110 min. Second, the reaction mix was removed, and the secondary FITC-labeled antibody was added. Development of the FITC fluorescence signal was detected. Signal intensities were analyzed in the BioNavigator software (PamGen) as a function of time and expressed as LFC versus DMSO treatment after 6 or 48 hours, respectively.

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