FIGURE 5.

URB1 regulates activating transcription factor 4 (ATF4) through the transcriptional control of X‐box binding protein 1 (XBP1). A, B, Microarray screening, RT‐PCR, and western blotting were used to investigate the potential regulation of XBP1 by URB1. C, XBP1 binding site on the ATF4 promoter region was predicted by consulting the JASPAR database. D, Schematic illustration of the XBP1 binding site on WT and mutant (Mut) ATF4 promoter sequences. E, F, Dual luciferase reporter assays and RT‐PCR confirmed the specific binding of XBP1 with the ATF4 promoter and the activation of ATF4 mRNA transcription. G, Subcellular colocalization of XBP1 and ATF4 in RKO cells was evaluated by immunofluorescence staining. H, Interaction of XBP1 with ATF4 promoter region was validated by CHiP assay. Scale bar = 50 μm (×100 magnification). Data are expressed as mean ± SD (n = 3). **P < .01, ***P < .001. ns, not significant; sh‐NC, negative control