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. 2015 Sep 28;2(11):1767–1784. doi: 10.1016/j.ebiom.2015.09.042

Fig. 4.

Fig. 4

DLL4 and NOTCH1 mediate VEGFC-induced angiogenic events. A. An equal amount of protein from control and various time periods of VEGFC (100 ng/ml)-treated HRMVECs were analyzed by Western blotting for DLL1, DLL4, Jagged1, NOTCH1, NOTCH2 and NOTCH4 levels using their specific antibodies. The DLL4 blot was normalized to β-tubulin. B. Cells were transduced with Ad-GFP or Ad-KCREB (40 moi), growth-arrested, treated with and without VEGFC (100 ng/ml) for the indicated time periods and cell extracts were prepared and analyzed for DLL4 and NOTCH1 levels using their specific antibodies and the blot was sequentially reprobed for CREB, and β-tubulin levels to show the overexpression of KCREB and normalization. C. An equal amount of protein from control and various time periods of VEGFA (40 ng/ml)-treated HRMVECs were analyzed by Western blotting for pCREB, CREB, DLL4 and cNOTCH1 levels using their specific antibodies and the DLL4 blot was normalized to β-tubulin. D. HRMVECs were transfected with control, or DLL4 siRNA, growth-arrested, treated with and without VEGFC (100 ng/ml) for 2 h, cell extracts were prepared and analyzed by Western blotting for NOTCH1 and DLL4 levels using their specific antibodies and normalized to β-tubulin. E. HRMVECs were transfected with control, DLL4 or NOTCH1 siRNA and 48 h later either cell extracts were prepared and analyzed by Western blotting for DLL4 and NOTCH1 levels using their specific antibodies and normalized to β-tubulin. F–H. All the conditions were the same as in panel E except that after transfection, cells were quiesced and subjected to VEGFC (100 ng/ml)-induced DNA synthesis (F), migration (G), or tube formation (H). I. All the conditions were the same as in panel E except that after transfection cells were subjected to sprouting assay as described in Fig. 1, panel J. The bar graphs represent quantitative analysis of 3 independent experiments. The values are presented as Mean ± SD. * p < 0.01 vs control; ** p < 0.01 vs control siRNA + VEGFC. Scale bar represents 50 μm in panel F.