9 Internalization and activation status of VEGFR2 in response to VEGF165a. were expressed as a percentage of responses obtained using 30?nM VEGF165a-TMR (100%) or vehicle (0%). Data were fitted using non-linear least squares regression (variable slope) using GraphPad Prism. All data were expressed as mean??S.E.M and pooled from 8 independent experiments. For 120?min time course data of VEGF165a-TMR internalization, VEGFR2 activation (measured by anti p1175 or pY1214 labeling) or VEGFR2-Halo internalization, data were normalised as a percentage of peak responses measured at 20?min stimulation (100%) or with no agonist stimulation (0%) respectively. Data for VEGF165a-TMR internalization were fitted to a mono exponential association function: =?All data obtained from the NFAT luciferase reporter gene assays were normalised to 10?nM VEGF165a responses and fitted with non linear regression using the equation as described previously [10]. Statistical analysis used unpaired All data were expressed as percentage fold increases in cell count following VEGF165a or VEGF165a-TMR treatment when normalised to vehicle treatment alone (100%). All data were expressed as mean??S.E.M. Statistical analysis using one way ANOVA was performed to compare vehicle with ligand treatments (P?JI051 and the uncovered N terminal cysteine [34]. This purification and labeling reaction were performed in a physiological buffer under reducing conditions (100?M tris(2-carboxyethyl)phosphine; TCEP). The purified and labeled VEGF165a (VEGF165a-TMR) was collected and dialyzed to allow final formation of the di-sulphide linked anti-parallel VEGF165a homodimer under non-reducing conditions. Open in a separate window Fig. 1 Synthesis and characterisation of purified VEGF165a-TMR. (a) Synthetic strategy for purification and labeling of VEGF165a-TMR. (b) Fluorescent SDS-PAGE analysis (of VEGF165a-TMR (Eex?=?532?nm; Eem?=?580?nm) in the presence or absence of 100?mM DTT and with or without deglycosylation by PNGase. (c) Influence of bovine serum albumin (0.1% BSA) and 10?mM DTT on VEGF165a-TMR concentrations measured using fluorescence correlation spectroscopy (FCS). Data are from 3 impartial experiments and expressed as mean??SEM. (d) Stimulation of NFAT luciferase production by HEK293T cells stably expressing VEGFR2 by VEGF165a (R&D Systems; closed circles), VEGF165a prepared identically to the fluorescent analogue (open circles) or fluorescent VEGF165a-TMR (red circles). Values represent mean??SEM from 4 independent experiments from which quadruplicate determinations were made. Data are expressed as a percentage of the response to 10?nM VEGF165a (R&D Systems) obtained in each individual experiment. (e) Effect of Tetracosactide Acetate VEGF165a and VEGFR165a-TMR on proliferation of human umbilical endothelial cells (HUVECs). Following stimulation with JI051 VEGF165a or VEGF165a-TMR (3 or 30?nM) for 24 or 48?h, HUVECs were fixed using 3% PFA/PBS and the nuclei stained using “type”:”entrez-nucleotide”,”attrs”:”text”:”H33342″,”term_id”:”978759″,”term_text”:”H33342″H33342 (2?mg/ml). Cells were imaged using a IX Micro widefield platereader at 4 magnification and nuclei were counted using a granularity algorithm (MetaXpress, Molecular Devices). Data are presented as fold increases in proliferation compared to vehicle treatment (mean??S.E.M) and are pooled from 5 individual experiments. One way ANOVA was used to determine the statistical significance of ligand treatment when compared to vehicle only for both.
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