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Novus Biologicals map2 mab, mouse antibody
Map2 Mab, Mouse Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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map2 mab, mouse antibody - by Bioz Stars, 2026-09

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Article Title: Synaptic activity bidirectionally regulates a novel sequence-specific S-Q phosphoproteome in neurons
Article Snippet: Antibodies Antibodies were obtained from Novus (Map2 MAb, mouse), Thermo-Scientific (PSD95 MAb, mouse), Santa Cruz biotechnology (B-Tubulin MAb, Mouse), Cell Signaling (pSQ MAb, Rabbit), Millipore (pS1981, Mab), Sigma (ATM MAb, Mouse), and Abcam (ATM MAb, Mouse).

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Journal: Frontiers in Neuroanatomy

Article Title: Analysis of the expression and distribution of protein O-linked mannose β1,2- N -acetylglucosaminyltransferase 1 in the normal adult mouse brain

doi: 10.3389/fnana.2022.1043924

Figure Lengend Snippet: Primary antibodies used in this work.

Article Snippet: MAP2 , Mouse, mAb , Signalway, Shanghai, China , — , — , 1:200.

Techniques: Immunohistochemistry-IF

Cellular localization of POMGNT1. (A–I) Dual immunostaining of POMGNT1 (red) and neuronal glial cell subtype-specific markers (green) in the coronal brain sections of the cerebral cortex, including MAP2 for mature neurons (A) , S100B for resting and activated astrocytes (B) , GFAP for activated astrocytes (C) , MBP for oligodendrocytes (D) , or Iba-1 for microglia (E) , VGLUT1 for Glutamatergic neurons (F) , and GAD65 for GABAergic neurons (G) . POMGNT1 expressed in some neuronal glial cell subtype-specific marker-positive cells, including MAP2, S100B, MBP, Iba-1, GAD65, and VGLUT1, suggesting POMGNT1 may distribute in mature neurons, astrocytes, oligodendrocytes, microglia, glutamatergic neurons, and GABAergic neurons. Nearly no POMGNT1 expressed in the GFAP-positive cells suggesting POMGNT1 may not distribute in the activated astrocytes. N = 6. Scale bars = 20 μM. (H) Quantification of the percentage of the marker labeling cells containing POMGNT1 shown in panels (A–G) . N = 6. Data are presented as the mean ± SEM; ** P < 0.01; *** P < 0.001 versus the MAP2 group (one-way ANOVA); # P < 0.05 versus the VGLUT1 group (Student’s t -test). (I) The protein expression of POMGNT1 in neurons or glial cells using in vitro culturing, including HT22, MA-c, MOPC, and BV2, was detected by Western blot. β-actin as the loading control. (J) The results of the western blot were quantified. N = 4. Data are presented as the mean ± SEM; ** P < 0.01; **** P < 0.0001 versus the HT22 group (one-way ANOVA).

Journal: Frontiers in Neuroanatomy

Article Title: Analysis of the expression and distribution of protein O-linked mannose β1,2- N -acetylglucosaminyltransferase 1 in the normal adult mouse brain

doi: 10.3389/fnana.2022.1043924

Figure Lengend Snippet: Cellular localization of POMGNT1. (A–I) Dual immunostaining of POMGNT1 (red) and neuronal glial cell subtype-specific markers (green) in the coronal brain sections of the cerebral cortex, including MAP2 for mature neurons (A) , S100B for resting and activated astrocytes (B) , GFAP for activated astrocytes (C) , MBP for oligodendrocytes (D) , or Iba-1 for microglia (E) , VGLUT1 for Glutamatergic neurons (F) , and GAD65 for GABAergic neurons (G) . POMGNT1 expressed in some neuronal glial cell subtype-specific marker-positive cells, including MAP2, S100B, MBP, Iba-1, GAD65, and VGLUT1, suggesting POMGNT1 may distribute in mature neurons, astrocytes, oligodendrocytes, microglia, glutamatergic neurons, and GABAergic neurons. Nearly no POMGNT1 expressed in the GFAP-positive cells suggesting POMGNT1 may not distribute in the activated astrocytes. N = 6. Scale bars = 20 μM. (H) Quantification of the percentage of the marker labeling cells containing POMGNT1 shown in panels (A–G) . N = 6. Data are presented as the mean ± SEM; ** P < 0.01; *** P < 0.001 versus the MAP2 group (one-way ANOVA); # P < 0.05 versus the VGLUT1 group (Student’s t -test). (I) The protein expression of POMGNT1 in neurons or glial cells using in vitro culturing, including HT22, MA-c, MOPC, and BV2, was detected by Western blot. β-actin as the loading control. (J) The results of the western blot were quantified. N = 4. Data are presented as the mean ± SEM; ** P < 0.01; **** P < 0.0001 versus the HT22 group (one-way ANOVA).

Article Snippet: MAP2 , Mouse, mAb , Signalway, Shanghai, China , — , — , 1:200.

Techniques: Immunostaining, Marker, Labeling, Expressing, In Vitro, Western Blot, Control

In vivo localization of CBF-A in the adult mouse brain. (A–F) Coronal sections. (A and B) CBF-A is localized in neuronal nuclei and in small clusters in their proximity. (C and D) CBF-A clusters are associated with MAP2-positive dendrites in a synaptic pattern. (E and F) CBF-A clusters match the distribution of the presynaptic marker synapsin I but are not overlapping, suggesting preferential postsynaptic localization of CBF-A. The rectangle is shown in F. Scale bars: A and B = 50 μm, C and D = 20 μm, E = 10 μm, F = 2.5 μm.

Journal: Molecular Biology of the Cell

Article Title: In neurons, activity-dependent association of dendritically transported mRNA transcripts with the transacting factor CBF-A is mediated by A2RE/RTS elements

doi: 10.1091/mbc.E10-11-0904

Figure Lengend Snippet: In vivo localization of CBF-A in the adult mouse brain. (A–F) Coronal sections. (A and B) CBF-A is localized in neuronal nuclei and in small clusters in their proximity. (C and D) CBF-A clusters are associated with MAP2-positive dendrites in a synaptic pattern. (E and F) CBF-A clusters match the distribution of the presynaptic marker synapsin I but are not overlapping, suggesting preferential postsynaptic localization of CBF-A. The rectangle is shown in F. Scale bars: A and B = 50 μm, C and D = 20 μm, E = 10 μm, F = 2.5 μm.

Article Snippet: The mouse mAbs against Map2, NeuN, synapsin I, and PSD95 were, respectively, obtained from Sigma (St. Louis, MO), Chemicon (now Millipore; Billerica, MA), Cell Signaling Technology (Danvers, MA), and Abcam (Cambridge, UK).

Techniques: In Vivo, Marker

Dendritic localization of CBF-A in rat hippocampal neurons. (A) Consistent with the in vivo distribution, CBF-A is found in dendrites of hippocampal neurons, as revealed by coimmunostaining with anti-CBF-A (ICCI) and MAP2 antibodies. Scale bar, 10 μM. Magnifications of boxed areas are approximately fivefold in comparison to the corresponding overviews. (B) Double immunofluorescence staining with the anti–CBF-A antibodies ICCI and SAK22 reveals considerable overlap in nucleus and dendrites of rat hippocampal neurons. Scale bar, 20 μM. (C) Quantification of individual dendritic granules shows a linear correlation between the signals obtained with ICCI and SAK22 antibodies against CBF-A. More than 80% of the individual granules are positively labeled with both CBF-A antibodies.

Journal: Molecular Biology of the Cell

Article Title: In neurons, activity-dependent association of dendritically transported mRNA transcripts with the transacting factor CBF-A is mediated by A2RE/RTS elements

doi: 10.1091/mbc.E10-11-0904

Figure Lengend Snippet: Dendritic localization of CBF-A in rat hippocampal neurons. (A) Consistent with the in vivo distribution, CBF-A is found in dendrites of hippocampal neurons, as revealed by coimmunostaining with anti-CBF-A (ICCI) and MAP2 antibodies. Scale bar, 10 μM. Magnifications of boxed areas are approximately fivefold in comparison to the corresponding overviews. (B) Double immunofluorescence staining with the anti–CBF-A antibodies ICCI and SAK22 reveals considerable overlap in nucleus and dendrites of rat hippocampal neurons. Scale bar, 20 μM. (C) Quantification of individual dendritic granules shows a linear correlation between the signals obtained with ICCI and SAK22 antibodies against CBF-A. More than 80% of the individual granules are positively labeled with both CBF-A antibodies.

Techniques: In Vivo, Comparison, Double Immunofluorescence Staining, Labeling

CBF-A association with RTS-containing Arc, BDNF, and CaMKIIα mRNAs is sensitive to postsynaptic receptor stimulation. CBF-A accumulates in dendrites of hippocampal neurons upon synaptic stimulation with the agonists NMDA or AMPA. (A) Merged images and fivefold magnification of rectangular areas obtained from (A–C) untreated neurons, (D–F) APV-treated neurons, and (G–J) NMDA-treated hippocampal neurons immunostained with antibodies to CBF-A and MAP2. (B and C) Quantification of NMDA and AMPA effects on the distribution of CBF-A. CBF-A intensities measured from randomly selected dendritic areas taken from neurons treated with the indicated reagents (NMDA or APV; AMPA or CNQX) were plotted in bar diagrams with standard deviations. CBF-A levels are specifically and significantly enriched in dendrites upon NMDA treatment in comparison to untreated or AMPA-treated cells (p values were calculated by Student's t test). AU, arbitrary units. (D) NMDA stimulation of hippocampal neurons induces an increased level of Arc, CaMKIIα, and BDNF mRNAs. Total RNA from untreated, NMDA-, or APV-treated neurons was reverse-transcribed with oligo(dT) primers and the cDNA analyzed by qRT-PCR with primers amplifying Arc, CaMKIIα, BDNF, and GAPDH mRNAs. The bar diagram shows relative amounts of the indicated RNAs toward GAPDH mRNA determined over three independent experiments. Error bars depict standard error estimated by Student's t test. (E) RNA coimmunoprecipitated with CBF-A from lysates of neurons treated with NMDA or APV was analyzed by qRT-PCR. Arc, BDNF, and CaMKIIα mRNA levels are specifically and significantly enriched following NMDA stimulation (p values were calculated by Student's t test). Error bars, standard deviations.

Journal: Molecular Biology of the Cell

Article Title: In neurons, activity-dependent association of dendritically transported mRNA transcripts with the transacting factor CBF-A is mediated by A2RE/RTS elements

doi: 10.1091/mbc.E10-11-0904

Figure Lengend Snippet: CBF-A association with RTS-containing Arc, BDNF, and CaMKIIα mRNAs is sensitive to postsynaptic receptor stimulation. CBF-A accumulates in dendrites of hippocampal neurons upon synaptic stimulation with the agonists NMDA or AMPA. (A) Merged images and fivefold magnification of rectangular areas obtained from (A–C) untreated neurons, (D–F) APV-treated neurons, and (G–J) NMDA-treated hippocampal neurons immunostained with antibodies to CBF-A and MAP2. (B and C) Quantification of NMDA and AMPA effects on the distribution of CBF-A. CBF-A intensities measured from randomly selected dendritic areas taken from neurons treated with the indicated reagents (NMDA or APV; AMPA or CNQX) were plotted in bar diagrams with standard deviations. CBF-A levels are specifically and significantly enriched in dendrites upon NMDA treatment in comparison to untreated or AMPA-treated cells (p values were calculated by Student's t test). AU, arbitrary units. (D) NMDA stimulation of hippocampal neurons induces an increased level of Arc, CaMKIIα, and BDNF mRNAs. Total RNA from untreated, NMDA-, or APV-treated neurons was reverse-transcribed with oligo(dT) primers and the cDNA analyzed by qRT-PCR with primers amplifying Arc, CaMKIIα, BDNF, and GAPDH mRNAs. The bar diagram shows relative amounts of the indicated RNAs toward GAPDH mRNA determined over three independent experiments. Error bars depict standard error estimated by Student's t test. (E) RNA coimmunoprecipitated with CBF-A from lysates of neurons treated with NMDA or APV was analyzed by qRT-PCR. Arc, BDNF, and CaMKIIα mRNA levels are specifically and significantly enriched following NMDA stimulation (p values were calculated by Student's t test). Error bars, standard deviations.

Techniques: Comparison, Reverse Transcription, Quantitative RT-PCR

CBF-A silencing impairs dendritic mRNA localization in hippocampal neurons. (A) Rat hippocampal neurons transfected with CBF-A specific or control siRNA oligonucleotides. Silencing is monitored by immunostaining with anti–CBF-A antibodies and with a mAb against MAP2. Arrows point to examples of cells in which CBF-A is specifically knocked down. Scale bar, 10 μm. (B) qRT-PCR was performed on cDNA derived from total RNA isolated from CBF-A–silenced or control hippocampal neurons. Significant down-regulation of relative CBF-A mRNA levels was observed upon CBF-A gene silencing in comparison to untreated or control siRNA treated cells (p values were calculated by Student's t test). Error bars represent standard deviations. (C) In CBF-A–silenced hippocampal neurons, there is a drop in the levels of CaMKIIα mRNA in dendrites as revealed by immuno-FISH with antibodies against CBF-A and an RNA probe hybridizing with CaMKIIα mRNA. Analysis is by confocal microscopy. Scale bars, 10 μm. (D–F) Fivefold magnifications of boxed areas in A–C. (J–L) Fivefold magnifications of boxed areas in G–I. Overall, these experiments were repeated in triplicate. (D) The bar diagrams display signal intensities for both CBF-A and CaMKIIα mRNA obtained in immuno-FISH experiments on untreated, control siRNA-, and CBF-A siRNA–treated hippocampal neurons. In all cases, signal intensities were measured over as many as 20–30 hippocampal neurons.

Journal: Molecular Biology of the Cell

Article Title: In neurons, activity-dependent association of dendritically transported mRNA transcripts with the transacting factor CBF-A is mediated by A2RE/RTS elements

doi: 10.1091/mbc.E10-11-0904

Figure Lengend Snippet: CBF-A silencing impairs dendritic mRNA localization in hippocampal neurons. (A) Rat hippocampal neurons transfected with CBF-A specific or control siRNA oligonucleotides. Silencing is monitored by immunostaining with anti–CBF-A antibodies and with a mAb against MAP2. Arrows point to examples of cells in which CBF-A is specifically knocked down. Scale bar, 10 μm. (B) qRT-PCR was performed on cDNA derived from total RNA isolated from CBF-A–silenced or control hippocampal neurons. Significant down-regulation of relative CBF-A mRNA levels was observed upon CBF-A gene silencing in comparison to untreated or control siRNA treated cells (p values were calculated by Student's t test). Error bars represent standard deviations. (C) In CBF-A–silenced hippocampal neurons, there is a drop in the levels of CaMKIIα mRNA in dendrites as revealed by immuno-FISH with antibodies against CBF-A and an RNA probe hybridizing with CaMKIIα mRNA. Analysis is by confocal microscopy. Scale bars, 10 μm. (D–F) Fivefold magnifications of boxed areas in A–C. (J–L) Fivefold magnifications of boxed areas in G–I. Overall, these experiments were repeated in triplicate. (D) The bar diagrams display signal intensities for both CBF-A and CaMKIIα mRNA obtained in immuno-FISH experiments on untreated, control siRNA-, and CBF-A siRNA–treated hippocampal neurons. In all cases, signal intensities were measured over as many as 20–30 hippocampal neurons.

Techniques: Transfection, Control, Immunostaining, Quantitative RT-PCR, Derivative Assay, Isolation, Comparison, Confocal Microscopy

Fig. 1. The cytoplasmic tail of Caspr2 contains a signal for axonal targeting and somatodendritic internalization. (A) Schematic representation of Caspr2 mutant constructs. The Caspr2 extracellular region contains a discoidin domain, four laminin G and two EGF-like domains and a fibrinogen domain. The Caspr2 cytoplasmic region contains a 4.1B-binding domain and a C-terminal PDZ-binding sequence. A HA epitope was inserted downstream of the signal peptide in Caspr2-HA. The control construct GFP-NrCAMcyt contains the NrCAM signal peptide, the HA epitope, GFP and the transmembrane and cytoplasmic regions of NrCAM. The GFP-Caspr2cyt contains the NrCAM signal peptide, the HA epitope, GFP and the transmembrane and cytoplasmic regions of Caspr2. (B-I) DIV8 hippocampal neurons transfected with Caspr2 (B,C), Caspr2Δcyt (D,E), GFP-NrCAMcyt (F,G) or GFP-Caspr2cyt (H,I). (B,D,F,H) Neurons were surface-labeled for the extracellular epitope HA (red), and stained for the somatodendritic marker MAP2 (green, pseudocolor in F and H). Soma of transfected neurons are indicated with asterisks and axons with white arrows. (J) Fluorescence intensities of axonal and somatodendritic surface labeling for HA were measured and the ratio calculated (A/SD ratio). Values are means ± s.e.m. Caspr2 and GFP-Caspr2cyt are targeted to the axonal surface whereas Caspr2cyt and GFP-NrCAMcyt display uniform axonal and somatodendritic surface labeling. (C,E,G,I) Fixed and permeabilized cells were double-stained for HA (red) and EEA1 (green, pseudocolor in G and I). Double-stained early endosomes are indicated with white arrows. (K) The percentage of early endosomes positive for EEA1 only (green) or double-labeled for the transfected protein (yellow) was measured. Caspr2 and GFP-Caspr2cyt colocalized with the early endosomes marker EEA1 within the somatodendritic compartment, whereas Caspr2cyt and GFP-NrCAMcyt did not. ANOVA indicates a significant difference between means. *P<0.05; ***P<0.001. Scale bars: 15 μm (B,D,F,H) and 7 μm (C,E,G,I).

Journal: Journal of cell science

Article Title: Axonal targeting of Caspr2 in hippocampal neurons via selective somatodendritic endocytosis.

doi: 10.1242/jcs.050526

Figure Lengend Snippet: Fig. 1. The cytoplasmic tail of Caspr2 contains a signal for axonal targeting and somatodendritic internalization. (A) Schematic representation of Caspr2 mutant constructs. The Caspr2 extracellular region contains a discoidin domain, four laminin G and two EGF-like domains and a fibrinogen domain. The Caspr2 cytoplasmic region contains a 4.1B-binding domain and a C-terminal PDZ-binding sequence. A HA epitope was inserted downstream of the signal peptide in Caspr2-HA. The control construct GFP-NrCAMcyt contains the NrCAM signal peptide, the HA epitope, GFP and the transmembrane and cytoplasmic regions of NrCAM. The GFP-Caspr2cyt contains the NrCAM signal peptide, the HA epitope, GFP and the transmembrane and cytoplasmic regions of Caspr2. (B-I) DIV8 hippocampal neurons transfected with Caspr2 (B,C), Caspr2Δcyt (D,E), GFP-NrCAMcyt (F,G) or GFP-Caspr2cyt (H,I). (B,D,F,H) Neurons were surface-labeled for the extracellular epitope HA (red), and stained for the somatodendritic marker MAP2 (green, pseudocolor in F and H). Soma of transfected neurons are indicated with asterisks and axons with white arrows. (J) Fluorescence intensities of axonal and somatodendritic surface labeling for HA were measured and the ratio calculated (A/SD ratio). Values are means ± s.e.m. Caspr2 and GFP-Caspr2cyt are targeted to the axonal surface whereas Caspr2cyt and GFP-NrCAMcyt display uniform axonal and somatodendritic surface labeling. (C,E,G,I) Fixed and permeabilized cells were double-stained for HA (red) and EEA1 (green, pseudocolor in G and I). Double-stained early endosomes are indicated with white arrows. (K) The percentage of early endosomes positive for EEA1 only (green) or double-labeled for the transfected protein (yellow) was measured. Caspr2 and GFP-Caspr2cyt colocalized with the early endosomes marker EEA1 within the somatodendritic compartment, whereas Caspr2cyt and GFP-NrCAMcyt did not. ANOVA indicates a significant difference between means. *P<0.05; ***P<0.001. Scale bars: 15 μm (B,D,F,H) and 7 μm (C,E,G,I).

Article Snippet: Rat anti-HA monoclonal antibody (mAb) was purchased from Roche, mouse anti-MAP2 mAb and rabbit anti-EEA1 antibody from Sigma, and mouse anti-Ankyrin-G mAb from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Construct, Binding Assay, Sequencing, Control, Transfection, Labeling, Staining, Marker, Fluorescence

Fig. 2. Dynamin-dependent endocytosis underlies the removal of Caspr2 from the somatodendritic surface. (A) DIV8 hippocampal neuron transfected with Caspr2. Internalization of Caspr2 was mediated using anti-HA mAb for 1 hour at 37°C. Surface labeling (HA surface, green) was performed using Alexa Fluor 488 anti-rat immunoglobulins at 18°C. Neurons were fixed, permeabilized and the internalized pool of HA-tagged Caspr2 was stained (HA endo, red) using Alexa Fluor 568 anti-rat immunoglobulins. Neurons were stained for MAP2 (blue). The lower panels are enlargements of the boxed areas of upper panels, showing internalization of Caspr2 in axonal and dendritic compartments. (B) Quantification of the number of Caspr2-internalized vesicles (red and not green) per neuron in axonal (A) and somato-dendritic (SD) compartments is shown on the left. Graph on the right shows the ratio of vesicles per neurite length in axons (A) and dendrites (D) for 10 neurons. (C-F) DIV8 hippocampal neurons cotransfected with Caspr2 and wild-type Dynamin-1 (Dynamin-1-WT) (C,E), or mutant Dynamin-1-K44A (D,F). (G,H) DIV8 hippocampal neurons transfected with Caspr2, treated with Dynasore 30 hours after transfection, and immunostained 18 hours after treatment. Neurons were surface-labeled for HA (red) and stained for MAP2 (blue) (C,D,G) or permeabilized and double-stained for HA (red) and EEA1 (blue) (E,F,H). The fluorescence of GFP-tagged Dynamin-1 was detected directly (green). White arrows indicate axons and asterisks indicate the soma of transfected neurons in C,D,G and early endosomes double-labeled for HA-tagged Caspr2 in E,F,H. (I) A/SD ratio; means ± s.e.m. (ANOVA, **P<0.01). Cotransfection of Dynamin-1-WT with Caspr2 did not modify the polarized expression of Caspr2 nor its somatodendritic endocytosis (C,E), whereas in the presence of Dynamin-1-K44A or after treatment with Dynasore, Caspr2 was highly expressed at the somatodendritic surface (D,G) and the number of EEA1-positive vesicles was dramatically reduced (F,H). Scale bars: 10 μm (A), 15 μm (C,D,G) and 7 μm (E,F,H)

Journal: Journal of cell science

Article Title: Axonal targeting of Caspr2 in hippocampal neurons via selective somatodendritic endocytosis.

doi: 10.1242/jcs.050526

Figure Lengend Snippet: Fig. 2. Dynamin-dependent endocytosis underlies the removal of Caspr2 from the somatodendritic surface. (A) DIV8 hippocampal neuron transfected with Caspr2. Internalization of Caspr2 was mediated using anti-HA mAb for 1 hour at 37°C. Surface labeling (HA surface, green) was performed using Alexa Fluor 488 anti-rat immunoglobulins at 18°C. Neurons were fixed, permeabilized and the internalized pool of HA-tagged Caspr2 was stained (HA endo, red) using Alexa Fluor 568 anti-rat immunoglobulins. Neurons were stained for MAP2 (blue). The lower panels are enlargements of the boxed areas of upper panels, showing internalization of Caspr2 in axonal and dendritic compartments. (B) Quantification of the number of Caspr2-internalized vesicles (red and not green) per neuron in axonal (A) and somato-dendritic (SD) compartments is shown on the left. Graph on the right shows the ratio of vesicles per neurite length in axons (A) and dendrites (D) for 10 neurons. (C-F) DIV8 hippocampal neurons cotransfected with Caspr2 and wild-type Dynamin-1 (Dynamin-1-WT) (C,E), or mutant Dynamin-1-K44A (D,F). (G,H) DIV8 hippocampal neurons transfected with Caspr2, treated with Dynasore 30 hours after transfection, and immunostained 18 hours after treatment. Neurons were surface-labeled for HA (red) and stained for MAP2 (blue) (C,D,G) or permeabilized and double-stained for HA (red) and EEA1 (blue) (E,F,H). The fluorescence of GFP-tagged Dynamin-1 was detected directly (green). White arrows indicate axons and asterisks indicate the soma of transfected neurons in C,D,G and early endosomes double-labeled for HA-tagged Caspr2 in E,F,H. (I) A/SD ratio; means ± s.e.m. (ANOVA, **P<0.01). Cotransfection of Dynamin-1-WT with Caspr2 did not modify the polarized expression of Caspr2 nor its somatodendritic endocytosis (C,E), whereas in the presence of Dynamin-1-K44A or after treatment with Dynasore, Caspr2 was highly expressed at the somatodendritic surface (D,G) and the number of EEA1-positive vesicles was dramatically reduced (F,H). Scale bars: 10 μm (A), 15 μm (C,D,G) and 7 μm (E,F,H)

Techniques: Transfection, Labeling, Staining, Mutagenesis, Fluorescence, Cotransfection, Expressing

Fig. 3. Vesicular transport and insertion of Caspr2 at the plasma membrane in hippocampal neurons. Caspr2-GFP construct (top): GFP was inserted at the N-terminus of Caspr2 downstream of the signal peptide. Neurons were transfected at DIV9 with Caspr2-GFP and live imaging recorded at DIV10. Frames were collected at the rate of 1 per second for 120 seconds and the trajectories of Caspr2-GFP vesicles analyzed using the Metamorph tracking software. Representative frames in axons (A) or dendrites (B) corresponding to supplementary material Movies 1 and 4, respectively. Examples of anterograde (white) or retrograde (R, blue) vesicle tracking, with the mean velocity indicated. (C) Mean velocity of Caspr2-GFP vesicles, the number of tracked vesicles is indicated. (D) Percentage of anterograde and retrograde moving vesicles in axons and distal dendrites. (E-G) Cell-surface distribution of Caspr2 after Brefeldin A removal. DIV7 neurons were transfected with Caspr2 and treated 4 hours after transfection with 0.75 μg/ml Brefeldin A for 16 hours. (E) Upon Brefeldin A removal, neurons were fixed, permeabilized and double-stained for HA (red) and MAP2 (green). After 1 hour (F) and 2 hours (G) of Brefeldin A recovery, neurons were surface-labeled for HA (red) and double-stained for MAP2 (green). White arrows indicate axons. Note the uniform surface staining for Caspr2 of the axonal and somatodendritic compartments after 1 hour of recovery (F). 20% of cells exhibit a polarized expression of Caspr2 after 2 hours of recovery (G). Scale bars: 10 μm (A,B) and 15 μm (E-G).

Journal: Journal of cell science

Article Title: Axonal targeting of Caspr2 in hippocampal neurons via selective somatodendritic endocytosis.

doi: 10.1242/jcs.050526

Figure Lengend Snippet: Fig. 3. Vesicular transport and insertion of Caspr2 at the plasma membrane in hippocampal neurons. Caspr2-GFP construct (top): GFP was inserted at the N-terminus of Caspr2 downstream of the signal peptide. Neurons were transfected at DIV9 with Caspr2-GFP and live imaging recorded at DIV10. Frames were collected at the rate of 1 per second for 120 seconds and the trajectories of Caspr2-GFP vesicles analyzed using the Metamorph tracking software. Representative frames in axons (A) or dendrites (B) corresponding to supplementary material Movies 1 and 4, respectively. Examples of anterograde (white) or retrograde (R, blue) vesicle tracking, with the mean velocity indicated. (C) Mean velocity of Caspr2-GFP vesicles, the number of tracked vesicles is indicated. (D) Percentage of anterograde and retrograde moving vesicles in axons and distal dendrites. (E-G) Cell-surface distribution of Caspr2 after Brefeldin A removal. DIV7 neurons were transfected with Caspr2 and treated 4 hours after transfection with 0.75 μg/ml Brefeldin A for 16 hours. (E) Upon Brefeldin A removal, neurons were fixed, permeabilized and double-stained for HA (red) and MAP2 (green). After 1 hour (F) and 2 hours (G) of Brefeldin A recovery, neurons were surface-labeled for HA (red) and double-stained for MAP2 (green). White arrows indicate axons. Note the uniform surface staining for Caspr2 of the axonal and somatodendritic compartments after 1 hour of recovery (F). 20% of cells exhibit a polarized expression of Caspr2 after 2 hours of recovery (G). Scale bars: 10 μm (A,B) and 15 μm (E-G).

Techniques: Clinical Proteomics, Membrane, Construct, Transfection, Imaging, Software, Staining, Labeling, Expressing

Fig. 4. The 4.1-binding domain contains a determinant implicated in the somatodendritic internalization of Caspr2. (A) Mutations and deletions in the 4.1- binding domain of Caspr2. The 4.1- binding domain is indicated in violet and the C-terminal PDZ-binding sequence in orange. (B-K) DIV8 hippocampal neurons transfected with Caspr2Δ4.1 (B,C), Caspr2-Y1293F (D,E), Caspr2-H1294A (F,G), Caspr2Δ1293-1305 (H,I) or Caspr21284-1292 (J,K). (B,D,F,H,J) Neurons were surface-labeled for HA (red), and stained for MAP2 (green). Soma of transfected neurons are indicated with asterisks and axons with white arrows. (L) A/SD ratios; means ± s.e.m. **P<0.01, compared with control Caspr2 (ANOVA). (C,E,G,I,K) Cells permeabilized and double-stained for HA (red) and EEA1 (green). Double-stained early endosomes are indicated with white arrows. The percentage of early endosomes positive for EEA1 only (green) or double-labeled for the transfected protein (yellow) was determined (M). ***P<0.001 compared with control Caspr2 (ANOVA). Deletion of the 4.1- binding domain (B,C) or of the N- terminus of this domain (residues 1284- 1292) (J,K) prevented the somatodendritic elimination of Caspr2. The point mutations Y1293F and H1294A had no effect (D,E,F,G). Deletion of residues 1293-1305 did not modify the axonal targeting (H) or somatodendritic endocytosis (I) of Caspr2. Scale bars: 15 μm (B,D,F,H,J) and 7 μm (C,E,G,I,K).

Journal: Journal of cell science

Article Title: Axonal targeting of Caspr2 in hippocampal neurons via selective somatodendritic endocytosis.

doi: 10.1242/jcs.050526

Figure Lengend Snippet: Fig. 4. The 4.1-binding domain contains a determinant implicated in the somatodendritic internalization of Caspr2. (A) Mutations and deletions in the 4.1- binding domain of Caspr2. The 4.1- binding domain is indicated in violet and the C-terminal PDZ-binding sequence in orange. (B-K) DIV8 hippocampal neurons transfected with Caspr2Δ4.1 (B,C), Caspr2-Y1293F (D,E), Caspr2-H1294A (F,G), Caspr2Δ1293-1305 (H,I) or Caspr21284-1292 (J,K). (B,D,F,H,J) Neurons were surface-labeled for HA (red), and stained for MAP2 (green). Soma of transfected neurons are indicated with asterisks and axons with white arrows. (L) A/SD ratios; means ± s.e.m. **P<0.01, compared with control Caspr2 (ANOVA). (C,E,G,I,K) Cells permeabilized and double-stained for HA (red) and EEA1 (green). Double-stained early endosomes are indicated with white arrows. The percentage of early endosomes positive for EEA1 only (green) or double-labeled for the transfected protein (yellow) was determined (M). ***P<0.001 compared with control Caspr2 (ANOVA). Deletion of the 4.1- binding domain (B,C) or of the N- terminus of this domain (residues 1284- 1292) (J,K) prevented the somatodendritic elimination of Caspr2. The point mutations Y1293F and H1294A had no effect (D,E,F,G). Deletion of residues 1293-1305 did not modify the axonal targeting (H) or somatodendritic endocytosis (I) of Caspr2. Scale bars: 15 μm (B,D,F,H,J) and 7 μm (C,E,G,I,K).

Techniques: Binding Assay, Sequencing, Transfection, Labeling, Staining, Control

Fig. 5. The somatodendritic internalization of Caspr2 requires PKC phosphorylation. (A) The Caspr2 sequence implicated in endocytosis contains a putative substrate motif KGT for the phosphorylation of T1292 by PKC. Calphostin C is an inhibitor for PKC activity. (B-E) DIV8 hippocampal neurons transfected with Caspr2 (B-D) or the Nav1.2-C-terminus 1871 fused with the CD4 reporter (NavCter) (E). Neurons were treated with Calphostin C (B,E), H89 (C), or DMSO (D), surface-labeled for HA (red), and stained for MAP2 (green). Soma of transfected neurons are indicated with asterisks and axons with white arrows. Control DMSO treatment had no effect on the polarized expression of Caspr2 (D). PKA inhibition by H89 did not modify the axonal targeting of the protein (C). Calphostin C (Calph) induced the nonpolarized expression of Caspr2 in the somatodendritic and axonal compartments (B), whereas it had no effect on the polarized expression of NavCter (E). (F,G) DIV8 hippocampal neurons transfected with Caspr2 were untreated (F) or treated with Calphostin C (G) and incubated for 1 hour at 37°C with Alexa-Fluor-594-conjugated transferrin (red) and anti-HA mAb to mediate internalization of Caspr2. Neurons were surface-labeled for HA (green) using Alexa Fluor 488 anti-rat immunoglobulins at 18°C. Cells were fixed, permeabilized and the internalized pool of HA-tagged Caspr2 was stained (blue). Calphostin C strongly inhibited the internalization of Caspr2 but had no effect on transferrin uptake. Scale bars: 15 μm (B-E) and 5 μm (F,G). (H) GST and the cytoplasmic region of Caspr2 fused with GST were subjected to in vitro PKC phosphorylation followed by SDS-PAGE. Coomassie blue staining (left) and 32P-incorporation revealed by autoradiography (right) are shown.

Journal: Journal of cell science

Article Title: Axonal targeting of Caspr2 in hippocampal neurons via selective somatodendritic endocytosis.

doi: 10.1242/jcs.050526

Figure Lengend Snippet: Fig. 5. The somatodendritic internalization of Caspr2 requires PKC phosphorylation. (A) The Caspr2 sequence implicated in endocytosis contains a putative substrate motif KGT for the phosphorylation of T1292 by PKC. Calphostin C is an inhibitor for PKC activity. (B-E) DIV8 hippocampal neurons transfected with Caspr2 (B-D) or the Nav1.2-C-terminus 1871 fused with the CD4 reporter (NavCter) (E). Neurons were treated with Calphostin C (B,E), H89 (C), or DMSO (D), surface-labeled for HA (red), and stained for MAP2 (green). Soma of transfected neurons are indicated with asterisks and axons with white arrows. Control DMSO treatment had no effect on the polarized expression of Caspr2 (D). PKA inhibition by H89 did not modify the axonal targeting of the protein (C). Calphostin C (Calph) induced the nonpolarized expression of Caspr2 in the somatodendritic and axonal compartments (B), whereas it had no effect on the polarized expression of NavCter (E). (F,G) DIV8 hippocampal neurons transfected with Caspr2 were untreated (F) or treated with Calphostin C (G) and incubated for 1 hour at 37°C with Alexa-Fluor-594-conjugated transferrin (red) and anti-HA mAb to mediate internalization of Caspr2. Neurons were surface-labeled for HA (green) using Alexa Fluor 488 anti-rat immunoglobulins at 18°C. Cells were fixed, permeabilized and the internalized pool of HA-tagged Caspr2 was stained (blue). Calphostin C strongly inhibited the internalization of Caspr2 but had no effect on transferrin uptake. Scale bars: 15 μm (B-E) and 5 μm (F,G). (H) GST and the cytoplasmic region of Caspr2 fused with GST were subjected to in vitro PKC phosphorylation followed by SDS-PAGE. Coomassie blue staining (left) and 32P-incorporation revealed by autoradiography (right) are shown.

Techniques: Phospho-proteomics, Sequencing, Activity Assay, Transfection, Labeling, Staining, Control, Expressing, Inhibition, Incubation, In Vitro, SDS Page, Autoradiography

Fig. 6. The point mutation T1292A prevents the somatodendritic elimination of Caspr2. (A,C,D) DIV8 hippocampal neurons transfected with Caspr2-T1292A (A,D) or Caspr2 (C). (A) Neurons were surface-labeled for HA (red), and stained for MAP2 (green). (B) A/SD ratios. ANOVA indicates a significant difference between means (**P<0.01). (C,D) Neurons were incubated for 1 hour at 37°C with anti-HA mAb to mediate internalization of Caspr2. They were then surface-labeled for HA (green, surface), fixed, permeabilized, and the internalized pool of HA-tagged Caspr2 was stained (red, endo). C and D are enlarged images of the surface and internal overlays from C and D. (E) Quantification of the number of internalized vesicles per neuron for Caspr2, Caspr2cyt, Caspr21284-1292 and Caspr2T1292A. The point mutation T1292A prevented the somatodendritic elimination of Caspr2. Scale bars: 15 μm (A,C,D) and 6 μm (C,D).

Journal: Journal of cell science

Article Title: Axonal targeting of Caspr2 in hippocampal neurons via selective somatodendritic endocytosis.

doi: 10.1242/jcs.050526

Figure Lengend Snippet: Fig. 6. The point mutation T1292A prevents the somatodendritic elimination of Caspr2. (A,C,D) DIV8 hippocampal neurons transfected with Caspr2-T1292A (A,D) or Caspr2 (C). (A) Neurons were surface-labeled for HA (red), and stained for MAP2 (green). (B) A/SD ratios. ANOVA indicates a significant difference between means (**P<0.01). (C,D) Neurons were incubated for 1 hour at 37°C with anti-HA mAb to mediate internalization of Caspr2. They were then surface-labeled for HA (green, surface), fixed, permeabilized, and the internalized pool of HA-tagged Caspr2 was stained (red, endo). C and D are enlarged images of the surface and internal overlays from C and D. (E) Quantification of the number of internalized vesicles per neuron for Caspr2, Caspr2cyt, Caspr21284-1292 and Caspr2T1292A. The point mutation T1292A prevented the somatodendritic elimination of Caspr2. Scale bars: 15 μm (A,C,D) and 6 μm (C,D).

Techniques: Mutagenesis, Transfection, Labeling, Staining, Incubation

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