PERK regulates accumulation of cyclin D1. (A) NIH 3T3
cells infected with empty virus (lanes 1–4), virus encoding PERK
(lanes 5–6), or virus encoding Ire1β (lanes 7–8) were left
untreated or were treated with 0.5 μg/ml tunicamycin (lanes 3–4).
Equivalent amounts of cellular protein were resolved on denaturing
polyacrylamide gels and subjected to immunoblot analysis with the 9E10
antibody, which recognizes a c-myc epitope tag expressed at the C
terminus of both PERK and Ire1β or a cyclin D1-specific monoclonal
antibody. Sites of antibody binding were visualized by enhanced
chemiluminescence. (B and C) After
infection of NIH 3T3 cells with virus encoding PERK (lane 2), PERKΔC
(lane 3) or empty virus (lane 1), equivalent amounts of total protein
were resolved on a denaturing polyacrylamide gel and transferred to
nitrocellulose membrane. Membranes were subsequently probed with the
9E10 antibody (PERK and PERKΔC) or antibodies specific for cyclin D1,
serine 51-phosphorylated eIF2α, or total eIF2α. Sites of antibody
binding were visualized by enhanced chemiluminescence.