Figure 2

(A) The deletion of the HD1 (PYGO-interacting) domain of BCL9 and BCL9L induces a variation in the ?chain of adaptors? causing the loss of PYGO association with the Wnt/?-catenin transcriptional complex (Cantù et al., 2014). (B) Immunofluorescence staining of tumors collected from control or conditional Pygo1/2-KO and Bcl9/9l-KO mice. Prox1 (red) and DAPI (blue) are shown in in the top panels; Vimentin (green) and Laminin (red) in the bottom panels. (C) Quantitative RT-PCR of selected groups of targets (compare it with the same analysis of Pygo1/2-KO in Figure 1E) of RNA extracted from control or Bcl9/9l-?HD1 tumors. (D) Experimental outline of the tumor proteins pull-down and mass-spectrometry. TBX3 was identified among the proteins potentially interacting with BCL9 but not with BCL9-?HD1. (E) The IP proteins analyzed by mass spectrometry were in parallel subjected to SDS page electrophoresis and probed with an anti-TBX3 antibody (upper panel). The expression of Tbx3 in control compared to Bcl9/9l-?HD1 tumors (N = 4) was evaluated via qRT-PCR (bottom panel), to exclude that differential pull-down was due to lost expression in mutant tumors. (F) 13.5 dpc Bcl9/9l-?HD1 embryos display forelimb malformations and absence of digits (emphasized by dashed white lines) ? a characteristic Tbx3-mutant phenotype (upper panels). The limb defect is absent in Pygo1/2-KO embryos (bottom panels) underscoring that BCL9/9L act, in this context, independently of PYGO1/2.