IRE-1 but not ATF6 or PERK is activated by ATIII overexpression.
A, ATIII variants and A1AT NHK were expressed in CHO cells for 24 h. Control cells were treated with DMSO or Tm for 24 h. Cells were lysed, and RNA was collected. cDNA was generated and amplified via PCR using XPB-1–specific primers. B, ATIII variants and A1AT NHK were expressed in CHO cells for 24 h. Control cells were treated with either DMSO or Tm for 24 h. Cells were then lysed with MNT and TCA-precipitated. 5% whole-cell lysate was then resolved on a 9% SDS-PAGE and imaged by Western blotting using anti-Myc antibody (ATIII), anti-PERK antibody, and anti-A1AT antibody. C, ATIII variants and A1AT NHK were expressed in CHO cells for 24 h. Control cells were treated with either DMSO or Tm for 24 h. Cells were then lysed with MNT and TCA-precipitated. 5% whole-cell lysate was then resolved on a 9% SDS-PAGE and imaged by Western blotting using the indicated antibodies. D, quantification of B. Relative BiP expression was calculated by normalizing all BiP levels to DMSO, using GAPDH as a loading control. All experiments are representative of three independent experiments. Error bars, S.D.