FIG. 6.

A dominant-negative allele of PERK inhibits eIF2α phosphorylation in response to hypoxia and thapsigargin (Thaps.). (A) 3T3 cells infected with a retrovirus expressing a c-myc epitope-tagged C-terminal deletion of PERK (PERKΔC) and 3T3 cells infected with the empty vector (pBABEpuro) were exposed to hypoxia for the indicated times or treated with 1 μM thapsigargin for 2 h. (Top) Immunoblotting with an antibody against a c-myc tag (9E10), showing the expression of dominant-negative PERK. (Middle) Immunoblotting with the anti-ser51-P eIF2α antibody. (Bottom) Immunoblotting with a polyclonal antibody that recognizes total eIF2α. (B) Effects of hypoxia and thapsigargin on protein synthesis rates in 3T3 cells expressing a dominant-negative PERK allele. 3T3 cells stably infected with PERKΔC or the empty vector expressing only the puromycin resistance gene were exposed to hypoxia (0.01%) for the indicated times or treated with thapsigargin (1 μM) for 2 h. During the last 20 min of the treatments, cells were labeled with [³⁵S]methionine and TCA-precipitable counts were measured as described in Materials and Methods. The results shown are averages of three independent experiments ± the standard errors. Con, control; Incorp., incorporation.