Fig. 4

Xbp1s regulates the expression of Atf4. a, b Western blotting analysis of the effects of GSK2606414 or MKC3946 on bortezomib-activated Atf4 and Xbp1s in mMSCs (left panel) and MC3T3-E1 cells (right panel). Confluent MSCs or MC3T3-E1 cells were treated without or with bortezomib (1 nM) in the absence or presence of the PERK inhibitor GSK2606414 (2.5 nM) or IRE1α inhibitor MKC3946 (10 nM) for 24 h. c RT-PCR analysis of the effects of MKC3946 on the bortezomib-induced formation of Xbp1s mRNA. MSCs or MC3T3-E1 cells were treated as the abovementioned. d A schematic of the promoter region of the Atf4 gene. One putative Xbp1s-binding site (white rectangle, − 83/− 80) is shown. e ChIP assays using control IgG, antibodies against Xbp1s. Cell extracts were collected from mMSCs treated with or without bortezomib (2.5 nM) for 16 h. Protein/DNA complexes from cells were precipitated without antibody (input) or with an Xbp1s antibody or IgG. PCR was performed using a set of primers covering the promoter region containing the putative Xbp1s binding site. PCR using input DNA was used as a positive control. PCR using primers covering the promoter region of the Atf4 gene (− 1218/− 1066) was used as a negative control. Data shown are representative of 3 independent experiments