Extended Data Fig. 8: Distinct mechanisms of regulation of c-ISR and s-ISR in response to inhibitors of eIF4E or CK2 activity.

(a) Representative western blot analysis of the indicated proteins in ΔuORF1 and WT mouse ES Cells treated with the inhibitor of PP1 phosphatase (containing GADD34 as a subunit), Salubrinal (15 μM, 5 h; (n = 3 independent experiments). (b,f) Western blot analysis of the indicated proteins in MEFs expressing Con (shCon) or Eif2s2 (shEif2s2) shRNAs and treated with Tg (400 nM) (b) or Torin1 (250 nM) (f) for the specified times. Representative blots are shown (n = 3 independent experiments). (c-d) Representative western blot analyses of the indicated proteins in MEFs treated with Tg (400 nM) for the specified durations, or MEFs expressing the indicated shRNAs, without (c) or with Tg-treatment (d) (n = 3 independent experiments). Tg-treated MEFs in (c) were used as a positive control for induction of the c-ISR. (e) Representative western blot analysis of the indicated proteins in MEFs expressing control (shCon) or Eif2b5 shRNA (shEif2b5) and treated with (Tg-400 nM) or Torin1 (250 nM) of the specified durations (n = 3 independent experiments). (g-h) Western blot analysis for the indicated proteins in MEFs expressing control (shCon) or Eif2b5 shRNA (shEif2b5) and treated with 4EGI-1 (200 μM) (g) Tg (400 nM) or SGC-CK2-1 (5 μM) (h) for the specified times. Representative western blots are shown (n = 2 independent experiments). Lower eIF2α-p levels are consistent with GADD34 induction (h, 4^th lane). (i) Representative western blot analyses of the indicated proteins in MEFs treated with glucose (0 or 2.5 mM, 16 h). LiCl (10 or 25 mM) or Torin1 (250 nM) were used for the specified final hours of the 16 h treatment with medium containing 2.5 mM glucose (n = 3 independent experiments).