Xenbase Image ID: 159354

Figure 4. Syndecans Are Receptors for nANGPTL4 and Cooperate in Wnt Inhibition (A) Cell-surface binding assay in HEK293T cells. Cells were transfected with the indicated receptor constructs and treated with conditioned media containing alkaline phosphatase (AP) fusion proteins of ANGPTL4, and the positive control ligands RSPO3DC (ligand for LGR4, 5 and SDC1–4) or dkk1 (ligand for LRP6). Ligand binding was detected using a chromogenic AP substrate (red). (B) ELISA-based in vitro binding assay. ANGPTL4, RSPO3DC (positive control), and Dkk3 (negative control) were immobilized and incubated with dilutions of alkaline phosphatase (AP)-tagged soluble Syndecan-4 (SDC4DTMC). The fraction of bound SDC4 was determined using an AP detection kit. (C and D) Internalization assay in HepG2 cells. (C) Cells were treated with the indicated siRNAs or sodium chlorate and incubated with HRP-tagged full-length ANGPTL4 (green). (D) Cells were incubated with the indicated myc-tagged Angptl4 constructs. Protein internalization was detected by tyramide signal amplification (C) or anti-myc staining (D). (E) In vitro binding assay of Streptag-HA-SDC4DTMC with Flag-tagged Angptl4 N terminus (nAngptl4), C terminus (cAngptl4), dkk1, or Dkk3 recombinant proteins. Pull-down with streptavidin beads shows specific binding of SDC4 to nAngptl4 by immunoblot. (F) Wnt luciferase reporter assay in H1703 cells stimulated with Wnt3a or control (Co) conditioned medium in the presence of limiting doses of the indicated siRNAs. Data are displayed as means ± SD and show one representative of multiple independent experiments with three biological replicates. RLA, relative luciferase activity. ***p < 0.001. (G and H) qRT-PCR analysis of gsc (G, H) and noggin (H) in animal cap explants at neurula (st.18) from X. tropicalis embryos injected as indicated. Low dose of angptl4 Mo1 (1 ng) was used in this experiment. Data show one representative of multiple independent experiments with at least three biological replicates. See also Figure S4 and Table S1. Image published in: Kirsch N et al. (2017) Copyright © 2017. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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