FIG 2.

erbB3 activation is erbB2 dependent. (A) The anti-erbB2 antibody, 2C4, reduces HRG-dependent erbB3 phosphorylation in SKBR3 cells. SKBR3 lysates from cells that were untreated (−), treated for 1 h with 100 nM 2C4 (2C4), stimulated 2 min with 3.2 nM HRG (+HRG), or treated for 1 h with 100 nM 2C4 and stimulated 2 min with 3.2 nM HRG (2C4+HRG) were separated by SDS-PAGE and immunoblotted with antibodies against phospho or total erbB3 and erbB2. The graphs in panels A and B report the ratio of p-erbB3 to total erbB3 based upon quantitative analysis of band intensity. (B) Lapatinib inhibition of erbB3 phosphorylation in SKBR3 cells. SKBR3 lysates from cells treated with or without 500 nM lapatinib for 4 h and then with or without 12 nM HRG for 2 min were immunoblotted as in panel A. (C) Pretreatment of SKBR3 cells with lapatinib reduces erbB2 and erbB3 kinase activity. SKBR3 cells were treated as in panel B, followed by immunoprecipitation with anti-erbB2 or erbB3 antibodies. Note that erbB2 kinase activity was only reduced by ∼50%, likely due to the reversible nature of lapatinib inhibition (54). (D) 2C4 pretreatment, as in panel A, reduces erbB3 phosphorylation after 3.2 nM HRG stimulation in CHO erbB3^wt-mCit cells. (E) Lapatinib treatment inhibits erbB3 phosphorylation in CHO erbB3^wt-mCit cells. Cells were serum starved, pretreated with 1 μM lapatinib for 30 min, and then stimulated with 12 nM HRG for 2 min. Western blot analyses were performed, and bands were quantified as in panel A.