(A) Ire1 luminal domain (LD; regions II–IV) and His6-tagged BiP ATPase domain proteins, both individually and in 1:1 molar ratio mixture, were visualized as control lanes on a 4–12% Bis-Tris SDS-PAGE gel. The same proteins were then subjected to EGS cross linker for 1 hr, after which the reaction was quenched and samples were visualized along side control lanes. In the presence of cross linker, Ire1 LD forms dimer and tetramer species; when in a mixture with BiP ATPase, there is a reduction in the corresponding tetramer band (*). Also, a band appears that is consistent in size with a trimer species. (B) Samples from (A) were immunoblotted using anti-His6 antibody, which detects His6-tagged BiP ATPase domain protein. Since BiP ATPase protein is monomeric in the absence or presence of cross linker, BiP ATPase forms hetero dimer and hetero trimer with Ire1 LD. The binding of BiP to Ire1 reduces the size of the tetramer (*) that is exclusively formed by Ire1 LD, leading to the conclusion that BiP inhibits Ire1 LD tetramer formation, by preventing formation of the dimer species.