Native structure and composition of the TCR complex expressed in γδ– × hδTg T cells. (A) The TCR complex was immunoprecipitated with antiphosphotyrosine antibodies from digitonin lysates of pervanadate-stimulated WT and γδ– × hδTg thymocytes, and subsequently eluted with phenylphosphate. After separation by BN-PAGE, proteins were transferred to a nitrocellulose membrane and blotted with an anti-CD3ζ antibody. The positions of the complete TCR complex (αβγϵδϵζζ in WT and αβ(hδϵ)2ζζ in γδ– × hδTg thymocytes), free CD3ζ, and a partial TCR complex (arrowhead) are indicated. (B) Phenylphosphate-eluted TCR complexes from WT and γδ– × hδTg splenocytes were incubated with the indicated amounts of the antibodies 17A2 (anti-CD3γϵ) or 2C11 (anti-CD3γϵ/δϵ), and subsequently resolved by BN-PAGE. The Western blot membrane was developed with anti-CD3ζ antibodies. The number of antibody-mediated shifts of the TCR complex is indicated to the right.
