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. 2020 May 7;11(5):325. doi: 10.1038/s41419-020-2522-0

Fig. 5. VEGFR3 mediates VEGFA-induced angiogenic events in HRMVECs.

Fig. 5

a Western blot analysis of control and the indicated time periods of VEGFA (40 ng/ml)-treated HRMVECs for VEGFR3 and β-tubulin levels. b,c The effect of siControl, siPKCθ and siJunB (100 nM) on VEGFA (40 ng/ml)-induced (2 h) VEGFR3 levels. The blots were sequentially reprobed for PKCθ and β-tubulin levels or JunB and β-tubulin levels to show the specificity and efficacy of siRNA on its target and off target molecules. d Upper panel: Retinal ECs were isolated from WT and PKCθ−/− mice and tested for the effect of VEGFA on PKCθ phosphorylation and JunB and VEGFR3 expression. Lower panel: Retinal ECs from PKCθ−/− mice were transfected with empty vector or JunB expression vector and two days later cell extracts were prepared and analyzed by western blotting for JunB, VEGFR3 and β-tubulin levels. e Upper panel: western blot analysis of VEGFR2, VEGFR3 and β-tubulin levels to show the specificity and efficacy of siControl and siVEGFR3 (100 nM) in HRMVECs. Bottom panel: The effect of siControl, siVEGFR3 and MAZ51 (5 μM) on VEGFA (40 ng/ml)-induced HRMVEC migration. f–h All the conditions were same as in (e) except that cells were treated with and without VEGFA (40 ng/ml) and DNA synthesis (f), sprouting (g) or tube formation (h) were measured. The bar graphs represent quantitative analysis of three independent experiments. The values are presented as mean ± SD. *p < 0.01 vs vehicle control or siControl; **p < 0.01 vs siControl + VEGFA. Scale bars in (g) and (h) are 50 and 200 μm, respectively.