Figure 5.
Figure 5. EphrinB2 can suppress the proliferative effect of bFGF on HUAECs via the syndecan-1 ectodomain. (A) To understand whether shedding of the syndecan-1 ectodomain in HUAECs was increased after the addition of ephrinB2, HUAEC cultures were treated with preclustered ephrinB2 (0 to 500 ng/mL) for 24 hours. The conditioned media were collected and concentrated with Centricon according to the manufacturer's instruction. Under gentle vacuuming, 1.5 μL of each concentrate was applied to the methanol-moistened PVDF membrane. After blocking, monoclonal antiectodomain of syndecan-1 was added and incubated as the protocol of Western blot. The membrane was finally developed with enhanced chemiluminescence (ECL). Another secreted peptide of endothelial cells, VEGF, was used as an internal control because its expression was not regulated by ephrinB2 based on our cDNA microarray and RT-PCR. (B) To test whether overproduced syndecan-1 was consistently associated with FGFR1, a co-IP assay was employed. HUAEC cultures were treated with varying concentrations of preclustered ephrinB2 (0 to 500 ng/mL) for 24 hours. Membrane portions of cell lysates were isolated and processed for IP with monoclonal mouse anti-FGFR1 antibody.After centrifugation, the supernatant and pellet were separately collected and processed for Western blotting with polyclonal rabbit anti-FGFR1 or goat antisyndecan-1 antibodies. (C) To examine whether dispersed syndecan-1 ectodomain was responsible for inhibiting the bFGF-induced proliferative effect, the conditioned media of HUAEC cultures with or without pretreatment of ephrinB2 were collected and concentrated to 2 mg/mL. The methodology of immunoprecipitation was used to deplete either syndecan-1 or syndecan-4 ectodomain from the media. The control samples received protein A/G Plus–Agarose beads alone. A dot blot assay was used to confirm the depletion of syndecan-1 or -4 in the leftover fluid. Then the fluid was added, in a protein concentration of 500 μg/mL, to fresh M199 medium with 10 ng/mL bFGF to undergo MTT proliferation assay as previously described. Each group was tested in sextuplicate, and tests were performed in triplicate. Each data set was normalized with the average value of the control group. Bars represent the mean ± SD from 6 data sets of 1 representative experiment. eB2 indicates ephrinB2. *P < .05 compared with bFGF alone group (first bar from the left).

EphrinB2 can suppress the proliferative effect of bFGF on HUAECs via the syndecan-1 ectodomain. (A) To understand whether shedding of the syndecan-1 ectodomain in HUAECs was increased after the addition of ephrinB2, HUAEC cultures were treated with preclustered ephrinB2 (0 to 500 ng/mL) for 24 hours. The conditioned media were collected and concentrated with Centricon according to the manufacturer's instruction. Under gentle vacuuming, 1.5 μL of each concentrate was applied to the methanol-moistened PVDF membrane. After blocking, monoclonal antiectodomain of syndecan-1 was added and incubated as the protocol of Western blot. The membrane was finally developed with enhanced chemiluminescence (ECL). Another secreted peptide of endothelial cells, VEGF, was used as an internal control because its expression was not regulated by ephrinB2 based on our cDNA microarray and RT-PCR. (B) To test whether overproduced syndecan-1 was consistently associated with FGFR1, a co-IP assay was employed. HUAEC cultures were treated with varying concentrations of preclustered ephrinB2 (0 to 500 ng/mL) for 24 hours. Membrane portions of cell lysates were isolated and processed for IP with monoclonal mouse anti-FGFR1 antibody.After centrifugation, the supernatant and pellet were separately collected and processed for Western blotting with polyclonal rabbit anti-FGFR1 or goat antisyndecan-1 antibodies. (C) To examine whether dispersed syndecan-1 ectodomain was responsible for inhibiting the bFGF-induced proliferative effect, the conditioned media of HUAEC cultures with or without pretreatment of ephrinB2 were collected and concentrated to 2 mg/mL. The methodology of immunoprecipitation was used to deplete either syndecan-1 or syndecan-4 ectodomain from the media. The control samples received protein A/G Plus–Agarose beads alone. A dot blot assay was used to confirm the depletion of syndecan-1 or -4 in the leftover fluid. Then the fluid was added, in a protein concentration of 500 μg/mL, to fresh M199 medium with 10 ng/mL bFGF to undergo MTT proliferation assay as previously described. Each group was tested in sextuplicate, and tests were performed in triplicate. Each data set was normalized with the average value of the control group. Bars represent the mean ± SD from 6 data sets of 1 representative experiment. eB2 indicates ephrinB2. *P < .05 compared with bFGF alone group (first bar from the left).

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