BT474 cells were treated in biological duplicates with 100 nM TZB, 100 nM DARPin 6L1G, 10 μM HER2 inhibitor ARRY, and the combination of 100 nM TZB and 10 μM ARRY (termed A+T), each treatment for 6 and 48 hours. Cell pellets were lysed in 8 M urea supplemented with phosphatase inhibitors on ice, and cell extract concentrations were determined by BCA assays (Pierce). Cell lysates were reduced with 1 M dithiothreitol in ammonium acetate at pH 8.9, and 55 mM iodoacetamide was added to quench reformation of disulfide bridges. Afterward, ~400 μg of sample was digested with trypsin overnight at room temperature, and the reaction was stopped by adding acetic acid to a final concentration of about 90%. Samples were loaded on Sep-Pak columns, washed with 90% acetonitrile/0.1% acetic acid, and eluted in 25% acetonitrile/0.1% acetic acid.

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