Figure 2.

ERdj4 Promotes a BiP-IRE1 Complex

(A) Schema of the IRE1^LD-GST protein containing the entire human IRE1α luminal and transmembrane domains (residues 19–486, solid) fused to GST (striped).

(B) Representative immunoblots of IRE1^LD-GST and endogenous BiP, recovered by glutathione affinity chromatography or in lysate of transfected ΔERdj4 cells.

(C) Ratio of BiP to IRE1^LD-GST signal from 4 experiments, as in (B). Mean ± SD. ^∗∗p = 0.0048, parametric ratio paired Student’s t test).

(D) As in (B); compares IRE1^LD-GST to PERK^LD-GST. R(B/LD) notes the ratio of the BiP signal to the LD-GST species from the representative experiment shown.

(E) As in (B); compares ERdj4 to ERdj6.

(F) As in (B); compares IRE1^LD-GST to IRE1^CLD-GST.

(G) As in (B); prior to elution with sample buffer, the indicated glutathione Sepharose beads were incubated for 5 min with 3 mM ATP at room temperature.

(H) Immunoblot of endogenous IRE1α and BiP recovered from CHO cells of the indicated genotype by immunoprecipitation of IRE1α. Prior to elution with sample buffer, the indicated protein-A Sepharose beads were incubated with ATP (as in G). The bottom panel shows the input of BiP in the two samples.