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. 2015 Jun 29;212(7):1061–1080. doi: 10.1084/jem.20141601

Figure 3.

Figure 3.

Wnt3a induces CXXC5 expression and enhances CXXC5-Dvl interaction. (A and B) Human dermal fibroblasts (n = 2–3 cells) were treated with or without Wnt3a (50 or 200 ng/ml). (A) WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, α-SMA, collagen I, or α-tubulin and to RT-PCR analyses with primers for CXXC5 or GAPDH (n = 2 independent experiments). (B) Relative densitometry values are shown underneath blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). The cells were also immunocytochemically stained for β-catenin, CXXC5, or collagen I. Representative ICC images are shown (top), and mean intensity quantitation was performed (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (C) Human dermal fibroblasts were incubated with 50 ng/ml Wnt3a for 1, 6, 12, 24, 48, or 72 h, and WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, or α-tubulin (n = 2 independent experiments). Relative densitometric ratios of each protein to α-tubulin are shown. (D and E) Human dermal fibroblasts were treated with 50 ng/ml Wnt3a for 2 d after treatment with control siRNA (Con si) or CXXC5 siRNA (CXXC5 si). (D) Western blot analysis (n = 3 independent experiments) of WCLs was performed with antibodies against β-catenin, α-SMA, collagen I, CXXC5, or α-tubulin. Relative densitometry values are shown underneath the blots as ratios relative to the levels of α-tubulin. ICC analysis (n = 3 independent experiments) of samples in D and E was performed with β-catenin or CXXC5. (E) Representative ICC images are shown (left), and mean intensity values were calculated (right; ***, P < 0.0005). (B and E) Bars, 50 µm. Means ± SD. (F) Human dermal fibroblasts were transfected with pcDNA3.1, pcDNA3.1-CXXC5-Myc, or pcDNA3.1-CXXC5ΔDBM-Myc. WCLs or cell lysates immunoprecipitated with anti-Myc were analyzed by immunoblotting to detect Dvl-1, Myc, β-catenin, or α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots. (G) WCLs from human dermal fibroblasts treated with (Wnt3a) or without (Con) 50 ng/ml Wnt3a for 2 d were subjected to immunoprecipitation with anti–Dvl-1 antibody, and Western blot analyses were subsequently performed to detect Dvl-1, CXXC5, β-catenin, and α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots.