Figure 6.

Coimmunoprecipitation assay demonstrating conditional membrane recruitment with a nontoxic rapamycin derivative, rap* (9R). T-antigen expressing Jurkat cells were cotransfected with 0.5 μg of MF3 and 4 μg of FRB kinase (lanes 1–4) or FRB* kinase (lanes 5–8). After 48 hr, the cells were incubated for 30 min with ligand [20 nM rapamycin, lanes 1 and 5; 20 nM C16-(R)-methallyl rapamycin (rap*), lanes 2 and 6; no ligand, lanes 3 and 7], washed with cold PBS, and lysed in PINT buffer (18) (0.3% Triton X). Aliquots were removed for the cell lysate (lanes 4 and 8), and the remainder was pelleted at 14,000 rpm for 10 min to separate the nuclear fraction, and immunoprecipitated with anti-hemagglutinin antibody and protein A-conjugated to beads. The immunoprecipitates were washed extensively with PINT buffer and the bound proteins were eluted with soluble peptide corresponding to the Flu epitope. The eluted proteins were separated by SDS/PAGE and detected by Western blot analysis with anti-Flag M2 antibody. The structure of rap* (9R) is shown below the Western blot.