Figure 2.

5× ATF6 reporter activation is defective in IRE1α-null MEFs. (A) 5× ATF6 reporter gene expression in wild-type and IRE1α-null MEFs. The reporter plasmids containing the luciferase gene under control of 5× ATF6 binding sites and β-galactosidase under control of the CMV promoter were cotransfected into wild-type and IRE1α-null MEFs. The transfected cells were treated with 2 μg/mL tunicamycin for 16 h prior to harvest. The luciferase activities are presented relative to CMV β-galactosidase activities. Similar results were obtained from two independent experiment. (B–D) Wild-type and IRE1α-null MEFs were transfected as in A in the presence of vector alone or vector encoding wild-type IRE1α, kinase-defective (K599A) IRE1α, RNase defective (K907A) IRE1α, C-terminal-deleted IRE1α (IRE1αΔC), ATF2, ATF4, ATF6, processed form of ATF6 (ATF6 50-kD), c-Jun, or c-Fos as indicated. The vector used for IRE1α expression was pEDΔC. The empty vectors used as controls were pEDΔC (B,D), pcDNA3 (Vector 1), and pCMV-HA (Vector 2) (C). MEFs were transfected by either Effectine (B,D) or FuGENE6 (C) according to the manufacturers' recommended procedures. The transfected cells were treated with 10 μg/mL tunicamycin for 6 h (B,D) or 2 μg/mL tunicamycin for 16 h (C) prior to harvest. Similar results were obtained from four independent experiments.