mouse mabs against map2 antibody  (Millipore)

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

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.

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, Comparison, Double Immunofluorescence Staining, Labeling

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.

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: Comparison, Reverse Transcription, Quantitative RT-PCR

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.

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