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Ceramide-Specific Glycosyltransferase

However, the nature of the machinery controlling the trafficking of signaling endosomes containing activated neurotrophin receptors has not yet been completely elucidated

However, the nature of the machinery controlling the trafficking of signaling endosomes containing activated neurotrophin receptors has not yet been completely elucidated. of cell lysates exhibited that all four reagents proved to be effective in downregulating VAMP2. Presentation1.ZIP (1.3M) GUID:?45912579-EE27-456A-881C-7655EAD1CC8E Supplementary Figure 2: Derivation of motor neurons from mouse embryonic stem cells. (A) Circulation diagram describing the protocol utilized BAY 41-2272 for the differentiation of motor neurons from mouse HB9-GFP ES cells. After growth in suspension in differentiating medium for 3 days, the ES cells form embryoid body (EBs) comprising neuronal progenitor cells (NPC). These NPCs are then induced to commit to a motor neuron fate (indicated by GFP expression) by BAY 41-2272 the addition of retinoic acid (RA) and Smoothened Agonist (SAG), a small molecule activator of sonic hedgehog signaling. At the end of the fourth day in the presence of these differentiating brokers, the EBs are dissociated and motor neurons plated. Level bar, 10 m. (B) HB9-GFP ES cell derived motor neurons (GFP, green) 4 days after plating and immunostained for GFP (reddish) and the pan neuronal Rabbit Polyclonal to AurB/C marker, III tubulin (blue). Note the characteristically long motor neuron axon (arrowhead), arising for the soma (asterisk). Level bar, 50 m. Presentation1.ZIP (1.3M) GUID:?45912579-EE27-456A-881C-7655EAD1CC8E Supplementary Figure 3: Kinetics of HCT and BAY 41-2272 p75NTR antibody internalization. (A) HCT accumulation kinetics in motor neurons derived from HB9-GFP ES cells in the presence of p75NTR and the presence (reddish) or absence (black) of EHNA (= 3). Note that HCT accumulation was significantly decreased in the presence of EHNA ( 0.0001, Two-Ways ANOVA). (B) Accumulation kinetics of the p75NTR antibody (p75NTR) when co-incubated with HCT in motor neurons derived from HB9-GFP ES cells in the absence (black) or presence (reddish) of EHNA (= 3). Note that the receptor accumulation profiles generated from EHNA-treated samples diverge from your control plot only after 75 min ( 0.0001, Two-Ways ANOVA). Importantly, in the absence of HCT, the internalization kinetics of the p75NTR antibody (blue) overlapped with the internalization profile observed for this probe when it was co-incubated with HCT and EHNA. Presentation1.ZIP (1.3M) GUID:?45912579-EE27-456A-881C-7655EAD1CC8E Supplementary Physique 4: Assessment of knockdown efficiency of the siRNA screen. Five wells from each plate of the siRNA screen were treated with non-targeting control siRNA and five wells with VAMP2 siRNA. Protein lysates from each set of samples for each plate were pooled and analyzed by western blot to assess VAMP2 knockdown efficiency with III tubulin providing as a loading control. Presentation1.ZIP (1.3M) GUID:?45912579-EE27-456A-881C-7655EAD1CC8E Supplementary Physique 5: Internalization kinetics of HCT and p75NTR antibody in wild type, = 3. Results are shown s.e.m.). (B) p75NTR antibody internalization kinetics were assessed in wild type, = 3. Results are shown s.e.m. Presentation1.ZIP (1.3M) GUID:?45912579-EE27-456A-881C-7655EAD1CC8E DataSheet1.DOCX (121K) GUID:?3FA2205E-D5B4-47C5-A2EA-3135047A7A84 Abstract Neurons rely on the long-range transport of BAY 41-2272 several signaling molecules such as neurotrophins and their receptors, which are required for neuronal development, function and survival. However, the nature of the machinery controlling the trafficking of signaling BAY 41-2272 endosomes made up of activated neurotrophin receptors has not yet been completely elucidated. We aimed to identify new players involved in the dynamics of neurotrophin signaling endosomes using a medium-throughput unbiased siRNA screening approach to quantify the intracellular accumulation of two fluorescently tagged reporters: the binding fragment of tetanus neurotoxin (HCT), and an antibody directed against the neurotrophin receptor p75NTR. This screen performed in motor neurons differentiated from mouse embryonic stem (ES) cells recognized a number of candidate genes encoding molecular motors and motor adaptor proteins involved in regulating the intracellular trafficking of these probes. Bicaudal D homolog 1 (BICD1), a molecular motor adaptor with pleiotropic functions in intracellular trafficking, was selected for further analyses, which revealed that BICD1 regulates the.