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. 2018 May 1;9:1747. doi: 10.1038/s41467-018-04042-w

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — FZD8 associates with TGF-β receptors. a Western blots of anti-1D4 immunoprecipitates (IP) and extracts (inputs) from PC-3M cells transfected for 24 h with 1D4-tagged FZD₈, Flag-tagged TGFβRI, and HA-tagged TGFβRII plasmids were probed for TGFβRII (HA), TGFβRI (Flag), and FZD₈ (1D4); blots are representative of three independent experiments. b Confocal immunofluorescence analysis of PC-3M cells transfected with 1D4-tagged FZD₈ (red) and Flag-tagged TGFβRI or HA-tagged TGFβRII (green) for 24 h. Images are representative of three independent experiments; 1D4 epitope or goat anti-FZD₈ (Aviva) were used to detect FZD₈ and Flag and HA epitope tag antibodies were used to detect TGFβRI and TGFβRII, respectively; blue staining shows cell nuclei (DAPI); scale bar 25 µm. c Western blots of Protein A/G-agarose pull-downs (IP IgG) and extracts (inputs) from PC-3M cells transfected for 24 h with FZD₈-CRD-IgG, LRP6-IgG, Flag-tagged TGFβRI, and HA-tagged TGFβRII plasmids were probed for TGFβRII (HA), TGFβRI (Flag), and LRP6 or FZD₈ (IgG); blots are representative of three independent experiments. d Western blots of Protein A/G-agarose pull-downs (IP IgG) and extracts (inputs) from PC-3M cells transfected for 24 h with FZD₈-CRD-IgG, Flag-tagged TGFβRI, and Flag-tagged TGFβRI extracellular domain (ΔTGFβRI) plasmids were probed for TGFβRI (Flag) and FZD₈ (IgG); blots are representative of three independent experiments. e Western blots of anti-1D4 immunoprecipitates (IP) and extracts (inputs) from PC-3M cells transfected for 24 h with plasmids encoding 1D4-tagged FZD₈, Flag-tagged TGFβRI, and HA-tagged TGFβRII, treated −/+1 ng ml⁻¹ TGF-β for 30 min were probed for TGFβRII (HA), TGFβRI (Flag), and FZD₈ (1D4); extracts were also probed for pSMAD3 and GAPDH as a loading control. Blots are representative of three independent experiments; graph shows average TGFβRII (HA) and TGFβRI (Flag) levels in 1D4 IPs, as determined by densitometry, in control (−) and TGF-β-treated (+) cells transfected with all three receptors, normalized to 1D4 from three independent experiments (*p < 0.05 by Student's t-test). f Cartoon depicting crosstalk of Wnt-11 and TGF-β signaling at the level of the receptors and transcription factors. Dashed arrow indicates potential regulation of SMAD2/3 by ATF-2, based on other published studies (see text)