Fig. 4

Wnt signaling modulates LARGE2-dependent O-glycosylation of α-DG in CRC. A) Immunoblot analysis of O-glycosylated α-DG (WGA-AE purified). APC, β-DG, and α-tubulin were analyzed on WCL from HT-29 cells after silencing of APC for 72 h. B,C) Immunoblot analysis and laminin overlay (OL) on WGA-AE purified α-DG from HT-29 cells after silencing of APC(B) or ectopic expression of CTNNB1-S33Y (C). β-DG and α-tubulin were analyzed on WCL. D) qRT-PCR analysis of indicated genes in HT-29 cells, carrying a DOX-inducible shRNA of APC, after CRISPR/Cas9-mediated targeting of the TCF7L2-BS (L2_BSg1 and g2) in LARGE2 intron 1. Error bars indicate SD (n=3). E) Immunoblot analysis of O-glycosylated α-DG (WGA-AE purified) in HT-29 wild-type or mutant for the TCF7L2-binding site in LARGE2 intron 1 (L2_BSg2) after silencing of APC for 96 h. WCLs were used to detect β-DG and tubulin. F,G) Immunoblot analysis of WGA-AE purified O-glycosylated α-DG in LS174T-NE (F) and SW480-NE cells (G) relative to their ERT2 controls upon treatment with 400 nM 4-OHT for 72 h. WCLs were used to detect β-DG and tubulin. H,I) Immunoblot analysis on WGA-AE purified α-DG from SW480 (H) and PDTO1 (I) cells upon CRISPR/eCas9-mediated targeting of the TCF7L2-BS in the first intron of LARGE2 via guideRNAs. β-DG and α-tubulin were analyzed on WCL