Extended Data Fig. 7: Structure-function analysis of XBP1 interaction with proteins of the b-catenin signaling pathway.
(a-b) Assessment of XBP1 interaction with b-catenin (a) or TLE1(L) (long isoform, L) (b) by co-immunoprecipitation (co-IP) in HEK293T cells. *, residual band from TCF4 immunoblot after membrane stripping. (c) Co-IP between XBP1 and TCF4 in HEK293T cells. HIF1a,which was previously shown to bind XBP1, was used as a positive control. d) Representative confocal microscopy images of LSK cells from Ern1+/+Flt3ITD/+ or IRE1α-deficient (Ern1koFlt3ITD/+) mice stained (red) with XBP1 antibody (clone 9D11A43; BioLegend). Nuclear DNA (green) was counter-stained with Hoechst dye. Outline of cell membrane are highlighted in the merged images. Scale bar: 25 mm. (e-g) Co-IP of XBP1 and truncation mutants of TCF4 protein (e). Whereas XBP1 co-immunoprecipitated with the HMG/DNA binding ___domain of TCF4 (residues 349-496) alone (f, lane 6), it failed to interact with a C-terminus truncated TCF4 (residues 1-356) protein (f, lane 7; g, lane 4). *, non-specific band. (h) Hypothetical model of XBP1-mediated repression of b-catenin-driven TCF/LEF transcriptional activity. TCF/LEF factors are constitutively repressed by TLE proteins (i) but become activated when b-catenin displaces TLE1 from its binding to TCF/LEF proteins downstream of canonical Wnt ligands or growth factor signaling (ii). XBP1-mediated suppression is triggered upon IRE1α activation and represses TCF/LEF even in the presence of b-catenin potentially by recruiting co-repressor proteins (iii). Data (panels a, b, c, f and g) are representative of at least three independent experiments.
