Figure 4.

Inefficient EGFR autophosphorylation in serum-deprived MCA is associated with cyclin D1 expression. (A) Serum-starved HSC-3 cells were detached and incubated on poly-HEMA coated dishes in DMEM without serum. Cells at varying levels of aggregation were collected, lysed, and analyzed with various phospho-specific EGFR antibodies and total EGFR as indicated (right panel: densitometry analysis of the phosphorylated tyrosine residues represented in arbitrary units). Samples from serum-starved monolayer cells, treated with or without EGF (10 ng/ml) for 15 min, are included as positive references for the different phospho-EGFR antibodies used. (B) Pre-formed MCAs were incubated in DMEM alone and with or without EGF (10 ng/ml). After different time durations, MCAs were collected, lysed, and immunoblotted with p-Y1086 antibody. The membrane was stripped and re-blotted for total EGFR. (C) Pre-formed MCAs as in Figure 3 were incubated in DMEM containing no serum or with 1 or 10% FBS. PD168393 and AG1478 (EGFR inhibitors) were included at 1 μM to MCAs supplemented with 10% FBS. After 6 h, MCAs were collected, lysed, and processed for immunoblotting as indicated. A representative membrane was stripped and reprobed with anti-EGFR. Tubulin was monitored for equivalent protein load. Representative result from three independent experiments is shown.