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. 2011 Jul 1;6(7):e20444. doi: 10.1371/journal.pone.0020444

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Grandclement et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

PMC Copyright notice

A, smad2/3 and phospho-smad2/3 expression was assessed in HT29^ctrl, HT29^NRP2, Colo320^siRNA-ctrl, Colo320^siRNA-NRP2 by intra-cellular flow-cytometry. Relative Fluorescence Intensity (RFI) was calculated. B, Western-blotting experiments were performed on nuclear extracts of HT29^ctrl and HT29^NRP2 cultured in serum free conditions or with 10 ng/mL of TGF-β1 for 16 hours. Histone-H1 was used as a control of protein loading. Blotted proteins have been quantified with the BIO-1D advanced software and reported to the Histone-H1 level. While exogenous TGF-β1 is mandatory for smad2 phosphorylation detection in HT29, Smad2 is constitutively phosphorylated in HT29^NRP2 cells without any previous exposition to exogenous TGF-β1. C, TGF-β1 Cignal reporter assay kit was used for the quantification of TGF-β1-induced smad2/3 signaling in HT29^ctrl, HT29^NRP2, Colo320^siRNA-ctrl and Colo320^siRNA-NRP2 cells. An up-regulation of approximatively 17 times of the smad response is observed in HT29^NRP2 cells compared to HT29^ctrl after TGF-β1 stimulation. At the opposite, smad dependent response is significantly decreased in Colo320 cells treated by siRNA targeting NRP2 compared to Colo320 cells treated by siRNA-control after TGF-β1 stimulation. Results are presented as the ratios between the firefly luciferase activity and the renilla luciferase activity (Ren/Luc) for each conditions. This experiment was realized 3 times, each time in triplicates. D, Expression of TGFRI and TGFRII in HT29^ctrl, HT29^NRP2, Colo320^siRNA-ctrl, Colo320^siRNA-NRP2 colon cancer cells. Relative Fluorescence Intensity (RFI) was calculated. Colo320 cells don't express TGFRII. E, Surface plasmon resonance studies were performed to explore TGF-β1 interactions with NRP₂. Fc-NRP2 proteins were covalently grafted on a chemically activated self assembled protein chip. Injections of TGF-β1, BSA (Control -), VEGF (Control +) were performed at 250 nM in PBS-Tween 0.05%, before biacore analysis. These experiments were reproduced two times and showed a specific binding of TGFβ1 to Fc-NRP₂ protein. F, NRP2 interacts with TGFRI in co-immunoprecipitaion experiments using HT29^NRP2 cells.

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