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. 2005 Sep 8;24(19):3470–3481. doi: 10.1038/sj.emboj.7600777

Figure 1.

Figure 1

Extreme hypoxia induces ATF4 and CHOP upregulation in a PERK-dependent manner. PERK+/+ and PERK−/− MEFs were exposed to ⩽0.02% O2 or treated with 1 μM thapsigargin for the times indicated. (A) Immunoblots with anti-ATF4, anti-CHOP, anti-BiP and anti-β-actin (loading control). Different time points are used since induction of ATF4 occurs much earlier than CHOP or BiP. (B) Northern blot analysis of UPR mRNAs following hypoxia and thapsigargin treatments. PERK+/+ and PERK−/− MEFs were treated as in (A). Blotting for α-tubulin was used as a loading control. (C) Translation efficiency of CHOP mRNA following hypoxia. PERK+/+ and PERK−/− MEFs were treated as in (A) and lysates were subjected to sucrose gradient sedimentation. Polysome profiles shown are from cell lysates of aerobic and hypoxic PERK+/+ cells. The positions of the 40S and 60S subunits as well as the polysomal RNA are indicated. (D) The amounts of CHOP, β-actin and 18S mRNA were determined by real-time quantitative PCR in each of the polysome fractions. The gray column indicates the polysome fraction used for quantitative PCR. Values plotted represent the total amount of CHOP mRNA found within the polysomes relative to β-actin. Also shown is the total cellular amount of CHOP found in the PERK+/+ and PERK−/− MEFs normalized to β-actin as determined by quantitative RT–PCR.