goat polyclonal antibody against dner  (R&D Systems)

Journal: Journal of neurochemistry

Article Title: Polarized targeting of DNER into dendritic plasma membrane in hippocampal neurons depends on endocytosis.

doi: 10.1111/j.1471-4159.2010.06714.x

Figure Lengend Snippet: Fig. 1 Somtatodendritic targeting of DNER and its mutants. Primary hippocampal neurons were co-transfected with plasmids containing EGFP and DNER constructs shown as schematic diagrams in (a). Boldface sequences represent the tyrosine-based motif and the atypical dileucine-based motif. The italicized sequences represent putative sorting signals. (b) GFP signals (left panels) reveal the mor- phology of transfected neurons including a long, thin axon (arrow- heads) and several branched, tapering dendrites (arrows). Intracellular (middle panels) and surface (right panels) expression of transfected DNER mutants was detected by a modified sandwich protocol. Insets are enlarged views of axons in boxed regions. On the surface (right panels), the full-length HA-DNER is localized to the somatodendritic compartment (arrows) and is excluded from the axon (arrowheads). DNER/D676-end is evenly distributed to the axon and dendrites. DNER/D722-end is partly distributed to the axon. DNER/Y677A mostly resides in the somatodendritic compartment. Scale bar, 50 lm. (c) The average axon-to-dendrite ratio (ADR) of the full-length and mutant DNER. Values are mean ± SEM. **Significantly different (p < 0.01); *significantly different (p < 0.05); #not significantly different (p > 0.1).

Article Snippet: Other primary and secondary antibodies used for immunofluorescence were as follows; rabbit polyclonal antibody against GFP (Molecular Probes), goat polyclonal antibody against DNER (R&D Systems, Minneapolis, MN, USA), and mouse monoclonal antibody against GFP (Molecular Probes).

Techniques: Transfection, Construct, Expressing, Mutagenesis

Journal: Journal of neurochemistry

Article Title: Polarized targeting of DNER into dendritic plasma membrane in hippocampal neurons depends on endocytosis.

doi: 10.1111/j.1471-4159.2010.06714.x

Figure Lengend Snippet: Fig. 4 Interaction of DNER cytoplasmic region with AP-2 complex. (a) Left; GST-fusion constructs of DNER cytoplasmic domain and its mutants: Right; Summary of GST-pulldown assay results. (b) Mouse brain lysates were incubated with GST fusion proteins shown in (a) and subjected to western blotting using anti a-adaptin antibody (up- per). The expression of each protein was verified by Coomassie blue (CBB) staining (middle). A quantitative comparison of the AP-2 binding activities of the constructs (lower). The ratio of the intensity of the band for GST-pulldown assay to that for CBB staining was calculated, and the percentage of the AP-2-binding by C/FL is presented. The entire experiments were performed three times.

Article Snippet: Other primary and secondary antibodies used for immunofluorescence were as follows; rabbit polyclonal antibody against GFP (Molecular Probes), goat polyclonal antibody against DNER (R&D Systems, Minneapolis, MN, USA), and mouse monoclonal antibody against GFP (Molecular Probes).

Techniques: Construct, GST Pulldown Assay, Incubation, Western Blot, Expressing, Staining, Comparison, Binding Assay

Journal: Journal of neurochemistry

Article Title: Polarized targeting of DNER into dendritic plasma membrane in hippocampal neurons depends on endocytosis.

doi: 10.1111/j.1471-4159.2010.06714.x

Figure Lengend Snippet: Fig. 5 Cell surface expression of DNER is regulated by clathrin-dependent and non- clathrin-dependent endocytosis. (a) Hippo- campal neurons were transfected with indicated DNER mutants with GFP or GFP- ED95/295, and stained for cell surface (left) and intracellular (right) pools of DNER. Scale bar, 5 lm. (b) Hippocampal neurons were transfected with HA-DNER for 24 h and incubated with or without 5 mM MbCD for 30 min before fixation, and stained for cell surface (left) and intracellular (right) pools of DNER. (c) Quantitation of cell surface expression of DNER mutants in the presence or absence of GFP-ED95/295 or MbCD. Values are mean ± SEM. *p < 0.05; **p < 0.01; #not significantly different (p > 0.1).

Article Snippet: Other primary and secondary antibodies used for immunofluorescence were as follows; rabbit polyclonal antibody against GFP (Molecular Probes), goat polyclonal antibody against DNER (R&D Systems, Minneapolis, MN, USA), and mouse monoclonal antibody against GFP (Molecular Probes).

Techniques: Expressing, Transfection, Staining, Incubation, Quantitation Assay

Journal: Journal of neurochemistry

Article Title: Polarized targeting of DNER into dendritic plasma membrane in hippocampal neurons depends on endocytosis.

doi: 10.1111/j.1471-4159.2010.06714.x

Figure Lengend Snippet: Fig. 6 Somatodendritic targeting of DNER requires clathrin-independent endocytosis. (a) Subcellular localization of HA-DNER and its mutants in the presence or absence of GFP-ED95/295 or MbCD. Cells were stained in unpermeabilized conditions. Arrowheads indicate the axon visualized by GFP expression. Insets are magnified views of boxed regions. Scale bar, 50 lm. (b) The average ADR of experiments shown in (a). MbCD markedly increased axonal localization, while GFP-ED95/295 had little or no effect on DNER polarization. Values are mean ± SEM. #Not significantly differ- ent (p > 0.1); *significantly different (p < 0.01).

Article Snippet: Other primary and secondary antibodies used for immunofluorescence were as follows; rabbit polyclonal antibody against GFP (Molecular Probes), goat polyclonal antibody against DNER (R&D Systems, Minneapolis, MN, USA), and mouse monoclonal antibody against GFP (Molecular Probes).

Techniques: Staining, Expressing