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Before analyzing the phosphorylation dynamics of ephrinB2, ephrinB2/G and ephrinB2/∆V, we tested EphB4/Fc stimulation on MCF7 Tet-ephrinB2 cells using WB analysis (described in section 3.1.2.2) (data not shown). However, ephrinB2 phosphorylation levels were barely detectable, possibly due to lower amounts of ephrinB2 protein on the cell surface compared to the stable MCF7-ephrinB2 cell line. To overcome this issue we performed immunoprecipitation (IP) using anti-Flag mAb prior to WB analysis.

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3.2.1.1

EphrinB2, ephrinB2/G and ephrinB2/∆V phosphorylation dynamics

MCF7 tetracycline inducible cell lines were stimulated with doxycycline for 48 hours in order to achieve maximum cell surface expression levels prior to EphB4/Fc stimulation for 0, 15, 30, 60, 120 and 240 minutes. Flag-tagged ephrinB2, ephrinB2/G and ephrinB2/∆V were immunoprecipitated from cell lysates and analyzed by WB using phospho- and total-ephrinB antibodies. As illustrated in figure 3.10A, phosphorylation dynamics of ephrinB2 wild type in MCF7 tetracycline inducible cells was different compared to the MCF7-ephrinB2 stable cell line (figure 3.3A, section 3.1.2.2). Specifically, in MCF7 tetracycline inducible cells, the basal phosphorylation level of ephrinB2 under non-stimulated conditions was relatively high (lane 1) and only modestly increased upon EphB4/Fc stimulation, peaking at 30 minutes (lane 3) and sharply decreasing at 240 minutes (lane 6). Syntenin-1 depletion in ephrinB2 expressing cells did not seem to alter dynamics of phosphorylation (figure 3.10B). By contrast, ephrinB2/G and ephrinB2/∆V mutants showed a different phosphorylation pattern (figure 3.10A and 3.10B respectively), with hardly any increase in phosphorylation detected throughout the time-course of EphB4/Fc stimulation. However, the sharp decrease in ephrinB2 phosphorylation at 240 minutes (lane 6, panel A) was also observed for ephrinB2/G (lane 12, panel A) and ephrinB2/∆V (lane 6, panel B). The corresponding decrease in ephrinB2, ephrinB2/G and ephrinB2/∆V total expression levels suggests that protein degradation is responsible for the decrease of phosphorylation. Furthermore, we noticed that the decrease in ephrinB2/G (lane 12, panel A) and ephrinB2/ ΔV (lane 6, panel B) total levels at 240 minutes seemed more pronounced when compared to the wild type, possibly indicating increased protein degradation. Interestingly, the highly glycosylated form of ephrinB2 (identified as the higher molecular weight band in ephrinB2 blots) showed a different pattern

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of phosphorylation, which increased steadily throughout the time-course of EphB4/Fc stimulation and up to the final 240 minutes time-point. The corresponding highly glycosylated forms of ephrinB2/G and ephrinB2/∆V showed a lower increase in phosphorylation throughout the time-course compared to the wild type. It is possible that highly glycosylated forms of ephrinB2 are more resistant to de-phosphorylation and degradation following prolonged periods of EphB4/Fc stimulation. Moreover, the effect of PDZ binding motif mutations on the phosphorylation of the glycosylated form of the ligand seemed less pronounced compared to the non-glycosylated form. Syntenin-1 depletion had no effect.

Densitometry analysis on both phospho and total ephrinB2 levels of three independent WB experiments conducted on each cell line allowed us to perform a quantitative analysis of ephrinB2, ephrinB2/G and ephrinB2/∆V phosphorylation dynamics. Within each cell line, we calculated the ratios of phospho/total levels for each time-point and presented the data in a histogram as fold increase of phosphorylation (figure 3.10C). Thus presented, these results clearly indicate that ephrinB2 phosphorylation upon EphB4/Fc stimulation increased over time and that syntenin-1 depletion did not affect this process. On the other hand, ephrinB2/G and ephrinB2/∆V phosphorylation remained mostly unaltered throughout the time-course of stimulation. At the 240 minute time-point, the difference between ephrinB2 and ephrinB2/G, ephrinB2/ΔV phosphorylation levels becomes statistically significant. This suggests that syntenin-1 is not directly involved in ephrinB2 phosphorylation, which is affected by mutations in the PDZ binding-motif. Yet, our results indicate that a PDZ domain-containing partner/s of ephrinB2 is/are involved in EphB4-induced ligand phosphorylation. Additionally, densitometry analysis revealed that ephrinB2 phosphorylation kept increasing over time up to the last time-point, instead of peaking at 30 minutes and then decreasing as previously

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observed in WB analysis. This discrepancy is probably due to ephrinB2 internalization and degradation, which is triggered in order to terminate signalling.

A)

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C)

Figure 3.10: EphB4/Fc-induced phosphorylation of ephrinB2 is impaired in the presence of /G and /ΔV mutations, but not when syntenin-1 is knocked down.

Phosphorylation dynamics of ephrinB2, ephrinB2/G and ephrinB2/∆V upon EphB4/Fc stimulation in MCF7 Tet- ephrinB2 (panel A, left), MCF7 Tet-ephrinB2/G (panel A, right), MCF7 Tet-ephrinB2/∆V (panel B, left) and MCF7 Tet-ephrinB2/shSyntenin-1 (panel B, right) were analysed by immunoprecipitation followed by WB. Cells were stimulated with 1 µg/ml doxycycline for 48 hours prior to EphB4/Fc stimulation. Cells were then starved in serum free medium for 4 hours and subsequently stimulated with 5 µg/ml EphB4/Fc (pre-clustered for 1 hour with anti-Fc Ab) for 15, 30, 60, 120 and 240 minutes. Cells were lysed in 1% TritonX-100 buffer and lysates were incubated with anti-Flag antibody-conjugated agarose beads. Immunoprecipitated proteins (IP) were resolved on SDS-PAGE and phospho and total levels of ephrinB2 were detected with phospho-specific anti- ephrinB (Tyr325/329) pAb and anti-ephrinB pAb, respectively. C) The histogram shows the quantitative analysis performed on the densitometry data obtained from the blots of three independent experiments for each cell line. The ratio between densitometry values of phospho and total blots was calculated for every time-point and presented in relation to the non-stimulated control (0 minute time-point) as fold increase in ligand phosphorylation. Mean values and standard deviations of fold increase were then calculated and plotted on the histogram. Statistical significance was determined by the two-way ANOVA multiple comparison test with the Tukey-Kramer post-test, using GraphPad Prism software, whereby significance was set at 0.05 (** P<0.01; *** P<0.001). Note: densitometry measurements of phospho bands include both glycosylated and non-glycosylated forms of ephrinB2 proteins.

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Time points of EphB4/Fc stimulation

Phosphorylation levels of ephrinB2 and mutants