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. 2000 Dec 12;97(26):14340–14345. doi: 10.1073/pnas.011511898

Figure 4.

Figure 4

Cytoplasmic–nuclear shuttling of FRAP regulates downstream signaling. Myc-tagged FRAP proteins with various translocation signals were coexpressed with HA-p70s6k or FLAG-4E-BP1 in HEK293 cells. Various FRAP proteins are designated as follows: WT, wild-type; NLS, NLS-FRAP; NLS′, NLS′-FRAP; NES, NES-FRAP; NES′, NES′-FRAP; KD, kinase-dead. All FRAP constructs, including wild type, contained the S2035T mutation. All cells were pretreated with 100 nM rapamycin for 30 min before serum stimulation for 1 h. LMB treatment was carried out for 12 h before lysis. (A) In vitro kinase assays were performed with immunoprecipitated HA-p70s6k. Results are shown as activities relative to that for wild type. Expressions of Myc-FRAP and HA-p70s6k were monitored by Western blotting, by using epitope tag antibodies. (B) Phosphorylation of FLAG-4E-BP1 as reflected by mobility shift was examined by Western blotting, using M2 anti-FLAG antibody.