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. 2001 Nov 1;15(21):2852–2864. doi: 10.1101/gad.912401

Figure 3.

Figure 3

Phosphospecific antibodies directed against Ser 65 and Thr 70 confirm the serum sensitivity of the phosphorylation at these sites. (A) Western blotting using an antibody to total 4E-BP1 (top), or a phosphospecific antibody directed against Ser 65 (bottom) was performed on extracts from HEK 293T cells transfected with HA–4E-BP1 wt (lane 1) or HA–4E-BP1 Ser 65-Ala (lane 2), or extracts from HEK 293 cells that were serum starved (lane 3), or serum starved, and then serum stimulated for 30 min (lane 4). (B) Western blotting was performed as in A, but using an HA–4E-BP1 Thr 70-Ala protein in lane 2, and a phosphospecific antibody directed against Thr 70 (bottom). The positions of the endogenous 4E-BP1, and the transfected proteins are indicated.