Figure 3

EGF signaling network remains EGF-dependent in C/EBPbeta-2 overexpressing MCF10As. (A) Kinetworks™ phospho-site screen (KPSS) 2.1 immunoblot analyses of cell extracts prepared from uninfected control MCF10A cells and LZRS-His-C/EBPbeta-2 overexpressing cells (C/EBPbeta2/MCF10A) grown in the presence or absence of EGF for 3 days. Each lane was incubated with a specific mixture of antibodies, and scans of the ECL signals detected with a multi-imager are shown. The identity of specific phosphoylated bands on tyrosine (Y), threonine (T), or Serine (S) residues are as indicated: A SrcY529, B EGFR Y1068, C ERK1 T202/Y204, D ERK2 T185/Y187, E EGFR Y1148, F FAK Y576, G Src Y418, H MEK2 T394, I IGFR Y1162/Y1163, J FAK S910, K Insulin R Y972, L CDK-1 T14/Y15, M IRS1 Y1179, N FAK Y577, O MEK1 S298, P1 Shc Y239/240 (66), P2 Shc Y239/240 (52), P3 Shc Y239/240 (46), Q ErbB2 1139, R FAK S722, S MEK1 T292, T MEK1 T386. (B) Immunoblot analyses of whole cell extracts prepared from uninfected MCF10A cells (lanes 1 & 2), or MCF10A cells infected with LZRS-His-C/EBPbeta-2 virus (lanes 3 & 4) which were deprived of EGF in medium containing 0.5% serum for 45 hrs (lanes 1 & 3) and then stimulated with EGF (20 ng/ml) for 30 min (lanes 2 & 4). Protein samples were subjected to 8% SDS PAGE and blotted with an antibodies that specifically detect EGFR phosphorylated on tyrosine 1173, MEK1/2 phosphorylated on serine 221, or ERK1/2 phosphorylated on tyrosine 204 as well antibodies that detect total EGFR, total MEK1/2 or total ERK1/2 respectively.