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. 2000 Oct 17;97(23):12625–12630. doi: 10.1073/pnas.220247197

Figure 4.

Figure 4

PERK is required for UPR-dependent cell-cycle arrest. (A) NIH 3T3 cells were infected with virus encoding PERKΔC and selected with 7.5 μg/ml puromycin for 4 days. Expression of PERKΔC was confirmed by immunoblot analysis. (B) Parental NIH 3T3 or cells expressing PERKΔC were treated with tunicamycin for 20 h and pulsed with BrdUrd during the last 2.5 h of tunicamycin treatment. The percentage of S-phase cells is expressed as the number of BrdUrd-positive cells vs. the total number of cells. (C) Equivalent amounts of total protein prepared from cells treated as above were resolved on a denaturing polyacrylamide gel and blotted with antibodies specific for BiP and cyclin D1. Sites of antibody binding were visualized by enhanced chemiluminescence. (D) Whole-cell lysates prepared from PERKΔC-3T3 cells treated with 0.5 μg/ml tunicamycin for the indicated intervals were precipitated with antibodies to CDK2. Denatured immune complexes were separated on gels and blotted with antibodies to p21Cip1 or p27Kip1.