The proliferation of HUVEC treated with increasing concentrations of rVEGF164 was assessed. After 72-hour treatment with rVEGF164, cells were incubated with a tetrazolium salt and the OD450-OD650 was determined.
The purity of recombinant rVEGF164 was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant rVEGF164 and staining overnight with Coomassie Blue.
Western blot analysis of extracts from HUVEC, untreated or treated with rVEGF164 for 15 minutes, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (upper) or Akt1 (C73H10) Rabbit mAb #2938 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 20 μg BSA per 1 μg rVEGF164. Carrier free: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2.
Stable in lyophilized state at 4°C for 1 year after receipt. Sterile stock solutions reconstituted with carrier protein are stable at 4°C for 2 months and at -20°C for 6 months. Avoid repeated freeze-thaw cycles.Maintain sterility. Storage at -20°C should be in a manual defrost freezer.
>98% as determined by SDS-PAGE of 6 μg reduced (+) and non-reduced (-) recombinant rVEGF164. All lots are greater than 98% pure.
Recombinant rVEGF164 contains no "tags" and the nonglycosylated protein has a calculated MW of 19,234. DTT-reduced protein migrates as a 24-31 kDa polypeptide. Lower mobility in SDS-PAGE is due to glycosylation. The non-reduced cystine-linked homodimer migrates as a 46-53 kDa protein. The expected amino-terminal APTTE of recombinant rVEGF164 was verified by amino acid sequencing.
The bioactivity of recombinant rVEGF164 was determined in a cell proliferation assay using HUVEC. The ED50 of each lot is between 1-5 ng/ml.
Less than 0.01 ng endotoxin/1 μg rVEGF164.
Recombinant rat VEGF164 (rVEGF164) Ala27-Arg190 (Accession #NP_001103803) was expressed in human 293 cells at Cell Signaling Technology.
VEGF164 is one of many splice variants of the VEGF-A gene, and is one amino acid shorter than its human counterpart, VEGF165 (1, 2). VEGF164 is produced by a number of cells including endothelial cells, macrophages, and T cells (1, 2). VEGF164 is involved in angiogenesis, vascular endothelial cell survival, growth, migration, and vascular permeability (1,2). Gene expression is induced by hypoxia, inflammatory cytokines, and oncogenes (1, 2). VEGF164 binds to heparan sulfate and is retained on the cell surface and in the extracellular matrix (1-3). VEGFR1 and VEGFR2 are the receptor tyrosine kinases for VEGF164 (2). NRP-1 and NRP-2 may function as co-receptors and enhance VEGFR2 signaling (2-3). Binding of VEGF164 to VEGFR1 and VEGFR2 leads to activation of the PI3K/AKT, p38 MAPK, FAK, and Paxillin (2).
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