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. Author manuscript; available in PMC: 2010 Aug 7.
Published in final edited form as: Cell. 2009 Aug 7;138(3):562–575. doi: 10.1016/j.cell.2009.07.017

Figure 2. Two Distinct Modes to Produce XBP1s Transcription Factor.

Figure 2

(A) EtBr-stained agarose gel of XBP1 cDNA amplicons after induction of IRE1α variants for 8 hrs, followed by 5µM 1NM-PP1 (or DMSO) for 4 hrs. The cDNA amplicon of unspliced XBP1 mRNA is cleaved by a PstI site within a 26 nucleotide intron to give 2U and 3U. IRE1α-mediated cleavage of the intron and re-ligation in vivo removes the PstI site to give the 1S (spliced) amplicon. *is a spliced/unspliced XBP1 hybrid amplicon. The ratio of spliced over (spliced + unspliced) amplicons—1S/(1S+2U+3U)—is reported as % spliced XBP1 amplicons in histograms. Three independent biological samples were used. Data are means +/− SD. P-values: ** <0.01 and *** <0.002. ns=not significant. (B) % spliced XBP1 amplicons in IRE1α (I642G)-expressing INS-1 cells as a function of [1NM-PP1]. (C) Curve fitting to triplicate 1NM-PP1 concentration-dependent splicing assays in IRE1α (I642G)-expressing T-REx 293 cells. (D) Time course of XBP1 mRNA splicing under “phosphotransfer activation”—defined as provision of Dox (0.1µg/ml) and 1NM-PP1 (5µM) to stable WT IRE1α-expressing cells. 1NM-PP1 is inert for WT IRE1α—Figure 2A, lane 3—but always added to maintain consistency with pseudokinase activation. (E) ER stress-mediated splicing of XBP1 mRNA in INS-1 CAT cells using tunicamycin—Tm—(5µg/ml) for 2 hrs. (F) Time course of XBP1 mRNA splicing under “pseudokinase activation”, defined as provision of Dox (0.1µg/ml) and 1NM-PP1 (5µM) to stable IRE1α (I642G)-expressing cells. Time courses shown used T-REx 293 cells and were identical in INS-1 cells (not shown). (G) Immunoblot of XBP1s transcription factor under phosphotransfer or pseudokinase activation in INS-1 cells, and INS-1 CAT cells under Tm.