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RT-PCR analysis confirmed that all three cell lines express VEGF-C and VEGFR3 (Figure 1A). Cl66 cells, which have moderate metastatic potential, expressed the lowest levels of VEGF-C while 4T1 cells which this website are highly aggressive and metastatic expressed the highest levels of VEGF-C. All three cell lines expressed two bands for VEGFR3 with 4T1 showing the highest expression of the larger band and Cl66 showing approximately equal expression of both sizes. Figure 1 Expression and phosphorylation of VEGFR3 in murine mammary adenocarcinoma cells A. Expression of VEGF-C, VEGFR3, and GAPDH specific mRNA transcripts in murine mammary adenocarcinoma cell lines differing in their metastatic potential: Cl66 (moderately ... To confirm expression at the protein level we analyzed expression by Western blotting. We detected expression of VEGF-C and VEGFR3 in all cell lines (Figure 1B). Similarly two bands were detected for VEGFR3 by antibody detection. However, expression of the smaller molecular weight protein was more abundant with the highest level in 4T1 cells. In addition, we confirmed expression of the receptor using immunofluorescent staining (Figure 1C). To assess the activity of the receptor in the cells we evaluated the phosphorylation status of the receptor. Using cells which find more were unstimulated, we performed immunoprecipitation using an anti-VEGFR3 antibody and probed with anti-VEGFR3 and anti-p-Tyr antibodies. Results confirm expression of the receptor and its constitutive activation in all three cell lines although the larger molecular weight protein was phosphorylated (Figure 1D). VEGF-C-VEGFR3/Flt4 pathway regulates mammary tumor cell viability These observations indicate the potential for autocrine VEGF-C/VEGFR3 signaling in this breast cancer model. To extend our previous study on the MMP23B role of VEGF-C in murine mammary tumors we selected Cl66 cells for our further analysis. To elucidate the role of VEGF-C, we examined the effect of inhibiting VEGF-C on viability. Cl66 cells were plated and antibody to VEGFR3 (10 ��g/ml) or VEGF-C (10 ��g/ml) was added. Cells were assayed for viability at 24, 48 or 72 hours. 24 hours after addition of antibody, the viability of cells treated with VEGFR3 antibody was significantly decreased as compared to cells treated with VEGF-C antibody (p