cells were washed three times with warmed PBS, incubated for 1–2 h in serum-free RPMI without phenol red and without glutamine and resuspended in the same medium at 0.8 × 10cells per ml. αHER2-eStcE, eStcE, StcE or equal volume PBS was added to a final concentration of 1 nM, and the cells were incubated overnight at 37 °C. Cells were centrifuged at 600for 5 min, and conditioned supernatant was collected and treated with protease inhibitors and 10 mM EDTA.
Pellets were resuspended in 100 µl of 6 M guanidine hydrochloride, and protein amounts were determined by a bicinchoninic acid protein assay kit ; 125 µg of total protein material was used for each sample, and samples were adjusted to a total volume of 175 µl with water. Samples were then adjusted to 100 mM HEPES before adding freshly prepared TCEP to a final concentration of 10 mM and incubating at 37 °C for 30 min.