FIG. 1.

Characterization of the phospho-Ser608-specific antibody. Recombinant p85α/p110α was subjected to an in vitro kinase assay in the presence or the absence of ATP. Reaction products were analyzed by SDS-PAGE and transferred to a PVDF membrane. (A) Blots were incubated either with preimmune serum or with the affinity-purified anti-phospho-Ser608 antibody diluted 1:1,000. In some experiments, the immune serum was incubated with the immunizing phosphopeptide or the corresponding dephosphopeptide at 1 mM for 1 h at 25°C before further processing. (B) In some experiments, wortmannin (wortm.) at 100 nM was included during the kinase assay. IB, immunoblot. (C) Recombinant p85α/p110α which had been subjected to an in vitro autokinase assay in the presence or absence of ATP (Sf9) or bacterially expressed p85α (E. coli) was analyzed by immunoblotting using the affinity-purified anti-phospho-Ser608 antibody.