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. 2018 May 22;39(10):2048–2060. doi: 10.1177/0271678X18777916

Figure 5.

Figure 5.

Annexin A2 (ANXA2) is a Robo4 ligand and modulates Robo4-paxillin-ADP-ribosylation factor 6 (ARF6) signaling. (a) Immunoprecipitation (IP) of isolated human brain microvascular endothelial cell (HBMVEC) membrane with anti-ANXA2 (A2) followed by immunoblotting with anti-Robo4 and anti-paxillin, n = 3 independent experiments. (b) Immunoprecipitation (IP) of isolated HBMVEC membrane with anti-Robo4 followed by immunoblotting with anti-ANXA2 and anti-paxillin under normal and ANXA2 knockdown (A2 KD) conditions, n = 3 independent experiments. (c) Immunoprecipitation (IP) of isolated HBMVEC membrane with anti-Robo4 followed by immunoblotting with anti-paxillin and anti-ANXA2 under hypoxia plus IL-1β insult with/without recombinant ANXA2 (rA2) treatment, n = 3 independent experiments. (d) The effect of control (Con) and ANXA2 short interfering RNA in ARF6 activation assessed by precipitation of ARF6-GTP (guanosine-5′-triphosphate) and quantification of the activated ARF6 levels by western blots. n = 4 independent experiments (*P < 0.05, unpaired Student's t-test). (e) The effect of rA2 in IL-1β-induced ARF6 activation elevation. n = 4 independent experiments (*P < 0.05 vs. control, #P < 0.05 vs. IL-1β group, one-way analysis of variance (ANOVA) followed by Tukey's tests). (f) The effect of rA2 in hypoxia plus IL-1β-induced ARF6 activation elevation. n = 4 independent experiments (*P < 0.05 vs. control, #P < 0.05 vs. hypoxia + IL-1β group, one-way ANOVA followed by Tukey's tests).