Fig. 6. Binding of paxillin and Pyk2 to synthetic peptides containing the C terminus of the integrin ß3 cytoplasmic domain. (A) Integrin-derived sequences of the peptides: the C terminus of the chicken integrin ß3 tail, two variants of this sequence containing single amino acid substitutions (S⁷⁵²P (SP), Y⁷⁵⁹A (YA)), and the corresponding region of the integrin ß1 tail were linked at their N termini to penetratin, which was itself biotinylated at its N terminus. (B) Affinity precipitation experiment performed with these peptides and cell lysates of chicken osteoclast precursors. Proteins bound to the peptides were eluted together with these peptides into reducing sample buffer for SDS-polyacrylamide electrophoresis, and analyzed by immunoblotting with antibodies against talin, Pyk2 and paxillin. Peptides (M_r approx. 4.5 kDa) were detected on separate Coomassie-stained 15% SDS-polyacrylamide gels (Coom.). Supernatants of the binding reaction were also analyzed (unbound). Note the partial depletion in the ß3-unbound fraction of Pyk2 and paxillin, but not of talin. (C) Affinity precipitation with wild-type and S⁷⁵²P-mutated integrin ß3-tail peptides performed with chicken osteoclast-like cells that had been lysed either after 2 hour suspension culture (Susp) or after 40 minutes adhesion to serum-coated plastic (Adh). Bound and non-bound fractions were analyzed by immunoblotting with an antiphosphotyrosine mAb (PY99), or with antibodies against paxillin and Pyk2. Note that equal amounts of paxillin and Pyk2 were bound, although the levels of tyrosine phosphorylation differed strongly in the lysates of suspended versus adhered cells. (D) Affinity precipitation with the ß3-tail peptide performed with lysates of chicken osteoclast-like cells. Half of the lysates had been subjected to two rounds of immunoprecipitation with an anti-paxillin monoclonal antibody before the incubation with the ß3-tail peptide. Lysates and bound fractions were analyzed by immunoblotting with antibodies against Pyk2 and paxillin. Note that equal amounts of Pyk2 bound from paxillin-containing and paxillin-depleted lysates.