MDA-MB-468 cells overexpressing either ErbB4 JM-a or JM-b were starved overnight and crosslinked with 1 mM DTBP. Cells were lysed with buffer containing 0.1% Triton X-100, 1 mM EDTA, 5 mM NaF, 10 mM Tris-HCl, pH 7.4, and a protease inhibitor cocktail (ThermoFisher Scientific) and anti-ErbB4 immonoprecipitation was performed. Proteins were eluted with 6 M guanidine hydrochloride, 5 mM tris(2-carboxyethyl)phosphine, 10 mM chloroacetamide and 100 mM Tris, pH 8.5 in 95 °C for 10 minutes. Guanidine
...hydrochloride was diluted to 2 M and eluted samples were digested with 1:50 (w/v) trypsin (Thermo Scientific) overnight at 37 °C. Following digestion, the reactions were quenched with 10% trifluoroacetic acid at a final concentration of approximately 0.5% (pH ~2), desalted on tC18 SepPak 96-well plate (Waters), and dried by vacuum centrifugation.[Show more]