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. 1999 Jun 1;13(11):1422–1437. doi: 10.1101/gad.13.11.1422

Figure 4.

Figure 4

Figure 4

FRAP/mTOR phosphorylates 4E-BP1 complexed with eIF4E. (A) 4E-BP1 was pre-incubated without eIF4E, with an equimolar quantity or with a twofold molar excess of eIF4E, and subjected to a kinase assay using FRAP/mTOR immunoprecipitated from rat brain (lanes 1–3) or recombinant ERK2 (lanes 4–8). Phosphorylated proteins were separated via SDS-15%-PAGE, transferred to nitrocellulose membranes, and subjected to autoradiography. The substrates used were as follows. (Lanes 1–6) A thrombin-cleaved and HPLC-purified GST–4E-BP1; (lane 7) an uncleaved GST–4E-BP1 wild-type protein; (lane 8) an uncleaved GST–4E-BP1 mutant lacking the eIF4E-binding site. (B) Tryptic/chymotryptic map of 4E-BP1 alone (A, lane 1). (C) Tryptic/chymotryptic map of 4E-BP1 complexed with a twofold molar excess eIF4E (A, lane 3).