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Electron microscopy of axon channels reveals that BoNT/A-Hc is retrogradely transported by autophagosomes. A, Representative axon bundles from unlabeled neurons with autophagosomes (a1 and a2, boxes) and mitochondria (arrowheads) along the microtubule tracks. Scale bar, 500 nm. B, C, Quantification of autophagosomes (B) and mitochondria (C) observed along the axon bundles in the indicated conditions; n = 3 independent microfluidic devices. *p < 0.05; n.s. not significant, Student’s t test. D, Representative electron microscopy of axon bundles of neurons cultured in microfluidic devices with (right) or <t>without</t> <t>(left)</t> <t>BoNT/A-Hc-HRP</t> added to the nerve terminal chambers under high K+ conditions. Autophagosomes are indicated by arrowheads. Scale bar, 500 nm. E, Quantification of BoNT/A-Hc-HRP-containing autophagosome as shown in D; n = 3. **p < 0.01, Student’s t test. F, Representative LC3 and βIII-tubulin immunostaining of hippocampal neurons cultured in microfluidic devices and fixed at indicated times after stimulation. Boxed regions are shown in G; arrowheads indicate nerve terminals. H, Quantification of the level of LC3 fluorescence normalized to βIII-tubulin; n = 6 from 2 independent cultures. ***p < 0.001; **p < 0.01, Student’s t test. I, Representative LC3 and β-actin (loading control) immunoblots. J, Quantification of I; n = 3. *p < 0.05, Student’s t test.
Bont A Hc Hrp, supplied by Cytiva Europe, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Electron microscopy of axon channels reveals that BoNT/A-Hc is retrogradely transported by autophagosomes. A, Representative axon bundles from unlabeled neurons with autophagosomes (a1 and a2, boxes) and mitochondria (arrowheads) along the microtubule tracks. Scale bar, 500 nm. B, C, Quantification of autophagosomes (B) and mitochondria (C) observed along the axon bundles in the indicated conditions; n = 3 independent microfluidic devices. *p < 0.05; n.s. not significant, Student’s t test. D, Representative electron microscopy of axon bundles of neurons cultured in microfluidic devices with (right) or without (left) BoNT/A-Hc-HRP added to the nerve terminal chambers under high K+ conditions. Autophagosomes are indicated by arrowheads. Scale bar, 500 nm. E, Quantification of BoNT/A-Hc-HRP-containing autophagosome as shown in D; n = 3. **p < 0.01, Student’s t test. F, Representative LC3 and βIII-tubulin immunostaining of hippocampal neurons cultured in microfluidic devices and fixed at indicated times after stimulation. Boxed regions are shown in G; arrowheads indicate nerve terminals. H, Quantification of the level of LC3 fluorescence normalized to βIII-tubulin; n = 6 from 2 independent cultures. ***p < 0.001; **p < 0.01, Student’s t test. I, Representative LC3 and β-actin (loading control) immunoblots. J, Quantification of I; n = 3. *p < 0.05, Student’s t test.

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Control of Autophagosome Axonal Retrograde Flux by Presynaptic Activity Unveiled Using Botulinum Neurotoxin Type A

doi: 10.1523/JNEUROSCI.3757-14.2015

Figure Lengend Snippet: Electron microscopy of axon channels reveals that BoNT/A-Hc is retrogradely transported by autophagosomes. A, Representative axon bundles from unlabeled neurons with autophagosomes (a1 and a2, boxes) and mitochondria (arrowheads) along the microtubule tracks. Scale bar, 500 nm. B, C, Quantification of autophagosomes (B) and mitochondria (C) observed along the axon bundles in the indicated conditions; n = 3 independent microfluidic devices. *p < 0.05; n.s. not significant, Student’s t test. D, Representative electron microscopy of axon bundles of neurons cultured in microfluidic devices with (right) or without (left) BoNT/A-Hc-HRP added to the nerve terminal chambers under high K+ conditions. Autophagosomes are indicated by arrowheads. Scale bar, 500 nm. E, Quantification of BoNT/A-Hc-HRP-containing autophagosome as shown in D; n = 3. **p < 0.01, Student’s t test. F, Representative LC3 and βIII-tubulin immunostaining of hippocampal neurons cultured in microfluidic devices and fixed at indicated times after stimulation. Boxed regions are shown in G; arrowheads indicate nerve terminals. H, Quantification of the level of LC3 fluorescence normalized to βIII-tubulin; n = 6 from 2 independent cultures. ***p < 0.001; **p < 0.01, Student’s t test. I, Representative LC3 and β-actin (loading control) immunoblots. J, Quantification of I; n = 3. *p < 0.05, Student’s t test.

Article Snippet: The conjugated BoNT/A-Hc-HRP was purified using a PD MiniTrap G-25 Column (GE Healthcare, catalog #28-9180-08) following the gravity protocol.

Techniques: Electron Microscopy, Cell Culture, Immunostaining, Fluorescence, Control, Western Blot