Consistent with this model, treatment of axons with the dynein inhibitor EHNA prevented the

reduction of axonal SMAD1/5/8 after protein synthesis inhibition (Figures 5A and S4E). Taken together, these data suggest LGK-974 concentration that SMAD1/5/8 is transported retrogradely from distal axons in a motor-dependent manner. To track the fate of axonally synthesized SMAD1/5/8, we used L-azidohomoalanine (AHA), a methionine analog that can be biotinylated using “click chemistry” (Kiick et al., 2002). E13.5 trigeminal ganglia neurons were cultured in microfluidic chambers, and AHA was added to the axonal compartment. AHA was allowed to incorporate into locally synthesized proteins, and the axonally synthesized, retrogradely trafficked proteins were collected by preparing lysates from the cell body compartment. pSMAD1/5/8 was immunoprecipitated and the presence of axonally derived AHA-labeled pSMAD1/5/8 was detected by anti-biotin western blotting. Biotinylated pSMAD1/5/8 was observed in cell bodies with axons treated with BMP4 after immunoprecipitation and click here click reaction (Figure 5B). This effect was blocked by including

anisomycin in the axon, demonstrating that the biotinylated pSMAD1/5/8 was synthesized in axons (Figure 5B). Together, these experiments show that endogenous, axonally derived SMAD1/5/8 is translocated to the cell body in its transcriptionally active phosphorylated form. To further examine the retrograde trafficking of axonal SMAD, we imaged Dendra2-SMAD1 in axons. Dendra2 or Dendra2-SMAD1 was photoconverted to the

red fluorescent form in the axon, and the distribution of the red signal was monitored over 50 s (Figure 5C). Red fluorescent Dendra2-SMAD1 preferentially localized to the proximal side of the photoconverted segment, consistent with the transport of SMAD protein in a retrograde manner (Figures S5B and S5C). The retrogradely oxyclozanide transported Dendra2-SMAD1 accumulates in the nucleus as we detected a significant increase of red signal in the nucleus after photoconverting Dendra2-SMAD1 in axon (Figure 5D). Collectively, these experiments suggest that axonal SMAD can be retrogradely trafficked back from the axon to the soma and accumulates in the nucleus. BMP4 receptors typically bind to SMADs through adaptor proteins (Moustakas and Heldin, 2009 and Shi et al., 2007). To determine if axonal SMAD associates with BMP4 receptors, we examined the localization of axonal SMAD1/5/8 with respect to signaling endosomes labeled with biotinylated BMP4. Following application of biotinylated BMP4 to the axonal compartment, biotinylated BMP4 exhibited significant colocalization with both axonal pSMAD1/5/8 and SMAD1/5/8 (Figure S5D), compared with biotinylated BSA. These data suggest that SMAD1/5/8 associates with BMP4 receptor complexes in axons. We next asked whether axonal SMAD is required for retrograde BMP4 signaling.