Soluble mouse LYVE-1 and VEGFR3 fusion construct cloning, purification of Fc fusion proteins, and intervention procedure
This protocol is extracted from research article:
Lymphangiogenesis in kidney and lymph node mediates renal inflammation and fibrosis
Sci Adv, Jun 26, 2019; DOI: 10.1126/sciadv.aaw5075

Fragments of murine LYVE-1 complementary DNA (cDNA) (position 241–924 in GenBank, clone AJ311501) encoding the extracellular domain of LYVE-1 and VEGFR3 cDNA (position 26–2363 in GenBank, clone L07296) encoding the extracellular domain of VEGFR3 were separately fused to Fc [mouse IgG2a (immunoglobulin G2a)] in the pcDNA3.1(+)/Fc (mouse IgG2a) vector (Futaibio, Jiangsu, China). The pcDNA3.1(+)/Fc (mouse IgG2a) vector was used as control. Constructs were transfected into 293T cells using calcium phosphate. Transfected cells were grown in UltraCHO medium (Lonza) for 3 days before harvesting culture supernatant. After adjustment of supernatant pH [using 2 M tris-HCl buffer (pH 8.0)], the fusion proteins were purified by affinity chromatography on a column (1-ml bed volume) of protein A–Sepharose (Sigma) eluted with 0.1 M glycine-HCl buffer (pH 3.0). Fractions containing Fc fusion proteins were neutralized by the addition of 0.05 volume of 2 M tris-HCl buffer (pH 8.0), and the purity was confirmed by SDS–polyacrylamide gel electrophoresis (6365). sVEGFR3-FC or sLYVE-1-FC was administered to wild-type mice by tail vein injection (0.5 μg/g) 24 hours before UUO or IRI surgery. sFC was injected as a control. UUO mice were administered VEGFR3-FC, sLYVE-1-FC, or sFC (0.5 μg/g) on days 0, 1, 3, and 5 and euthanized on day 7 after UUO. IRI mice were administered sVEGFR3-FC, sLYVE-1-FC, or sFC (0.5 μg/g) on days 0, 1, 3, 7, 10, and 13 and euthanized on day 14 after IRI.

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