Figure 3. The C-terminus of BCL9/B9L is required for transcriptional Wnt responses in human cells.

(A) SuperTOP assays in wt or KO HEK293T cells lacking BCL9 and/or B9L, as indicated, ±6 hr of Wnt stimulation (WCM, Wnt3a-conditioned-media); mean ± SEM (n = 3 independent experiments). (B) RT-qPCR assays in wt or DKO HEK293T cells, ±6 hr of 20 mM NaCl or LiCl as indicated, revealing relative transcript levels of AXIN2 or SP5; values were normalized to TBP (internal control), and are shown as mean ± SEM relative to unstimulated wt controls (set to 1); note that the uninduced levels of SP5 were unusually high in one of the two DKO clones, but neither of the DKO clones showed Wnt-inducible SP5. (C) SuperTOP assays in wt or DKO HEK293T cells as in (A), 24 hr after transfection with wt and mutant B9L as indicated (below, corresponding Western blots; α-ABC, active unphosphorylated β-catenin); ev, empty vector control.

DOI: http://dx.doi.org/10.7554/eLife.20882.007

Figure 3—figure supplement 1. CRISPR/Cas9-based gene editing strategies for BCL9 and B9L in HEK293T cells.

(A) Cartoon of BCL9 and B9L exon boundaries (with non-coding 5’-and-3’-UTR exons omitted); sgRNA targeting sites are marked for both genes. (B) sgRNA sequences, with PAM sites in bold. (C) Western blot of lysates from individual HEK293T clones with BCL9 or B9L single KO, or with BCL9/B9L DKO, probed with antibodies as indicated on the left. (D) Summary of BCL9/B9L KO and DKO lines generated in this study.

DOI: http://dx.doi.org/10.7554/eLife.20882.008