Figure 2.

FKpYEEI and SLFpYEEI form trimeric complexes with the Fyn SH2 domain and FKBP12. Recombinant GST-Fyn SH2 domain was expressed, bound to glutathione beads (7.5 μl beads, 0.55 nmol protein), and was incubated with 100 nM ³²P-labeled FKBP12 (10,000 cpm) in 100 μl of binding buffer (20 mM Tris, pH 7.2/150 mM NaCl). Increasing concentrations of FKpYEEI or SLFpYEEI were added and the reactions were rotated for 2 hr. The binding reactions were centrifuged in a pierced PCR tube to separate the beads from the supernatant. The radioactive protein associated with the beads was resuspended in 100 μl of PBS, which was added to EcoLite scintillation fluid (ICN) and counted in an LS5000CE liquid scintillation counter (Beckman).