Skip to main content
. 2022 Dec 1;11:e73105. doi: 10.7554/eLife.73105

Figure 2. Slc38a5 is a target gene of Wnt signaling in the vascular endothelium.

Figure 2.

(A) Slc38a5 mRNA levels were increased in human retinal microvascular endothelial cells (HRMECs) treated with Wnt ligands, recombinant Norrin, and Wnt3a-conditioned medium (Wnt3a-CM), compared with their respective vehicle controls (Ctrl, and Ctrl-CM), and suppressed by a Wnt inhibitor XAV939. (B–C) Protein levels of SLC38A5 in HRMECs were up-regulated by Wnt ligands Norrin (B) and Wnt3a-CM (C), and down-regulated by XAV939. Protein levels of SLC38A5 (52 kDa) and β-catenin (92 kDa) were quantified by Western blotting and normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 37 kDa) levels. n-p-β-catenin: non-phosphorylated β-catenin (92 kDa). (D) Three promoter regions upstream of Slc38a5 gene containing potential Wnt-responsive TCF (T-cell factor)-binding motifs (TTCAAAG) was identified based on sequence analysis. Three putative TCF-binding regions: P1 (–887 bp to –346 bp), P2 (–3925 bp to –3058 bp), and P3 (–5328 bp to –4423 bp) were cloned and ligated separately with a luciferase reporter, and co-transfected with an active β-catenin plasmid in HEK 293T cells, followed by measurement of luciferase activity. Data are expressed as mean ± SEM. (A-C) n=3–6 per group. (D) n=5 per construct group. *p≤0.05, **p≤0.01.

Figure 2—source data 1. Raw data for Figure 2.