Translation of TLK1B is dependent on phosphorylation of 4E-BP1 after IR and is sensitive to rapamycin. A. The phosphorylation of 4E-BP1 is induced by IR. Aliquots of cells were incubated for increasing time following IR (10 Gy), the proteins were separated on a 15% PAGE/SDS, transferred to a membrane, and probed with antibody to 4E-BP1 phosphorylated at Thr 46. B. The hyperphosphorylated form of 4E-BP1 is induced by IR and is sensitive to rapamycin. The cells were incubated for 1 h after IR with increasing concentrations of rapamycin. Total 4E-BP1 was analyzed by western blot with an antibody that recognizes all forms of 4E-BP1. The position of the 3 forms of 4E-BP1, from unphosphorylated (α) to hyperphosphorylated (γ) are indicated. C. The translational upregulation of TLK1B following IR is blocked by rapamycin or wortmannin. The cells were incubated for 1 h after IR with or without 20 nM rapamycin and then processed for western blot with the TLK1 antibody, which recognizes the large and small (TLK1B) spliced variants of TLK1 (left panel). The same experiment was carried out with 30 μM wortmannin (right panel).