As shown, using the STAT3 assay kits, a significant stimulation of STAT3 phosphorylation at Tyr705 is detected in A431 cells treated with EGF for 10 minutes compared with cells treated with AG1478, while a change in total STAT3 levels is also observed.
Day 1: A431 cells were seeded at 25K/well in a 96 well tissue culture plate in medium containing 10% FBS. Day 2: The cells were stimulated with various concentrations of EGF for 15 minutes. The media was removed, and the cells were lysed with 125 µL/well freshly prepared Lysis Mix, with shaking for 10 min. Lysates (50 µL) were transferred to replicate wells of a PhosphoTracer assay plate and analyzed for either phospho or total STAT3 using the standard PhosphoTracer protocol. Briefly, Antibody Mix specific for either phospho-STAT3, or Total STAT3 (50 µL/well), was added to the lysate, and the plate was incubated for 1 hr at room temp, with shaking. The plate was washed and Substrate Mix was added to the wells. The plate was covered in foil and incubated for 10 min with shaking. Signal in the wells was determined using a plate reader.