Fig. 6. Constitutively active ErbB4 mutants are expressed and are constitutively tyrosine phosphorylated in FR3T3 cells. ErbB4 expression and tyrosine phosphorylation were assayed in FR3T3 cells infected with retroviruses that direct the expression of wild-type ErbB4 or the ErbB4 mutants. Cells infected with the LXSN recombinant retrovirus vector control or with the ErbB2* retrovirus served as controls. Lysates were prepared from each of the cell lines, and ErbB receptors were precipitated from 1.5 mg of each lysate using protein A-Sepharose and either an anti-ErbB4 rabbit polyclonal antibody or an anti-ErbB2 rabbit polyclonal antibody. Samples were resolved by SDS-PAGE, electroblotted to nitrocellulose, and immunoblotted with an anti-phosphotyrosine antibody (upper panel). The blot was then stripped and probed with an anti-ErbB4 rabbit polyclonal antibody (lower panel). Arrows, positions of ErbB2 and ErbB4 on the blots.