Journal: PLoS ONE

Article Title: Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

doi: 10.1371/journal.pone.0075760

Figure Lengend Snippet: (A) CGNs immunostained for Sig-1R (red) and TrkB (green). Scale Bar: 10 µm. (B and C) CGNs were cultured for 24 h with or without PRE-084. As for cultures incubated with K252a, a pan-tropomyosin receptor kinase (trk) K252a was added at the same time as PRE was added to the culture. Representative images of CGNs are shown in (B). The neurite lengths were quantified from three independent experiments. The mean lengths of neurite are shown in the graph (C). Scale bar: 20 µm. (D) The same effects were observed when the cells were cultured in the presence of BDNF; n = 3, ** P <0.01, Scheffe’s test.

Article Snippet: For immunoprecipitations, western blots, and immunostainings, the following primary and secondary antibodies were used: goat anti-human Sigma-1 receptor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Goat TrueBlot: horseradish peroxidase (HRP)-conjugated anti-goat immunoglobulin G (IgG) (eBioscience, San Diego, CA, USA), rabbit neuronal class III ß-tubulin polyclonal antibody (Tuj1; Covance Laboratories, Emeryville, CA, USA), donkey anti-goat Alexa 568-conjugated IgG (Invitrogen, Carlsbad, CA, USA), goat anti-rabbit Alexa 488-conjugated IgG (Invitrogen), biotinylated anti-mouse TrkB antibody (R&D Systems, Minneapolis, MN, USA), streptavidin-HRP-conjugated IgG (Roche Diagnostic, Basel, Switzerland), phospho-TrkA (Tyr 674/675)/TrkB (Tyr 706/707) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), phospho-TrkA (Tyr 490)/TrkB (Tyr 516; Cell Signaling Technology) which detects phosphorylation of mouse Tyr 515, HRP-conjugated anti-rabbit IgG (Cell Signaling Technology), HRP-conjugated anti-mouse IgG (Cell Signaling Technology), c-Myc (9E10) (Santa Cruz), and rabbit polyclonal anti-HA tag (Abcam, Cambridge, MA, USA).

Techniques: Cell Culture, Incubation

Journal: PLoS ONE

Article Title: Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

doi: 10.1371/journal.pone.0075760

Figure Lengend Snippet: (A and B) Co-immunoprecipitation of Myc-tagged full-length sigma-1 receptor (Sig-1R-Myc) with HA-tagged full-length TrkB (HA-TrkB). HEK 293T cells were transiently transfected with the indicated plasmids and treated with or without PRE-084 (10 µM) for 1 h. Cell lysates were immunoprecipitated with the anti-Myc antibody (A) or anti-HA antibody (B). The immunoprecipitates (IP) and cell lysates (Lysates) were analyzed by immunoblotting with anti-HA and anti-Myc antibodies. (C) Association of endogenous Sig-1R with TrkB in the CGNs. The CGNs were treated with or without PRE-084 (20 µM) for 1 h. The lysates prepared from the CGNs were immunoprecipitated with anti-Sig-1R antibody followed by immunoblotting with anti-TrkB and anti-Sig-1R antibodies, respectively. The association between Sig-1R and TrkB was slightly enhanced in the PRE-084-treated cells. Control: PBS.

Article Snippet: For immunoprecipitations, western blots, and immunostainings, the following primary and secondary antibodies were used: goat anti-human Sigma-1 receptor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Goat TrueBlot: horseradish peroxidase (HRP)-conjugated anti-goat immunoglobulin G (IgG) (eBioscience, San Diego, CA, USA), rabbit neuronal class III ß-tubulin polyclonal antibody (Tuj1; Covance Laboratories, Emeryville, CA, USA), donkey anti-goat Alexa 568-conjugated IgG (Invitrogen, Carlsbad, CA, USA), goat anti-rabbit Alexa 488-conjugated IgG (Invitrogen), biotinylated anti-mouse TrkB antibody (R&D Systems, Minneapolis, MN, USA), streptavidin-HRP-conjugated IgG (Roche Diagnostic, Basel, Switzerland), phospho-TrkA (Tyr 674/675)/TrkB (Tyr 706/707) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), phospho-TrkA (Tyr 490)/TrkB (Tyr 516; Cell Signaling Technology) which detects phosphorylation of mouse Tyr 515, HRP-conjugated anti-rabbit IgG (Cell Signaling Technology), HRP-conjugated anti-mouse IgG (Cell Signaling Technology), c-Myc (9E10) (Santa Cruz), and rabbit polyclonal anti-HA tag (Abcam, Cambridge, MA, USA).

Techniques: Immunoprecipitation, Transfection, Western Blot, Control

Journal: PLoS ONE

Article Title: Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

doi: 10.1371/journal.pone.0075760

Figure Lengend Snippet: (A and B) The panels show representative western blots for detection of phosphorylation of Y515 (A) or Y706 (B) of TrkB and total TrkB. The graphs demonstrate relative phosphorylation level of Y515 (A) or Y706 (B) of TrkB. The Sig-1R activation by PRE-084 significantly promoted phosphorylation of Y515 of TrkB by 30%, whereas the phosphorylation level at Y706 significantly decreased by 10%. Without BDNF, PRE-084 alone did not increase TrkB phosphorylation. ND: not determined, (A) n = 5, (B) n = 3, ** P <0.01, Scheffe’s test. (C) Dominant negative knockdown of endogenous TrkB attenuated the effects of PRE-084 on neurite growth. The neurons transfected with control vector, or TrkB-Y515F were cultured for 24 h in the presence or absence of PRE-084. n = 3, ** P <0.01, Scheffe’s test.

Article Snippet: For immunoprecipitations, western blots, and immunostainings, the following primary and secondary antibodies were used: goat anti-human Sigma-1 receptor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Goat TrueBlot: horseradish peroxidase (HRP)-conjugated anti-goat immunoglobulin G (IgG) (eBioscience, San Diego, CA, USA), rabbit neuronal class III ß-tubulin polyclonal antibody (Tuj1; Covance Laboratories, Emeryville, CA, USA), donkey anti-goat Alexa 568-conjugated IgG (Invitrogen, Carlsbad, CA, USA), goat anti-rabbit Alexa 488-conjugated IgG (Invitrogen), biotinylated anti-mouse TrkB antibody (R&D Systems, Minneapolis, MN, USA), streptavidin-HRP-conjugated IgG (Roche Diagnostic, Basel, Switzerland), phospho-TrkA (Tyr 674/675)/TrkB (Tyr 706/707) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), phospho-TrkA (Tyr 490)/TrkB (Tyr 516; Cell Signaling Technology) which detects phosphorylation of mouse Tyr 515, HRP-conjugated anti-rabbit IgG (Cell Signaling Technology), HRP-conjugated anti-mouse IgG (Cell Signaling Technology), c-Myc (9E10) (Santa Cruz), and rabbit polyclonal anti-HA tag (Abcam, Cambridge, MA, USA).

Techniques: Western Blot, Phospho-proteomics, Activation Assay, Dominant Negative Mutation, Knockdown, Transfection, Control, Plasmid Preparation, Cell Culture

Journal: Nature Communications

Article Title: Deep visual proteomics uncovers nociceptor diversity and pain targets

doi: 10.1038/s41467-026-71418-8

Figure Lengend Snippet: a Schematic workflow showing patch clamp electrophysiology and ultra-low input proteomics of cultured sensory neurons. b IB4 labeling in magenta (scale = 20 µm). Independently repeated in at least three cultures from three different mice with similar results. c Representative traces of indentation-evoked, mechanically-activated currents in an IB4 − peptidergic nociceptor and the absence of such currents in an IB4 + non-peptidergic nociceptor. d Percentage of mechanically sensitive IB4 − and IB4 + neurons. The number of neurons is indicated in the bars. ** represents p = 0.0027, two-sided chi-sq test. e Representative action potential (AP) traces recorded from an IB4 − and an IB4 + nociceptor, with corresponding quantification of AP threshold, half peak duration, and afterhyperpolarization of the measured APs. IB4 − : n = 19, IB4 + : n = 13. Each dot represents a single neuron. Filled dots in the IB4 + group represent the two mechanosensitive non-peptidergic neurons. Data were obtained from at least three biological replicates, both male and female mice. * represents p = 0.04, unpaired two-tailed Student’s t test. f Representative images of sensory neurons cultured and stained on laser microdissection (LMD)-compatible membrane slides prior to LMD and mass spectrometry analysis (scale = 20 µm). g Numbers of precursors and proteins identified from peptidergic (IB4 − /TrkA + ) and non-peptidergic (IB4 + /TrkA − ) nociceptors and mechanoreceptors. N = 3. Each dot represents a biological replicate from a male mouse. h Principal component analysis of peptidergic and non-peptidergic nociceptors and mechanoreceptors. i Dynamic range of protein abundance in the two nociceptor subsets. j Volcano plot showing pairwise proteomic comparison between nociceptor subsets with marker proteins highlighted (two-sided Student’s t test). k Pathway enrichment analysis based on t test differences between nociceptor subsets. The top ten pathways with a Benjamini-Hochberg false discovery rate (FDR) < 0.05 are shown. Cyan = peptidergic, magenta = non-peptidergic, black = mechanoreceptor. Error bars in all panels represent s.e.m with the mean as the center. Source Data are provided as a Source Data file. Parts of Fig. 1a, f created in BioRender. Coscia, F. (2026) https://BioRender.com/a39426f .

Article Snippet: Before staining, slides were blocked in antibody diluent solution: 0.2% (v/v) Triton X-100, 5% (v/v) donkey serum and 1% (v/v) bovine serum albumin in PBS for 1 h at room temperature before overnight incubation at 4 °C with 1:500 polyclonal anti-goat TrkA (R&D systems, AF1056) primary antibody.

Techniques: Patch Clamp, Cell Culture, Labeling, Two Tailed Test, Staining, Laser Capture Microdissection, Membrane, Mass Spectrometry, Quantitative Proteomics, Comparison, Marker

Journal: Nature Communications

Article Title: Deep visual proteomics uncovers nociceptor diversity and pain targets

doi: 10.1038/s41467-026-71418-8

Figure Lengend Snippet: a Schematic workflow of sensory neurons cultured in an “inflammatory soup” containing nerve growth factor, NGF, and phorbol 12-myristate 13-acetate, PMA (top), along with representative indentation-evoked currents from a control (cyan) and an inflamed (red) peptidergic neuron. b Percentage of mechanically sensitive control and inflamed peptidergic nociceptors (left). * represents p = 0.027, two-sided chi-sq test. The number of neurons is indicated in the bars, along with their corresponding current amplitude (right). c Percentage of control and inflamed peptidergic nociceptors with residual current. * represents p = 0.045, two-sided chi-sq test. d Percentage of mechanically sensitive control (magenta) and inflamed (red) non-peptidergic nociceptors. The number of neurons is indicated in the bars. Volcano plots showing the pairwise proteomic comparison between control and inflamed neurons in non-peptidergic ( e ) and peptidergic ( f ) nociceptors with top regulated proteins highlighted (two-sided Student’s t test). g Bar plots showing raw protein levels of B3GNT2 in the control and inflamed groups of peptidergic (TrkA + , n = 3) and non-peptidergic (IB4 + , n = 3) nociceptors. Each dot represents a single mouse. h Schematic workflow of the validation experiment by knockdown of B3gnt2 using siRNA prior to inflammation and patch-clamp electrophysiology of peptidergic neurons. i Percentage of mechanically sensitive inflamed peptidergic nociceptors transfected with control siRNA (red) and B3gnt2-targeting siRNA (gray). The number of neurons is indicated in the bars. *** represents p = 0.0003, two-sided chi-sq test. j Current amplitudes (left) and percentage of neurons with residual currents in inflamed peptidergic nociceptors transfected with control siRNA (red, n = 26) and B3gnt2-targeting siRNA (gray, n = 11). Error bars in all panels represent s.e.m with the mean as the center. Source Data are provided as a Source Data file. Parts of Fig. 4a were created in BioRender. Coscia, F. (2026) https://BioRender.com/a39426f .

Article Snippet: Before staining, slides were blocked in antibody diluent solution: 0.2% (v/v) Triton X-100, 5% (v/v) donkey serum and 1% (v/v) bovine serum albumin in PBS for 1 h at room temperature before overnight incubation at 4 °C with 1:500 polyclonal anti-goat TrkA (R&D systems, AF1056) primary antibody.

Techniques: Cell Culture, Control, Comparison, Biomarker Discovery, Knockdown, Patch Clamp, Transfection

Journal: The EMBO Journal

Article Title: The TrkC-PTPσ complex governs synapse maturation and anxiogenic avoidance via synaptic protein phosphorylation

doi: 10.1038/s44318-024-00252-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Samples were incubated overnight at 4 °C with a combination of the following primary antibodies/stain: anti-NeuN-Alexa 647 conjugated (1:1000, monoclonal rabbit, ab190565, Abcam), anti-NF200 (1:1000, polyclonal rabbit, N4142, MiliporeSigma), anti-TrkB (1:500, polyclonal goat, AF1494, R&D Systems), anti-TrkC (1:500, polyclonal goat, AF1404, R&D Systems), anti-CGRP (1:1000, polyclonal goat, ab36001, Abcam), anti-VGLUT1 (1:1000, polyclonal guinea pig, AB5905, Sigma-Aldrich) and IsolectinB4 stain-Alexa 647 conjugated (1:1000, I32450, Life Technologies).

Techniques: Knock-In, Recombinant, Plasmid Preparation, Clone Assay, Staining, Sequencing, DC Protein Assay, Membrane, Western Blot, Electron Microscopy, Mass Spectrometry, Phospho-proteomics, Software, Fluorescence, Microscopy, Incubation

Journal: The Journal of biological chemistry

Article Title: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene.

doi: 10.1074/jbc.M609857200

Figure Lengend Snippet: FIGURE 1. A, relative transcript levels of the receptor tyrosine kinases TrkA, TrkB, and TrkC, and the low affinity neurotrophin receptor p75NTR in neuro- blastoma-derived Kelly cells. The cells were grown for 16 h either at 1 or 21% O2. The hypoxia mimetic DP (100 M) was added to the cultures at 21% O2 for 16 h. Messenger RNA levels were determined by a quantitative real time reverse transcriptase-PCR method and normalized to -actin transcripts. The cellular mRNA content at 21% O2 was set to 1. Values are mean S.D. of n 4 experiments, each performed in triplicate (*, p 0.01 versus the respective controls at 21% O2 in the absence of DP, Student’s paired t test). B, relative TrkB mRNA content in different cell lines grown either at 21% O2 in the pres- ence or absence of DP (100 M), or maintained in hypoxia (1% O2) for 16 h. Messenger RNA levels were quantified by real time reverse transcriptase-PCR and normalized to -actin transcripts. Note, that TrkB expression was stimu- latedsignificantlyinhypoxiaandinthepresenceofDP(21%O2)inallcelllines including HEK293 cells and U2OS osteosarcoma cells. Values are mean S.D. of n 5 experiments, each performed in triplicate (*, p 0.01 and #, p 0.05 versus the respective controls at 21% O2 without DP, Student’s paired t test).

Article Snippet: Immunocytochemistry—Immunostaining of acetone:methanol (3:2, v/v) fixed Kelly cells was performed with the following antibodies in ready-to-use diluent (catalog number 00–3218, Zymed Laboratories Inc., Berlin, Germany): goat polyclonal anti-TrkB antibody (catalog number sc-20542, Lot# F1505, Santa Cruz Biotechnology, 1:75 dilution) and polyclonal donkey Cy3-conjugated anti-goat IgG as secondary antibody.

Techniques: Derivative Assay, Reverse Transcription, Expressing

Journal: The Journal of biological chemistry

Article Title: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene.

doi: 10.1074/jbc.M609857200

Figure Lengend Snippet: FIGURE 2. A, representative graph showing the time-dependent increase of TrkB mRNA in Kelly cells grown at 1% O2 for different time intervals (4, 8, 16, 24, and 32 h). TrkB transcripts in normoxic (21% O2) Kelly cells were consid- ered as 1. B, immunoblot detecting the 145- and 95-kDa TrkB proteins, gp145TrkB and gp95TrkB, in membrane preparations of Kelly cells that had been incubated at 1% O2 for 16 h. Hypoxic stimulation of the neuroblastoma cells is indicated by their expression of HIF-1 protein. The two TrkB iso- forms could not be detected in membrane preparations of Kelly cells at normoxia (21% O2). Equal protein loading is demonstrated by the similar intensities of the -actin bands. C, TrkB immunostaining of Kelly cells after exposure for 16 h at 1% O2 (I and II) or 21% O2 (III). As a negative control the cells were incubated with normal goat serum instead of primary antibody (IV). Note the more intense labeling of hypoxic (I and II) versus normoxic (III) Kelly cells. Magnifications are 100 (I, III, and IV) and 320 times (II) the original. Counterstaining of the nuclei with 4,6-diamidino-2-phenylin- dole (blue) is shown in panel II, which represents a higher magnification of the boxed area in panel I.

Article Snippet: Immunocytochemistry—Immunostaining of acetone:methanol (3:2, v/v) fixed Kelly cells was performed with the following antibodies in ready-to-use diluent (catalog number 00–3218, Zymed Laboratories Inc., Berlin, Germany): goat polyclonal anti-TrkB antibody (catalog number sc-20542, Lot# F1505, Santa Cruz Biotechnology, 1:75 dilution) and polyclonal donkey Cy3-conjugated anti-goat IgG as secondary antibody.

Techniques: Western Blot, Membrane, Incubation, Expressing, Immunostaining, Negative Control, Labeling

Journal: The Journal of biological chemistry

Article Title: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene.

doi: 10.1074/jbc.M609857200

Figure Lengend Snippet: FIGURE 3. A, activity of a luciferase reporter construct carrying 2.1 kbp of the human TrkB promoter (pGL3-TrkB-2107). Twenty-four hours after transient transfection with the promoter-reporter construct the Kelly cells were incu- bated with specific siRNAs for HIF-1 and HIF-2, respectively (37). Another siRNAtargetingthefireflyluciferasecodingsequenceofthepGL2basicvector (luc-siRNA) served as negative control. The double-transfected cells were grown for 16 h at 21% O2 with or without addition of DP (100 M) to the culture medium. Graphs show the x-fold increase of normalized luciferase activity in DP-treated versus untreated cells (luciferase activity in untreated cells 1). Approximately 6-fold higher promoter activities were measured in DP-treated than in untreated Kelly cells transfected with the luc-siRNA (black columns). Note that TrkB promoter stimulation by the DP reagent was reduced significantly upon transfection of HIF-1 siRNA (*, p 0.01) but not of HIF-2 siRNA (p 0.05). Values are mean S.D. of n 6 experiments, each performed in duplicate. B, representative immunoblots demonstrating reduced HIF-1 and HIF-2 protein expression in DP-treated Kelly cells that had been transfected with the respective siRNAs. Notably, incubation with specific siRNAs did not completely suppress HIF accumulation in the presence of DP. Immuno- blotting of -actin was performed to confirm equal protein loading.

Article Snippet: Immunocytochemistry—Immunostaining of acetone:methanol (3:2, v/v) fixed Kelly cells was performed with the following antibodies in ready-to-use diluent (catalog number 00–3218, Zymed Laboratories Inc., Berlin, Germany): goat polyclonal anti-TrkB antibody (catalog number sc-20542, Lot# F1505, Santa Cruz Biotechnology, 1:75 dilution) and polyclonal donkey Cy3-conjugated anti-goat IgG as secondary antibody.

Techniques: Activity Assay, Luciferase, Construct, Transfection, Negative Control, Western Blot, Expressing, Incubation

Journal: The Journal of biological chemistry

Article Title: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene.

doi: 10.1074/jbc.M609857200

Figure Lengend Snippet: FIGURE 4. Activity of a luciferase reporter construct (pGL3-TrkB-2107) containing 2.1 kbp of the human TrkB promoter in wild-type (MEF HIF- 1/) and HIF-1-deficient (MEF HIF-1/) mouse embryonic fibro- blasts. The transiently transfected cells were incubated for 16 h either at 1 or 21% O2. Graphs show the x-fold increase of normalized luciferase activities in hypoxic versus normoxic cells (luciferase activity at 21% O2 1). Note that luciferase activity of the pGL3-TrkB-2107 construct was stimulated signifi- cantly at 1% O2 in wild-type but not in HIF-1-depleted MEF cells. The empty pGL3basic plasmid was transfected as control. Values are mean S.E. of n 5 experiments, each performed in duplicate. Statistical significance is indi- cated (*, p 0.05, Student’s paired t test).

Article Snippet: Immunocytochemistry—Immunostaining of acetone:methanol (3:2, v/v) fixed Kelly cells was performed with the following antibodies in ready-to-use diluent (catalog number 00–3218, Zymed Laboratories Inc., Berlin, Germany): goat polyclonal anti-TrkB antibody (catalog number sc-20542, Lot# F1505, Santa Cruz Biotechnology, 1:75 dilution) and polyclonal donkey Cy3-conjugated anti-goat IgG as secondary antibody.

Techniques: Activity Assay, Luciferase, Construct, Transfection, Incubation, Plasmid Preparation, Control

Journal: The Journal of biological chemistry

Article Title: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene.

doi: 10.1074/jbc.M609857200

Figure Lengend Snippet: FIGURE 5. A, luciferase activities of different sized TrkB promoter-reporter constructs in transiently transfected Kelly cells. The lengths of the reporter constructs relative to the transcription start site are indicated in the schematic drawing. The transfected cells were maintained at 1 and 21% O2 for 16 h. The hypoxia-sensitive regionintheTrkBpromotercouldbemappedtoa91-bpregionlocatedbetween969and878bpupstream of the transcription start site. The luciferase activities were normalized to -galactosidase, which was co- transfected as an internal control for transfection efficiencies. Values represent mean S.D. of n 3 experi- ments performed in duplicate. Statistical differences are indicated by asterisks (*, p 0.05, Student’s paired t test). B, sequence of the hypoxia-inducible region in the human TrkB promoter. The three predicted HREs are indicated. C, representative ChIP assay demonstrating interaction of HIF-1 with the TrkB promoter in hypoxic Kelly cells. An inverse image of the original agarose gel is shown for better visibility of the DNA bands. Note that PCR products reflecting the binding of HIF-1 to the TrkB promoter were obtained only with Kelly cells at 1% O2 but not at 21% O2. Binding of acetylated histone H3 to the TrkB promoter was similar in normoxic (21% O2) and hypoxic (1% O2) cells. Incubation with normal mouse IgG served as a negative control. The input samples (1/10 volume) are also shown. D, scheme of the 5-upstream region of the human NTRK2 gene, which encodes the TrkB neurotrophin receptor. The drawing illustrates the HIF-1 binding elements identified herein and the positionofthePCRprimersthatwereusedforamplificationofaDNAsequenceextendingbetween1045and 845 bp relative to the transcription start site (1) in the NTRK2 gene.

Article Snippet: Immunocytochemistry—Immunostaining of acetone:methanol (3:2, v/v) fixed Kelly cells was performed with the following antibodies in ready-to-use diluent (catalog number 00–3218, Zymed Laboratories Inc., Berlin, Germany): goat polyclonal anti-TrkB antibody (catalog number sc-20542, Lot# F1505, Santa Cruz Biotechnology, 1:75 dilution) and polyclonal donkey Cy3-conjugated anti-goat IgG as secondary antibody.

Techniques: Luciferase, Construct, Transfection, Control, Sequencing, Agarose Gel Electrophoresis, Binding Assay, Incubation, Negative Control

Journal: The Journal of biological chemistry

Article Title: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene.

doi: 10.1074/jbc.M609857200

Figure Lengend Snippet: FIGURE 6. A, EMSA demonstrating binding activity in nuclear extracts of hypoxic (1% O2, 5 h) Kelly cells to the predicted HREs in the human TrkB promoter. The binding reactions were carried out at room temperature for 30 min with 5-end labeled oligonucleotides and 5 g of nuclear extract. The samples were loaded on a non-denaturing 4% polyacrylamide gel, which was run for 4 h at 4 °C. Complex formation was visualized by auto- radiography of the dried gels. In addition to the appearance of nonspecific (ns) and constitutive signals (c), a retarded band (arrow, shift) was obtained with nuclear extract of hypoxic (1% O2) Kelly cells (lanes 3 and 11). This retardation band was not detected with normoxic (21% O2) cell nuclear extracts (lanes 2 and 10). The complexes could be supershifted (arrow, supershift) by incubation with specific anti-HIF-1 antibody (lanes 4 and 12). Disruption of the HIF recognition sequence in two mutant oli- gonucleotides (TrkB-HRE1mut and TrkB-HRE2/3mut) resulted in a loss of specific binding activity in the hypoxic Kelly cell nuclear extracts (lanes 7 and 15). B, sequence of the oligonucleotides that were used for the EMSA experiments. The HRE core sequences and the corresponding mutations are underlined and indicated in bold.

Article Snippet: Immunocytochemistry—Immunostaining of acetone:methanol (3:2, v/v) fixed Kelly cells was performed with the following antibodies in ready-to-use diluent (catalog number 00–3218, Zymed Laboratories Inc., Berlin, Germany): goat polyclonal anti-TrkB antibody (catalog number sc-20542, Lot# F1505, Santa Cruz Biotechnology, 1:75 dilution) and polyclonal donkey Cy3-conjugated anti-goat IgG as secondary antibody.

Techniques: Binding Assay, Activity Assay, Labeling, Incubation, Disruption, Sequencing, Mutagenesis

Journal: The Journal of biological chemistry

Article Title: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene.

doi: 10.1074/jbc.M609857200

Figure Lengend Snippet: FIGURE 7. Transcriptional activity of a 969-bp wild-type TrkB promoter construct (upper drawing) and two plasmids carrying mutations in the identified HREs. Kelly cells were transiently co-transfected with the indi- cated promoter constructs and a cytomegalovirus promoter-driven -galac- tosidase plasmid for normalization of transfection efficiencies. The trans- fected cells were incubated for 16 h at 1 and 21% O2. The combined mutation of HRE2 and HRE3 resulted in an insignificant reduction of promoter activity at 1% O2. Introducing mutations in all three HREs (HRE1 through HRE3, for sequence of the mutations, see Fig. 6B) abrogated TrkB promoter stimulation in hypoxia. Values represent mean S.D. of n 3 experiments performed in duplicate. Statistical differences are indicated by asterisks (*, p 0.05, Stu- dent’s paired t test).

Article Snippet: Immunocytochemistry—Immunostaining of acetone:methanol (3:2, v/v) fixed Kelly cells was performed with the following antibodies in ready-to-use diluent (catalog number 00–3218, Zymed Laboratories Inc., Berlin, Germany): goat polyclonal anti-TrkB antibody (catalog number sc-20542, Lot# F1505, Santa Cruz Biotechnology, 1:75 dilution) and polyclonal donkey Cy3-conjugated anti-goat IgG as secondary antibody.

Techniques: Activity Assay, Construct, Transfection, Plasmid Preparation, Incubation, Mutagenesis, Sequencing

Journal: The Journal of biological chemistry

Article Title: Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene.

doi: 10.1074/jbc.M609857200

Figure Lengend Snippet: FIGURE 8. In vitro migration assay of Kelly cells that had been preincu- batedfor24heitherat1or21%O2tostimulateTrkBexpression.Thecells (77,000 cells per insert) were seeded in serum-free tissue culture medium in the upper compartment of the transwell chambers (BioCoat Cell Culture Inserts, BD Biosciences) and maintained at 21% O2, 5% CO2. After 27 h the membranes (8 m pore size) were stained with toluidine blue, and the migrated cells were counted on the downside. Cell migration was increased 2-fold in ex-hypoxic versus normoxic Kelly cells. Note that the number of migrating cells was reduced significantly upon omission of the TrkB ligand, BDNF (50 ng/ml), from the medium in the lower transwell compartment. Like- wise, treatment with the tyrosine kinase inhibitor K252a (200 nM) suppressed the migratory potential of normoxic (21% O2) and ex-hypoxic (1% O2) Kelly cells. All experiments (n 5) were performed in triplicate. Statistical signifi- cance between the indicated normoxic and ex-hypoxic cells is marked by an asterisk (p 0.05, Student’s t test). The symbols (#, ) indicate statistical significance (p 0.05, Student’s t test) versus the respective controls at 21% (#) and 1% O2 ().

Article Snippet: Immunocytochemistry—Immunostaining of acetone:methanol (3:2, v/v) fixed Kelly cells was performed with the following antibodies in ready-to-use diluent (catalog number 00–3218, Zymed Laboratories Inc., Berlin, Germany): goat polyclonal anti-TrkB antibody (catalog number sc-20542, Lot# F1505, Santa Cruz Biotechnology, 1:75 dilution) and polyclonal donkey Cy3-conjugated anti-goat IgG as secondary antibody.

Techniques: In Vitro, Migration, Cell Culture, Pore Size, Staining

Journal: The Journal of Neuroscience

Article Title: Impact of the NGF Maturation and Degradation Pathway on the Cortical Cholinergic System Phenotype

doi: 10.1523/JNEUROSCI.1144-11.2012

Figure Lengend Snippet: Antibodies used for the labeling of cortical synaptic boutons

Article Snippet: The sections were washed 3× 10 min in PBS, mounted on gelatin subbed slides, dried overnight at 4°C, and coverslipped with Aqua Polymount (Polysciences). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Anatomical marker Primary antibody Serum used for blocking step and antibody dilutions Secondary antibody VAChT (expressed in cholinergic varicosities) Goat anti-VAChT; 1:10,000 (Millipore), polyclonal Normal rabbit serum (Jackson ImmunoResearch) Biotinylated rabbit anti-goat; 1:200 (Vector Labs) TH (expressed in both dopaminergic and noradrenergic varicosities) Rabbit anti-TH; 1:3000 (Millipore), polyclonal Normal goat serum (Jackson ImmunoResearch) Alexa Fluor 488-conjugated goat anti-rabbit; 1:800 (Invitrogen) DBH (expressed in noradrenergic varicosities) Mouse anti-DBH; 1:25 (MediMabs), monoclonal Normal donkey serum (Jackson ImmunoResearch) Rhodamine Red X-conjugated donkey anti-mouse; 1:200 (Jackson ImmunoResearch) VGluT1 (expressed in glutamatergic varicosities) Mouse anti-VGluT1; 1:100 (characterized in Bell et al. (2006) ; MediMabs), monoclonal Normal donkey serum (Jackson ImmunoResearch) Alexa Fluro 488-conjugated donkey anti-mouse; 1:800 (Invitrogen) GAD 65 (expressed in GABAergic varicosities) Mouse anti-GAD-65; 1:1000 (Millipore), monoclonal Normal donkey serum (Jackson ImmunoResearch) Alexa Fluor 488-conjugated donkey anti-mouse; 1:800 (Invitrogen) Neurotrophic TrkA high-affinity mNGF receptor (expressed on cholinergic axon) Polyclonal goat anti-TrkA; 1:100 (R&D Systems) Normal donkey serum (Jackson ImmunoResearch) Rhodamine Red X-conjugated donkey anti-goat; 1:200 (Jackson ImmunoResearch) Open in a separate window Antibodies used for the labeling of cortical synaptic boutons

Techniques: Labeling, Marker, Blocking Assay, Plasmid Preparation

Journal: Journal of Neuroscience

Article Title: Loss of Signal Transducer and Activator of Transcription 3 (STAT3) Signaling during Elevated Activity Causes Vulnerability in Hippocampal Neurons

doi: 10.1523/jneurosci.2940-12.2012

Figure Lengend Snippet: Figure 1. Neurons become TrkB dependent when network activity is elevated. A, Spontaneous activity of primary hippocampal neurons (DIV14), monitored by calcium responses using fluo-4 AM, were compared before and after appli- cation of 5 mM 4-AP. Ratio of change in fluorescence intensity (dF) to the intensity at t 0 (F) is plotted. Responses of five simultaneously monitored neurons before and after application of 4-AP are shown as examples. B, Immunostaining for the neuronal marker, MAP2. Hippocampal neurons (DIV14) were exposed to 5 mM 4-AP and/or 5 g/ml anti-TrkB antibody for 2 d. C, Numbers of neurons after the 2 d treatments. One micromolar TTX and 50 M PTX were used (*p 0.05, one-way ANOVA; n 6). Error bars indicate SEM.

Article Snippet: Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for functionblocking experiments.

Techniques: Activity Assay, Fluorescence, Immunostaining, Marker

Journal: Journal of Neuroscience

Article Title: Loss of Signal Transducer and Activator of Transcription 3 (STAT3) Signaling during Elevated Activity Causes Vulnerability in Hippocampal Neurons

doi: 10.1523/jneurosci.2940-12.2012

Figure Lengend Snippet: Figure2. NeuronsbecomedependentonAktsignalingwhentreatedwith4-AP.A,Westernblotanalyses.Neuronsweretreatedfor18hwith5mM4-AP,and/or5g/mlanti-TrkBantibody,or 1 mM TTX. B, Immunostaining for c-cas3 and cytochrome c, and DAPI staining. Neurons were treated for 2 d (from DIV12 to DIV14) with 5.0 mM 4-AP and/or 25 M LY294002. The arrows indicate apoptoticneuronswith(yellow)orwithout(red)activationofcaspase3.C,Numbersofneuronsafter2dtreatments.Fivemillimolar4-AP,25MLY294002,10ng/mlBDNFwereused(n4).*p 0.05; ns, p 0.05 (one-way ANOVA). D, Neurons (DIV9) were cotransfected with plasmids containing cDNAs of GFP and myristoylated Akt, and then incubated with 5 mM 4-AP (and 5 g/ml anti-TrkB)for2d(fromDIV12toDIV14).TransfectedneuronsweredetectedbyGFPfluorescence.Numbersofneuronsremainingafter2dtreatments(n6).*p0.05.ErrorbarsindicateSEM.

Article Snippet: Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for functionblocking experiments.

Techniques: Immunostaining, Staining, Incubation

Journal: Journal of Neuroscience

Article Title: Loss of Signal Transducer and Activator of Transcription 3 (STAT3) Signaling during Elevated Activity Causes Vulnerability in Hippocampal Neurons

doi: 10.1523/jneurosci.2940-12.2012

Figure Lengend Snippet: Figure3. PhosphorylationofSTAT3isimpairedfollowingtreatmentwith4-AP.A,WesternblotanalysesofPSer-727STAT3and total STAT3. Neurons were treated for 18 h with the indicated concentrations of 4-AP and 1 M TTX, 50 M PD98059, or the indicated concentrations of anti-TrkB antibody and LY294002. B, Effect of inhibition of various serine/threonine kinases on PSer- 727 STAT3. Neurons were incubated for 2 h with 20 ng/ml rapamycin, 50 M roscovitine, or 50 M PD98059. C, Immunoprecipi- tation of STAT3. Shown are Western blot analyses of STAT3 and Erk1/2.

Article Snippet: Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for functionblocking experiments.

Techniques: Inhibition, Incubation, Western Blot

Journal: Journal of Neuroscience

Article Title: Loss of Signal Transducer and Activator of Transcription 3 (STAT3) Signaling during Elevated Activity Causes Vulnerability in Hippocampal Neurons

doi: 10.1523/jneurosci.2940-12.2012

Figure Lengend Snippet: Figure 4. Ser727Glu STAT3 prevents neurons from becoming TrkB and Akt dependent during elevated activity. A, Neurons (DIV9)werecotransfectedwithplasmidscontainingcDNAsofGFPandSer727GluSTAT3,andthenincubatedwith5mM4-AP(and 5g/mlanti-TrkB,or25MLY294002)for2d(fromDIV12toDIV14).TransfectedneuronsweredetectedbyGFPfluorescence.B, Numbers of neurons remaining after 2 d treatments. *p 0.05, n 8 (one-way ANOVA). Error bars indicate SEM.

Article Snippet: Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for functionblocking experiments.

Techniques: Activity Assay

Journal: Journal of Neuroscience

Article Title: Loss of Signal Transducer and Activator of Transcription 3 (STAT3) Signaling during Elevated Activity Causes Vulnerability in Hippocampal Neurons

doi: 10.1523/jneurosci.2940-12.2012

Figure Lengend Snippet: Figure6. STAT3blockadebysiRNAinducesTrkBandAktdependencyinneurons.A,Immu- nostaining of GFP, STAT3, p53, and Bax. Neurons (DIV9) were cotransfected with a plasmid containingcDNAofGFPandSTAT3siRNA.B,Numberofneuronsafterincubationwith5g/ml anti-TrkB, or 25 M LY294002 (and 10 ng/ml BDNF) for 2 d (DIV12 to DIV14). Numbers of neurons in control were taken as 100% (n 5). *p 0.05. Error bars indicate SEM.

Article Snippet: Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for functionblocking experiments.

Techniques: Plasmid Preparation, Control

Journal: Journal of Neuroscience

Article Title: Loss of Signal Transducer and Activator of Transcription 3 (STAT3) Signaling during Elevated Activity Causes Vulnerability in Hippocampal Neurons

doi: 10.1523/jneurosci.2940-12.2012

Figure Lengend Snippet: Figure 5. Ser727Ala STAT3 induces TrkB and Akt dependency in neurons. Neurons (DIV9) were cotransfected with plasmids containing cDNAs for GFP, wild-type STAT3, or Ser727Ala STAT3,andthenincubatedwith5g/mlanti-TrkB,or25MLY294002for2d(fromDIV12to DIV14). Numbers of neurons in control were taken as 100% (n 10). *p 0.05, one-way ANOVA. Error bars indicate SEM.

Article Snippet: Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for functionblocking experiments.

Techniques: Control

Journal: Journal of Neuroscience

Article Title: Loss of Signal Transducer and Activator of Transcription 3 (STAT3) Signaling during Elevated Activity Causes Vulnerability in Hippocampal Neurons

doi: 10.1523/jneurosci.2940-12.2012

Figure Lengend Snippet: Figure 7. STAT3 is responsible for the change in vulnerability during development. A, Western blot analyses of pErk1/2, STAT3, p53, and Bax from hippocampal cultures (DIV7 and DIV14). B, Immunostainingofp53andBaxinyoungneuron(DIV7)expressingGFP(andwild-typeSTAT3).C,Youngneurons(DIV7)expressingGFP(andwild-typeSTAT3orSer727AlaSTAT3)wereincubated in 5 g/ml anti-TrkB antibody or 25 M LY294002 for 2 d. *p 0.05; n 10. Error bars indicate SEM.

Article Snippet: Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for functionblocking experiments.

Techniques: Western Blot

Journal: Journal of Neuroscience

Article Title: Loss of Signal Transducer and Activator of Transcription 3 (STAT3) Signaling during Elevated Activity Causes Vulnerability in Hippocampal Neurons

doi: 10.1523/jneurosci.2940-12.2012

Figure Lengend Snippet: Figure 8. Elevated excitability increases levels of p53 and Bax in vitro and in vivo. A, Left panel,Westernblotanalysesofp53andBaxfromrathippocampalculture(DIV14)treatedwith 5mM4-APovernight.Rightpanel,RT-PCRanalysisofp53.B,Leftpanel,Westernblotanalyses ofPErk1/2,PSer-727STAT3,p53,andBaxfromwild-typeandKv4.2KOmousehippocampus(8 weeks). Right top panel, RT-PCR analysis of p53. Right bottom panel, ChIP assay of STAT3 binding to the p53 promoter. C, Immunostaining of NeuN and c-cas3 in Kv4.2KO mouse hip- pocampusinjectedwithanti-TrkBantibody.Thearrowindicatesanapoptoticneuron.D,Num- berofc-cas3neuronsinwild-typeandKv4.2KOhippocampalslicesinjectedwithvehicleand anti-TrkB antibody. *p 0.001; n 12. Error bars indicate SEM.

Article Snippet: Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for functionblocking experiments.

Techniques: In Vitro, In Vivo, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Immunostaining