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. 2022 May 4;298(7):102010. doi: 10.1016/j.jbc.2022.102010

Figure 7.

Figure 7

FSTL1 increases the association of Wnt3a with FZD4.A, FSTL1 increases the level of Wnt3a pulled down by FZD4-Fc in a solution. Wnt3a protein (750 ng) was incubated overnight at 4 °C with and without FZD4-Fc protein (1 μg), and in the absence or the presence of FSTL1 protein (2 μg) in TBS buffer containing 0.1% Triton X-100 (500 μl). Input samples were collected, and remaining mixtures were incubated with protein A beads, which had been blocked with 0.1% BSA in TBS. Eluted proteins and inputs were analyzed by Western blotting as indicated. B, FSTL1 increases the binding of Wnt3a to FZD4 on cell surface. HEK293 cells were transfected with plasmids for SNAP-FZD4 (100 ng/ml) and the chaperone MESD (50 ng/ml). About 24 h after transfection, cells were serum starved overnight. Cells then were incubated for 3 h with Wnt3a conditioned medium (CM) in the absence or the presence of FSTL1-HA CM. Cells were incubated with 1 μM cell-impermeable biotinylated SNAP substrate (BG-PEG12-Biotin) for 15 min at room temperature. After washing with PBS, lysates were incubated with streptavidin–agarose, and the precipitates were used for Western blotting as indicated. C, working model showing how FSTL1 enhances renal fibrosis. In CKD, FSTL1 expression increases, and FSTL1 increases the presentation of Wnt ligands to FZD receptors, thus enhancing Wnt/β-catenin signaling and renal fibrosis. D–F, the EC domain enhances Wnt3a signaling. In E and F, HEK293T (D) or NRK-49F cells (E) were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids, either alone or with Wnt3a plasmid, in the absence or the presence of the plasmid for EC domain. In F, NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids, and increasing doses of EC plasmid before the cells were incubated with Wnt3a CM. ∗∗∗p < 0.001. BSA, bovine serum albumin; CKD, chronic kidney disease; EC, extracellular calcium–binding domain; FSTL1, follistatin-like 1; FZD, Frizzled; HA, hemagglutinin; HEK293 human embryonic kidney 293 cell line; MESD, mesoderm development LRP chaperone; NRK-49F, normal rat kidney 49 fibroblast cell; SNAP-FZD4, SNAP-tagged FZD4; TBS, Tris-buffered saline.