Epidermal growth factor (egf)

Uninfected or cells infected with WT or mutant TB40/E_GFP virus were washed two time with PBS and serum starved in serum-free media for 24h prior to EGF stimulation. After serum starvation, cells were washed with ice cold PBS and incubated on ice for 30 min. Cells were then incubated on ice with serum free media containing 10 nM EGF (Gold Biotechnology) for 30 min, except for no EGF control. Cells were then washed with ice cold PBS. 37°C serum free media was added and samples were incubated at 37°C for 15min to 24h, depending on experiment. Samples were then collected for immunoblotting or chromatin immunoprecipitation, depending of experiment.

NME cells were constructed via stable overexpression of EGFR in normal murine mammary gland (NMuMG) cells as previously described [60 (link)]. These cells were cultured in DMEM containing 10% FBS, 10 μg/mL insulin, penicillin, and streptomycin. For cell signaling assays, cells were plated for 24 h prior to removal of the FBS for an additional 24 h. During this serum starvation, PP2 (Sigma, 529576) or SrcDEL10-ester were added as indicated, and cells were subsequently stimulated with EGF (50 ng/mL) (Goldbio, 1150-04) for 30 min. After this stimulation cells were lysed in a modified RIPA buffer containing 50 mM Tris, 150 mM NaCl, 0.25% Sodium Deoxycholate, 1.0% NP40, 0.1% SDS, protease inhibitor cocktail, 10 mM activated sodium ortho-vanadate, 40 mM β-glycerolphosphate, and 20mM sodium fluoride. Equal aliquots of these lysates were separated by reducing SDS PAGE and transferred to PVDF membranes. These membranes were probed with antibodies against phospho-EGFR Y1068, total EGFR, phosho-STAT3-Y705, total STAT3, phospho-ERK1/2, total ERK1/2 (Cell Signaling Technologies), and tubulin (DSHB). Differential binding of these antibodies was detected using their appropriate secondary antibodies.