BiP/Grp78 stabilization on bortezomib treatment is ensured at a posttranscriptional level by increased chaperoning activity of Hsp90. (A) Jeko-1 and JBR cells were treated with 7.5 or 15nM bortezomib (bz). RNA and protein were isolated after 3 and 8 hours of incubation. BIP mRNA levels were quantified by reverse transcription–PCR. BiP/Grp78 protein levels were evaluated by Western blotting with the use of β-actin as equal loading control. (B) Microphotographs of BiP/Grp78 (green) and calnexin (red) labeling in JBR cells either untreated (ct) or treated for 8 hours with 15nM bortezomib. (C) JBR cells were treated as in panel B, and BiP immunoprecipitation was performed as described in “Methods.” Bound and unbound fractions were analyzed by Western blotting for the presence of BiP/Grp78, IRE-1, Grp94, and the cytosolic-activated Hsp90. (D) Jeko-1, JBR, Z-138, and ZBR cells were treated as indicated, and Hsp90/BiP complexes were analyzed as previously described by coimmunoprecipitation, followed by Western blot analysis of both factors.