FIGURE 1:

Changes in the endogenous complexes of IRE1α, PERK, and ATF6α during ER stress. (A) HEK293 cells were treated with 12 μM of thapsigargin (TG) for the indicated time points and lysed with digitonin. The lysates were separated by SDS–PAGE and immunoblotted for the indicated proteins. A phos-tag–based immunoblotting was performed for probing phosphorylated IRE1α. (B) The digitonin lysates from A were analyzed by BN–PAGE immunoblotting with IRE1α antibody, PERK antibody (C), or ATF6α antibody (D). (E–H) HEK293 cells were treated with 5 mM DTT for the indicated time points and analyzed as in A–D. (I) HEK293 cells were pretreated with a serine protease inhibitor AEBSF (250 μM) for 1 h and subsequently treated with DTT for the indicated time points to induce ER stress. The digitonin lysates were analyzed as in A and D. Experiments shown are representative of experiments repeated at least two times during different days.