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. 2015 Jul 24;4(9):1063–1076. doi: 10.1242/bio.010918

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© 2015. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 4. — The NRP1 cytoplasmic region was essential for VEGF-A-induced cancer cell proliferation. (A) The western blot shows the expression of NRP1 in shControl- or NRP1 shRNA-treated DJM-1 clones (No. 12 and No. 13). (B) The comparison of NRP1 expression levels among lentivirus-overexpressed NRP1WT or cytoplasmic region deletion mutants (NRP1ΔSEA or NRP1ΔCyto) in shNRP1 DJM-1 No.13 clone (upper lanes). The same proteins were re-immunoblotted with an anti-actin antibody (lower lanes). (C) The colony formation assay of the lentivirus-overexpressed NRP1WT, NRP1ΔSEA or NRP1ΔCyto in the shNRP1 DJM-1 No.13 clone and shControl DJM-1 clone. (A,C) Percentages from each mean relative to the shControl are shown below the graph. (D) Co-immunoprecipitation assay with GIPC1 (a) or Syx (b). NRP1, GIPC1, and Syx were expressed in HEK293T cells and treated without (−) or with (+) VEGF-A (100 ng/ml) for 15 min. (a) Increased NRP1/GIPC1 and GIPC1/Syx interactions in the presence of VEGF-A are indicated by asterisks. (b) The Syx/GIPC1 interaction was increased in the presence of VEGF-A (asterisk). On the other hand, the NRP1/Syx interaction was decreased (asterisk). A 10% input as the loading control of NRP1, GIPC1, and Syx co-expressed in HEK293T cell lysates are shown in the right panels. Percentages from each blotted protein amount relative to “VEGF-A (−)” are shown below each lane. (E) Confirmation of the siRNA effects for GIPC1 or Syx. GIPC1 or Syx was overexpressed in HEK293T cells treated with 20 nM siControl, siGIPC1, or siSyx. The inhibitory efficiency of each siRNA on the expression of GIPC1 or Syx was compared to the siControl. The inhibitory percentages relative to the siControl are shown below each lane. (F) Colony formation assay in siNRP1, siGIPC1, or siSyx treated-DJM-1 cells in the presence or absence of exogenous VEGF-A (1 µg/ml) indicated as white columns (+) or black columns (−), respectively. Percentages from each mean relative to the siControl are shown below the graph. These data represent the means±s.d. N.S., not significant; **P<0.005; ***P<0.001.