Specificity of anti-Fyn (428). (A) Control Western immunoblot. Anti-Fyn (428) was used to immunoprecipitate resting PBMC (5 × 10⁶ cell equivalents), the immunoprecipitated proteins fractionated by SDS-PAGE and Western immunoblotting used to check the specificity of the immunoprecipitating antibody to recognize src-family PTK. Total cell lysate was used to control for Fyn, Lck, c-Src, and c-Yes proteins. (BI) Control kinase assay. Lysate containing 25 × 10⁶ cell equivalents of Fyn⁺ Jurkat cells (lane 2) or Fyn⁻ C8 (lane 3) and S1T (lane 4) cell lines were immunoprecipitated with rabbit anti-Fyn and immune-complexes washed and incubated with 5 μCi [γ³²P] in kinase buffer containing acid-treated enolase (1.0 μg/condition). Proteins were separated by reduced SDS-PAGE and electrotransferred to Immobilon-P membranes and tyrosine-phosphorylated proteins visualized by autoradiography. Lane 1 is an immunoprecipitating antibody control that also serves as a control for background enolase phosphorylation. (BII) The Immobilon-P membrane in (BI) was blocked in 5% skim-milk, probed with a monoclonal anti-Fyn, followed by goat-antimouse IgG conjugated to horseradish peroxidase and enhanced chemiluminescence. The positions of the molecular weight standards are indicated and arrows show the location of fyn, enolase (E), and the immunoprecipitating antibody heavy chains [Ig(H)], respectively.