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Fig. 3. Preformed heterodimers of the ErbB receptors in the absence of bound ligand. (A) BiFC analysis of the ErbB receptor heterodimerization in living cells. CHO cells expressing the BiFC constructs indicated were serum-starved, stained with or without Hoechst 33342, and then observed by confocal microscopy. Bars, 10 µm. (B) CHO cells co-expressing the BiFC constructs indicated were serum-starved, treated with 100 ng/ml EGF (+EGF) or 1.0 nM NRG (+NRG) for 30 minutes at 37°C, and then observed by confocal microscopy. Bars, 10 µm. (C) Ligand-induced phosphorylation of preformed ErbB heterodimers. CHO cells co-expressing ErbB2-VN and EGFR-VC, or ErbB3-VN and ErbB2-VC were serum-starved, and then treated with (+) or without (–) 100 ng/ml EGF or 1.0 nM NRG for 30 minutes at 4°C. Whole cell lysates were subjected to immunoblotting using specific antibodies for phosphorylated forms (p-EGFR, p-ErbB2, p-ErbB3 and p-Akt) or for unphosphorylated forms of EGFR, ErbB2, ErbB3 and Akt.
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