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Fig. 3 | Journal of Biomedical Science

Fig. 3

From: Conformational alteration in glycan induces phospholipase Cβ1 activation and angiogenesis

Fig. 3

GHCer, but not SSEA3Cer, competed with PLCβ1 for binding to TRAX. A Competitive ELISA. Recombinant PLCβ1-C (upper panel) or TRAX (lower panel) protein was immobilized on microtiter plate for ELISA assays. GHCer (20 μM) competed with the binding between TRAX and PLCβ1-C, respectively. The amount of binding protein was determined, respectively, by anti-TRAX and PLCβ1-C antibodies followed by AP-conjugated secondary antibody. BSA protein was used as a negative control. The data were presented with mean ± SD. B Biacore analysis of PLCβ1-C binding to TRAX. The TRAX protein was immobilized on CM5 chips. The sensorgram showed that PLCβ1-C bound TRAX in a concentration-dependent manner. The Kd estimated by BIA evaluation was about 7.5 × 10–6 M. C Biacore analysis for competition between GHCer and PLCβ1-C for binding with TRAX. The CM5 chips coated with TRAX protein were used for binding assay for PLCβ1-C (360 nM), or in combination with 0, 1, and 9 μM of GHCer. D FRET assay using confocal microscopy for the colocalization of TRAX and PLCβ1. HUVECs were incubated with 20 μM GHCer or SSEA3Cer for 3 h and treated with mouse anti-PLCβ1 antibody followed by donor Alexa 488-tagged anti-mouse IgG (Green). In addition, the cells were also treated with rabbit anti-TRAX antibody, followed by acceptor (Alexa 555) -tagged anti-rabbit IgG (Red). FRET signal was determined by 555 nm laser power-off. Representative pictures of FRET channel (yellow) from three independent experiments were shown. E GHCer-induced calcium influx. After loading with the calcium indicator Fluo-4/AM (40 μM) for 45 min at 37 °C, the fluorescence intensity of intracellular Ca2+ in HUVECs was traced for 6 min every 5 s and presented as arbitrary units (a.u.). Experiments were performed upon exposure to PBS, 60 μM of GHCer, or SSEA3Cer. The data are presented as mean ± SD of triplicate determinations

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