Volume 13, Issue 6, Pages (June 2008)

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Volume 13, Issue 6, Pages 557-560 (June 2008) Further Evidence for Expression and Function of the VEGF-C/VEGFR-3 Axis in Cancer Cells Jen-Liang Su, Pai-Sheng Chen, Ming-Hsien Chien, PoShen B. Chen, Ya-Huey Chen, Chien-Chen Lai, Mien-Chie Hung, Min-Liang Kuo Cancer Cell Volume 13, Issue 6, Pages 557-560 (June 2008) DOI: 10.1016/j.ccr.2008.04.021 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Analysis of VEGFR-3 Expression and Cellular Function (A) Both 9D9F9 and MAB3491 antibodies recognize pure recombinant human VEGFR-3 protein. rhVEGFR-3 (50, 100, 200, or 500 ng) was resolved by SDS-PAGE and then immunoblotted using 9D9F9 (upper panel) or MAB3491 (lower panel) antibody. According to the instructions for rhVEGFR-3 from R&D Systems, the rhVEGFR-3 has a calculated molecular mass of approximately 112 kDa (∗). As a result of glycosylation, the recombinant protein migrates as an approximately 160 kDa protein (#) in SDS-PAGE. (B and C) VEGFR-3 expressed in HDMEC, H928, A549, MCF-7, MDA-MB-231, CL1-0, and CL1-5 cells is detected by immunoblotting (B) or by immunoprecipitation using 9D9F9 or MAB3491 as indicated followed by immunoblotting with 9D9F9 antibody (C). Cell lysates containing 1 mg of total proteins were subjected to immunoprecipitation by incubation with the primary antibody (2 μl 9D9F9 antibody or 2 μg MAB3491 antibody) at 4°C overnight and subjected to immunoblotting assay. The uncleaved, unglycosylated (precursor), and cleaved VEGFR-3 have calculated molecular masses of approximately 195 kDa (∗), 175 kDa (#), and 125 kDa (@), respectively. (D and E) Cell lysates (10 mg protein/condition) were subjected to immunoprecipitation by incubation with the primary antibody (7.5 μl of 9D9F9 antibody or 7.5 μg of MAB3491; 4°C overnight), and the SDS-PAGE gels were stained. The protein in the ≈125 kDa bands was identified by mass spectrometry. (F) A549 cells were transfected with scramble control siRNA or indicated specific siRNA. RT-PCR was performed to confirm the inhibitory effects of siRNA on VEGF-C and VEGFR-3 expression. (G–H) Immunoprecipitation-immunoblot assay was performed to analyze the inhibitory effects of siVEGF-C on VEGFR-3 phosphorylation (G). Thirty-six hours after transfection with scramble control siRNA or indicated specific siRNA, cells were harvested and assayed using migration (H) or invasion (I) assay in the presence or absence of rhVEGF-C. Bars represent means ± SD. Asterisks denote a statistically significant induction (∗p < 0.05) compared with values of lane 1. The # symbol indicates a significant induction compared with values of lane 4. Results represent the findings of three separate experiments, and all incubations were conducted in triplicate. Cancer Cell 2008 13, 557-560DOI: (10.1016/j.ccr.2008.04.021) Copyright © 2008 Elsevier Inc. Terms and Conditions