Figures & data
Figure 1. eIF4E and 4E-BP1 are dephosphorylated upon mTOR inhibition by PP242. HeLa cells were treated with 2.5 μM of PP242 for 1 h or left untreated. Whole cell lysates were incubated with m7GTP-beads (cap column), beads alone (beads) or subjected to immunoprecipitation with eIF4GI antibody (IP eIF4GI). Bound proteins and whole cell lysates (input) were separated by SDS-PAGE and analyzed by western blotting with the indicated antibodies. Data are representative of at least three independent experiments.
Figure 2. eIF4E phosphorylation is favored by 4E-BP downregulation but hindered by eIF4G silencing. (A) HEK cells expressing shRNA against 4E-BP1 and/or 4E-BP2 or Scramble shRNA were treated with 2.5 μM of PP242 for 1 h or left untreated in the presence of 10% FCS. Black arrowhead indicates 4E-BP2 specific signal. Asterisk shows non-specific signal from dephosphorylated 4E-BP1. (B) 4E-BP1/4E-BP2 double knockout (4E-BP DKO) and wild type (WT) mouse embryonic fibroblasts were treated as in (A). (C) MDA-MB231 cells expressing inducible sh-miRNA against eIF4GI and/or eIF4GII or control vector were grown with 5 μg/ml doxycycline for 3 d. Cell lysates were analyzed by western blotting using the indicated antibodies. Data are representative of at least three independent experiments.
Figure 3. low amount of 4E-BP favors eIF4E phosphorylation. (A) MiaPaca-2, Panc-1 and HEK cells lysates were analyzed by western blotting. (B) MiaPaca-2, Panc-1 were treated with 2.5 μM of PP242 for 1 h. Cell lysates were analyzed by western blotting using the indicated antibodies. Data are representative of at least three independent experiments.
Figure 4. 4E-BPs downregulation does not confer resistance to the MNK inhibitor CGP57380. (A) HEK cells were treated for 1 h with 10 or 20 µM of CGP57380 (CGP.) or cercosporamide (Cerco.) or left untreated. (B) HEK cells expressing shRNA against 4E-BP1 and/or 4E-BP2 or Scramble shRNA were treated with 20 μM of CGP57380 (CGP.) for 1 h or left untreated. (C) 4E-BP DKO and WT MEF were treated with 2.5 μM of PP242 or with 20 µM of CGP57380 for 1 h or left untreated. (D) MiaPaca-2 and Panc-1 cells expressing shRNA against the 3′UTR of MNK1 mRNA or Scramble shRNA were treated with 20µM of CGP for 1 h prior harvesting. Cell lysates were analyzed by western blotting using the indicated antibodies. Data are representative of at least three independent experiments.
Figure 5. 4E-BPs downregulation renders eIF4E phosphorylation insensitive to serum or amino acids starvation. (A) 4E-BP DKO and WT MEF were incubated overnight in DMEM containing low serum concentration (0.5% FCS) or incubated for 1 h in amino acid-free HBSS medium or left untreated (10% FCS). (B) MiaPaca-2 cells expressing shRNA to 4E- BP1 or Scramble shRNA were incubated for 2 h in amino acid-free HBSS medium or supplemented with 10% FCS and amino acids. Cell lysates were analyzed by western blotting using the indicated antibodies. (C) HEK cells expressing shRNA against 4E-BP1 and/or 4E-BP2 or Scramble shRNA were incubated for 1 h in amino acid-free HBSS medium (-AA) or left untreated in the presence of 10% FCS (+AA). Data are representative of at least three independent experiments.
