(A) In vitro phosphorylation of PI3K from immunodepleted lysates. Post-130,000g cytosolic fractions of lysates from resting platelets were serially immunodepleted (preclear) four times with either Protein G beads alone (lane 1), isotype-matched IgG₁ antibody (lane 2), p85 antibody (lane 3), or an irrelevant antibody (lane 4). All of the precleared lysates were then re-immunoprecipitated (IP) with equal amounts of PI3K antibody and the immune complexes subjected to an in vitro kinase assay with γ-³²P-ATP and Mn²⁺ as described in Materials and Methods. Immune complex proteins were eluted, separated by SDS-PAGE and visualized by autoradiography. Molecular mass markers in kilodaltons are indicated at the left. (B) The immune complexes immunoprecipitated from each lane in (A) were eluted from the Protein G beads, separated by SDS-PAGE and immunoblotted for p110 content with a p110-specific antibody. Numbers below the lanes indicate the particular preclearing step. Molecular mass markers in kilodaltons are indicated at the left.