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. 2004 Sep;24(17):7469–7482. doi: 10.1128/MCB.24.17.7469-7482.2004

FIG. 5.

FIG. 5.

PERK is required for induction of ATF4 in response to hypoxic stress. (A) High-molecular-weight polysomes from normoxic cells (PERK+/+, PERK−/−, PKR−/−, PERK−/− PKR−/−, HT29-A1, and HT29-Puro) were pooled (fractions 7 to 11), reverse transcribed, and quantified by real-time PCR. The quantity of ATF4 is described as the number of ATF4 transcripts isolated from polysomal RNA per cell. Each sample was independently normalized to a spiked internal control. Q-PCR analysis was repeated in triplicate. (B) High-molecular-weight polysomes from hypoxia-treated (16 h) or normoxic (0 h) PERK+/+ and PERK−/− cells were pooled (fractions 7 to 11), reverse transcribed, and quantified by real-time PCR. The efficiency of ATF4 translation during hypoxic treatment (16 h) and normoxia (0 h) was plotted as the percentage of total mRNA associated with polysomes: (quantity of poly mRNAtime x/quantity of total mRNAtime x) × 100%. Each sample was independently normalized to a spiked internal control. Q-PCR analysis was repeated in triplicate. (C) Immunoblot of ATF4 and eIF2α phosphorylated on Ser-51 [eIF2a(p)] in PERK+/+ and PERK−/− MEFs for the indicated period of time after hypoxic stress. Actin served as a loading control.