Expression of norrin signaling components. Panel A – RT-PCR for norrin signaling pathway components Fz4 (832 bp), norrin (400 bp), and LRP5 (300 bp) in normal colonic mucosa obtained fresh from colonoscopic biopsy. Molecular weight markers appear in lane 1. β-actin (544 bp) was utilized as a control to confirm RNA quality. Control PCR reactions without reverse transcriptase did not give any PCR products for any RNA sample with any pair of primers (data not shown). Identities of the PCR products were confirmed by sequence analysis. Panel B – Expression defined by RT-PCR of Fz4 (top row), norrin (3rd row) and LRP5 (bottom row) in various cell lines. Lane 1: no template control; lane 2: CSC1; lane 3: Colo205; lane 4: Colo320; lane 5: HCT116; lane 6: HCT116 with additional chromosome 3; lane 7: HT29; lane 8: NCM460 (normal mucosa-derived); lane 9: RKO; lane 10: EaHy926 (endothelial). Fz4, norrin and LRP5 mRNA was detected in all tested cell lines with the exception of Fz4 in Colo205. Actin control is depicted in 2nd row.