Effects of mutant 4EBP1 constructs and 4EGI-1 on cell survival. (A) After Jurkat cells were treated with diluent (lanes 1 and 5); OSI-027 at 5, 10, or 20 µM (lanes 2-4); MLN0128 at 0.25, 0.,5 or 1 µM (lanes 6-8); or 10 nM rapamycin (lane 9), whole cell lysates were subjected to immunoblotting. Dashed line indicates juxtaposition of nonadjacent lanes from the same X-ray films. (B-C) Cells treated for 48 hours with diluent (lane 2), 10 µM OSI-027 (lane 3), 1 µM MLN0128 (lane 4, B), 10 nM rapamycin (lane 5, B), or 25 µM 4EGI-1 (lane 4, C) were lysed with NP-40 buffer. After clarification, lysates were incubated with sepharose (lane 1) or 7Me-GTP-sepharose beads overnight and washed 5 times with NP-40 buffer before immunoblotting. (D) 4EBP1 constructs used in panels E-H. In the remainder of this figure, 4EBP1 T37A/T46A is designated 2A and 4EBP1 T37A/T46A/S65A/T70A is 4A. (E) Cells transfected with the indicated 4EBP1 construct were incubated for 72 hours and analyzed for annexin V binding. In panel E, *P = .03 relative to empty vector. (F) Seventy-two hours after transfection with S peptide-tagged 4EBP1 2A or 4A, qRT-PCR was performed to quantify BIM and PUMA messages, which were normalized to GAPDH mRNA. In panel F, *P = .006 relative to empty vector. (G-H) Cells transfected with the indicated 4EBP1 construct tagged with (G) S peptide or (H) EGFP were incubated for 72 hours and harvested for immunoblotting. In panel G, *nonspecific band. (I) qRT-PCR for BIM and PUMA mRNA after treatment with 25 µM 4EGI-1 or 10 µM OSI-027. In panel I, *P = .05 and **P = .002, relative to diluent. (J) Subdiploid cells, another indicator of apoptosis,^21,28  after treatment for 48 hours with 4EGI-1. Error bars in this panel (n = 3 independent experiments) are smaller than symbols. (K) Cells transfected with PUMA siRNA or nontargeting control were treated for 48 hours with diluent or 4EGI-1 and stained with annexin V. In panel K, *P = .036 relative to control. (K, inset) Immunoblot of whole cell lysates.