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. 2001 Dec;21(24):8385–8397. doi: 10.1128/MCB.21.24.8385-8397.2001

FIG. 8.

FIG. 8

Dominant-negative mutants of FAK, HA-FAKY397F, and HA-FRNK, but not HA-FAKD395A, blocked Etk activation in response to bombesin. (A) Cells were cotransfected with wild-type Etk or T7-Etk with one of the following plasmids: vector, wild-type FAK, HA-FAK, or dominant-negative mutant HA-FAKD395A, HA-FAKY397F, or HA-FRNK. After transfection, LNCaP cells were then treated with 100 nM bombesin (lanes B) or untreated control (lanes C) for 30 min as indicated and subsequently lysed. Tyrosine phosphorylation of Etk was analyzed by immunoprecipitation using anti-Etk antibody (IP:αEtk) followed by Western blotting (immunoblotting) with anti-pY antibody (IB:αp-Y) 4G10 (top blots). The membrane was analyzed further by Western blotting using T7 antibody [IB:αT7(Etk)] (bottom blots). Half of the cell lysates described for Etk were immunoprecipitated with HA antibody [IP:αHA(FAK)] followed by blotting with FAK polyclonal antibody (IB:αFAK) (middle blots). (B) LNCaP cells were transfected with FAKY397F or empty vector, pcDNA3, and the lysates were immunoprecipitated with monoclonal Src antibody (IP:αSrc) and immunoblotted with anti-pY antibody (IB:αp-Y) (top blot) and anti-Src polyclonal antibody (IB:αSrc) (bottom blot).