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

Figure 4.

Figure 4.

Netrin-1, but not BDNF, triggers DCC mobilization and DCC/Sytx1 colocalization. A, Confocal images of hippocampal axonal shafts treated with Netrin-1-conditioned media for 0, 15, and 30 min, immunolabeled for DCC (red) and Sytx1A (green). Note that DCC and Sytx1A do not colocalize in axonal shafts in either condition. B, Confocal images of hippocampal growth cones treated with Netrin-1-conditioned media for 0–30 min, immunolabeled for DCC and Sytx1. Note increased DCC/Sytx1 colocalization in growth cones incubated with Netrin-1. C, Quantification of DCC/Sytx1 colocalization signals in hippocampal growth cones (expressed as percentage of DCC/Sytx1 colocalization over total Sytx1 signals) in cultures treated with Netrin-1-conditioned (black bars) or control-conditioned (white bars) media. D, Quantification of DCC/Sytx1 coimmunoprecipitation in hippocampal cultures treated with Netrin-1. Sytx1 immunoprecipitation reveals an increase in coassociated DCC in neurons treated with Netrin-1 (black bars). Immunoprecipitation with anti-DCC antibodies shows a marked increase in Sytx1 signals (white bars). E, Quantification of DCC/Sytx1 colocalization signals in hippocampal growth cones (expressed as percentage of DCC/Sytx1 colocalization over total Sytx1 signal) in cultures treated with BDNF (gray bars) or control (white bars). Note that BDNF does not increase colocalization of DCC and Sytx1. F, Western blots from hippocampal cultures treated with BDNF for 15–30 min and immunoprecipitated with anti-DCC or anti-Sytx1A antibodies. Immunoblots reveal that BDNF does not increase the coassociation of DCC with Sytx1A. G, Western blots from hippocampal cultures treated with Netrin-1-conditioned (N) or control-conditioned (C) media for 0–30 min (left), and immunoprecipitated with anti-DCC (top panel) or anti-Sytx1 (bottom panel) antibodies. Immunoblots reveal increased association of DCC and Sytx1 in neuronal cultures treated with Netrin-1. Immunoprecipitations of brain lysates from newborn wild-type, heterozygous and homozygous netrin-1 mutant mice reveal decreased DCC/Sytx1 association in the null mutants (right). Note decreased coimmunoprecipitation of Sytx1 (top) and DCC (middle) in null-mutant brains. H, Quantification of DCC and Sytx1 coimmunoprecipitations in homogenates from newborn wild-type, heterozygous and homozygous netrin-1 mutant mice. Immunoprecipitations with either anti-DCC (white bars) or anti-Sytx1 (black bars) antibodies reveal a marked reduction in DCC/Sytx1 coassociation in netrin-1-null mutants. Significant differences are labeled by asterisks (*p ≤ 0.05; **p ≤ 0.001). Scale bar: A, B, 3 μm. Error bars indicate SEM.