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. 2011 Oct 12;31(41):14463–14480. doi: 10.1523/JNEUROSCI.3018-11.2011

Figure 5.

Figure 5.

Netrin-1 does not trigger coassociation of DCC with the SNAREs SNAP25 and VAMP2. A, Confocal images of hippocampal growth cones treated with Netrin-1-conditioned media for 0 and 15 min, immunolabeled for DCC and the SNAREs SNAP25 and VAMP2. Note low colocalization signals of DCC and SNAP25 or VAMP2, both in control conditions and after incubation with Netrin-1. B, C, Quantification of DCC/SNAP25 and DCC/VAMP2 colocalization signals in hippocampal growth cones (expressed as percentage of DCC/SNARE colocalization over total SNARE signals) in cultures treated with Netrin-1-conditioned (black bars) or control-conditioned (white bars) media. Note decreased DCC/SNARE colocalization signals after Netrin-1 treatment. D, Western blots from hippocampal cultures treated with Netrin-1- (N) or control- (C) conditioned media for 0–30 min, and immunoprecipitated with anti-DCC or anti-Sytx1A antibodies. Immunoblots reveal no coimmunoprecipitation of DCC with the SNAREs SNAP25 and VAMP2 after incubation with DCC. Note coimmunoprecipitation of Sytx1A with SNAP25 and VAMP2. E–G, Confocal images of hippocampal growth cones treated with Netrin-1-conditioned media for 0 and 15 min, and in the presence of BoNT/C1. Cultures were immunolabeled for DCC and stained with phalloidin. Note that the mobilization of DCC to the axonal membrane after incubation with Netrin-1 (F) is not altered by BoNT/C1 incubation (G). H, Quantification of DCC signals in the periphery of and inside growth cones treated with Netrin-1-conditioned media for 0, 15, and 30 min, showing mobilization of DCC to the axonal membrane after incubation with Netrin-1. Whereas no DCC mobilization is detected after incubation with BDNF, treatment with BoNT/C1 does not alter DCC mobilization. I–L, Confocal images of control growth cones (I) and cones incubated with Netrin-1-conditioned media for 15 min (J), and with Netrin-1/BoNT/C1 (K). Cultures were immunolabeled for DCC (red) and Sytx1 (green). Note that the increase in DCC/Sytx1 colocalization in J is blocked after incubation with BoNT/C1 (K). L, Histograms illustrating DCC/Sytx1 colocalization in several experimental conditions. Significant differences are labeled by asterisks (*p ≤ 0.05, **p ≤ 0.001). Scale bar: A, 3 μm. Error bars indicate SEM.