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. 2012 Apr 17;31(10):2322–2335. doi: 10.1038/emboj.2012.84

Figure 5.

Figure 5

BIM controls XBP-1 mRNA splicing possibly through a direct interaction involving the BH3 domain. (A) BIM and PUMA DKO were transiently transfected with expression vectors for BIMWT, BIML150E or empty vector in the presence of 50 μM zVAD-fmk. After 20 h, cells were stimulated with Tm for 16 h and XBP-1 mRNA splicing monitored by RT–PCR. Percentage of XBP-1 mRNA is indicated. Upper panel: As control, the expression of BIM was monitored by western blot. Middle panel: XBP-1 mRNA splicing was monitored over time and quantified. Of note, transient transfection leads to an enhancement of XBP-1 mRNA splicing after Tm treatment. This control was used to normalize the levels of XBP-1 mRNA splicing and calculate the contribution due to BIM expression as a fold induction from Mock control (lower panel). Bars represent the average and standard error of three independent experiments. Statistically significant differences are indicated (*P<0.01). Efficiency of transfection was measured by FACS for all experiments (∼30% efficiency). (B) The possible interactions between BIMWT, BIML150E with the cytosolic domain of IRE1α (IRE1ΔN) were tested using a two-hybrid system (see Materials and methods). Different serial dilutions of yeast cultures are presented in control (−Leu/−Trp) and selection media (−Leu/−Trp/−His/X-α-Gal). (+): Indicate a positive control for the assay. (C) Recombinant IRE1ΔN–HIS (1 μg) was incubated with IVTT BIMWT or BIML150E. Their interactions were tested after pulling down IRE1ΔN–HIS and performing a western blot analysis (see Materials and methods). (D) The endoribonuclease activity of 0.1 μg of IRE1ΔN–HIS (+) was monitored in vitro using the conditions described in Supplementary data in the presence of IVTT BIM-EL, PUMA or a mock preparation. Substrate was added (total mRNA) and after 1 h, mRNA was re-extracted and endoribonuclease activity of IRE1α was analysed by RT–PCR. Actin levels were monitored as loading control. As positive control, mRNA was treated with 1 μg IRE1ΔN–HIS (++). (E) In parallel, using the conditions described in (h), the effects on the IRE1α RNAse activity of with IVTT BIMWT or BIML150E were tested. Figure source data can be found with the Supplementary data.