wild type probdnf  (Alomone Labs)


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    Structured Review

    Alomone Labs wild type probdnf
    Proneurotrophins abolish retinal axon branching via p75 NTR . The outgrowth/branching assay was performed as described [ 6 ]. Cells from E8 nasal retina were electroporated with eGFP and plated on a merosin/laminin substrate. (A) Cultures were treated at 1 day in vitro with 5 ng/ml BDNF, or 5 ng/ml proneurotrophins in the presence of 5 ng/ml BDNF as indicated, and fixed and analysed for branch number per axon after 3 days in vitro . The basal level of branching was not affected by treatment of retinal cultures with proneurotrophins alone. However, both <t>proBDNF</t> and proNGF led to a downregulation of the BDNF-induced branching to basal levels. (B) The length of outgrowth of retinal axons is not affected by treatment with neurotrophins and/or proneurotrophins. Axon length is given in arbitrary units. (C) To show that the proBDNF effect is mediated via p75 NTR , retinal cultures were electroporated either with an RNAi vector resulting in the knockdown of p75 NTR , with an RNAi vector not affecting p75 NTR protein levels or with empty vector. After plating, the cultures were treated with pro/neurotrophins as described in (A). p75 NTR knockdown obliterates the branch-suppressing effect of proBDNF. Three independent experiments were performed. The statistical analysis was done using Kruskal-Wallis test and Dunn's multiple comparison test post hoc . *** P
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    Images

    1) Product Images from "Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance"

    Article Title: Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance

    Journal: Neural Development

    doi: 10.1186/1749-8104-5-30

    Proneurotrophins abolish retinal axon branching via p75 NTR . The outgrowth/branching assay was performed as described [ 6 ]. Cells from E8 nasal retina were electroporated with eGFP and plated on a merosin/laminin substrate. (A) Cultures were treated at 1 day in vitro with 5 ng/ml BDNF, or 5 ng/ml proneurotrophins in the presence of 5 ng/ml BDNF as indicated, and fixed and analysed for branch number per axon after 3 days in vitro . The basal level of branching was not affected by treatment of retinal cultures with proneurotrophins alone. However, both proBDNF and proNGF led to a downregulation of the BDNF-induced branching to basal levels. (B) The length of outgrowth of retinal axons is not affected by treatment with neurotrophins and/or proneurotrophins. Axon length is given in arbitrary units. (C) To show that the proBDNF effect is mediated via p75 NTR , retinal cultures were electroporated either with an RNAi vector resulting in the knockdown of p75 NTR , with an RNAi vector not affecting p75 NTR protein levels or with empty vector. After plating, the cultures were treated with pro/neurotrophins as described in (A). p75 NTR knockdown obliterates the branch-suppressing effect of proBDNF. Three independent experiments were performed. The statistical analysis was done using Kruskal-Wallis test and Dunn's multiple comparison test post hoc . *** P
    Figure Legend Snippet: Proneurotrophins abolish retinal axon branching via p75 NTR . The outgrowth/branching assay was performed as described [ 6 ]. Cells from E8 nasal retina were electroporated with eGFP and plated on a merosin/laminin substrate. (A) Cultures were treated at 1 day in vitro with 5 ng/ml BDNF, or 5 ng/ml proneurotrophins in the presence of 5 ng/ml BDNF as indicated, and fixed and analysed for branch number per axon after 3 days in vitro . The basal level of branching was not affected by treatment of retinal cultures with proneurotrophins alone. However, both proBDNF and proNGF led to a downregulation of the BDNF-induced branching to basal levels. (B) The length of outgrowth of retinal axons is not affected by treatment with neurotrophins and/or proneurotrophins. Axon length is given in arbitrary units. (C) To show that the proBDNF effect is mediated via p75 NTR , retinal cultures were electroporated either with an RNAi vector resulting in the knockdown of p75 NTR , with an RNAi vector not affecting p75 NTR protein levels or with empty vector. After plating, the cultures were treated with pro/neurotrophins as described in (A). p75 NTR knockdown obliterates the branch-suppressing effect of proBDNF. Three independent experiments were performed. The statistical analysis was done using Kruskal-Wallis test and Dunn's multiple comparison test post hoc . *** P

    Techniques Used: In Vitro, Plasmid Preparation

    Expression of proBDNF in chick RGC axons . Retinal single cell cultures derived from E6 chick retina were stained after 2 days in vitro using a monoclonal antibody against the pro-domain of proBDNF [ 14 ] according to protocols given in Yang et al . [ 14 ]. (A-C) An RGC growth cone stained with control antibody (B), and phalloidin (C) to show the location of actin. (A) The composite of (B) and (C). In all composites phalloidin is shown in green and proBDNF/control antibody in red. (D-F) An RGC axon stained with control antibody (E), and phalloidin (F). (D) The composite of (E) and (F). (G-I) An RGC axon stained with a proBDNF antibody (H) [ 14 ], and phalloidin (I). (G) The composite of (H) and (I). (J-L) . An RGC growth cone stained with proBDNF antibody (K), and phalloidin (L). (J) The composite of (K) and (L).
    Figure Legend Snippet: Expression of proBDNF in chick RGC axons . Retinal single cell cultures derived from E6 chick retina were stained after 2 days in vitro using a monoclonal antibody against the pro-domain of proBDNF [ 14 ] according to protocols given in Yang et al . [ 14 ]. (A-C) An RGC growth cone stained with control antibody (B), and phalloidin (C) to show the location of actin. (A) The composite of (B) and (C). In all composites phalloidin is shown in green and proBDNF/control antibody in red. (D-F) An RGC axon stained with control antibody (E), and phalloidin (F). (D) The composite of (E) and (F). (G-I) An RGC axon stained with a proBDNF antibody (H) [ 14 ], and phalloidin (I). (G) The composite of (H) and (I). (J-L) . An RGC growth cone stained with proBDNF antibody (K), and phalloidin (L). (J) The composite of (K) and (L).

    Techniques Used: Expressing, Derivative Assay, Staining, In Vitro

    Repellent axon guidance is disrupted in the presence of an anti-proBDNF antibody or a soluble extracellular domain of p75 NTR . (A) Quantification of axon growth preferences in the presence of an anti-proBDNF antibody [ 14 ] or a control antibody. Stripe assay experiments were performed in the presence of a proBDNF antibody (1:200) [ 14 ] or a control antibody (mouse monoclonal antibody for placental alkaline phosphatase; 1:200). The quantification of axon growth preferences shows an abolishment of repellent guidance in the presence of the proBDNF antibody (see also Additional file 4 ). Error bars represent the standard error of the mean. Statistics were performed using Kruskal-Wallis test and Dunn's multiple comparison test with *** P
    Figure Legend Snippet: Repellent axon guidance is disrupted in the presence of an anti-proBDNF antibody or a soluble extracellular domain of p75 NTR . (A) Quantification of axon growth preferences in the presence of an anti-proBDNF antibody [ 14 ] or a control antibody. Stripe assay experiments were performed in the presence of a proBDNF antibody (1:200) [ 14 ] or a control antibody (mouse monoclonal antibody for placental alkaline phosphatase; 1:200). The quantification of axon growth preferences shows an abolishment of repellent guidance in the presence of the proBDNF antibody (see also Additional file 4 ). Error bars represent the standard error of the mean. Statistics were performed using Kruskal-Wallis test and Dunn's multiple comparison test with *** P

    Techniques Used: Stripping Membranes

    2) Product Images from "Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator"

    Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    Journal: Scientific Reports

    doi: 10.1038/srep27171

    Exogenous proBDNF induces pain hypersensitivity and spinal cord activation in mice. ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P
    Figure Legend Snippet: Exogenous proBDNF induces pain hypersensitivity and spinal cord activation in mice. ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P

    Techniques Used: Activation Assay, Mouse Assay, Injection

    Polyclonal Ab-proBDNF pretreatment attenuates inflammatory pain in mice. ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P
    Figure Legend Snippet: Polyclonal Ab-proBDNF pretreatment attenuates inflammatory pain in mice. ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P

    Techniques Used: Mouse Assay, Injection

    Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P
    Figure Legend Snippet: Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Western Blot, Migration

    Upregulation of p75NTR and effect of local proBDNF injection on inflammatory reaction in mice. ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p
    Figure Legend Snippet: Upregulation of p75NTR and effect of local proBDNF injection on inflammatory reaction in mice. ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p

    Techniques Used: Injection, Mouse Assay, Western Blot, Expressing

    Upregulation of proBDNF in the local tissue in acute and persistent inflammatory pain in mice. ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p
    Figure Legend Snippet: Upregulation of proBDNF in the local tissue in acute and persistent inflammatory pain in mice. ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p

    Techniques Used: Mouse Assay, Western Blot, Injection

    3) Product Images from "N-Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures"

    Article Title: N-Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.009989

    Expression of wildtype ( WT ) proBDNF and its N121Q mutant ( NQ ) in stably transfected HEK293F cells. Protein expression was determined by Western blotting using an antibody, recognizing both BDNF and proBDNF, in the conditioned media ( A ) and cell lysates ( B ). Arrows point to (pro)BDNF forms: glycosylated proBDNF ( black arrow ), nonglycosylated proBDNF ( white arrow ), and mature BDNF ( gray arrow ). M indicates MagicMark molecular weight marker. Expression of mRNA was determined in WT and N121Q mutant by RT-PCR ( C ). ProBDNF/BDNF expression in cell lysates ( D ) or conditioned media ( E ) of HEK293F cells expressing N121Q mutant treated with proteasome inhibitor MG132 ( MG ), lysosomal inhibitor chloroquine ( CQ ), or chemical chaperone 4-phenylbutyric acid ( PBA ). The black arrow points to the position of nonglycosylated proBDNF.
    Figure Legend Snippet: Expression of wildtype ( WT ) proBDNF and its N121Q mutant ( NQ ) in stably transfected HEK293F cells. Protein expression was determined by Western blotting using an antibody, recognizing both BDNF and proBDNF, in the conditioned media ( A ) and cell lysates ( B ). Arrows point to (pro)BDNF forms: glycosylated proBDNF ( black arrow ), nonglycosylated proBDNF ( white arrow ), and mature BDNF ( gray arrow ). M indicates MagicMark molecular weight marker. Expression of mRNA was determined in WT and N121Q mutant by RT-PCR ( C ). ProBDNF/BDNF expression in cell lysates ( D ) or conditioned media ( E ) of HEK293F cells expressing N121Q mutant treated with proteasome inhibitor MG132 ( MG ), lysosomal inhibitor chloroquine ( CQ ), or chemical chaperone 4-phenylbutyric acid ( PBA ). The black arrow points to the position of nonglycosylated proBDNF.

    Techniques Used: Expressing, Mutagenesis, Stable Transfection, Transfection, Western Blot, Molecular Weight, Marker, Reverse Transcription Polymerase Chain Reaction

    Kinetics of cleavage of glycosylated and nonglycosylated proBDNF by furin. Mass difference between glycosylated and nonglycosylated proBDNF enabled densitometric quantification of each proBDNF form: A, representative blot of the cleavage kinetic at the indicated time intervals; B, densitometric analysis of proBDNF intensities at the indicated time intervals. Results are expressed as percent of the proBDNF concentration (mean ± S.D., n = 3) at the beginning of the reaction. Note that proBDNF without C-terminal Myc-FLAG tag was used in the reaction as described under “Experimental procedures.”
    Figure Legend Snippet: Kinetics of cleavage of glycosylated and nonglycosylated proBDNF by furin. Mass difference between glycosylated and nonglycosylated proBDNF enabled densitometric quantification of each proBDNF form: A, representative blot of the cleavage kinetic at the indicated time intervals; B, densitometric analysis of proBDNF intensities at the indicated time intervals. Results are expressed as percent of the proBDNF concentration (mean ± S.D., n = 3) at the beginning of the reaction. Note that proBDNF without C-terminal Myc-FLAG tag was used in the reaction as described under “Experimental procedures.”

    Techniques Used: Concentration Assay, FLAG-tag

    ProBDNF stably expressed in HEK293F cells is glycosylated. N -Glycosylation of proBDNF and the prodomain is documented by mass shift following deglycosylation with PNGase F: A, detection of proBDNF and BDNF by an antibody recognizing the BDNF region; B, detection of proBDNF and the prodomain by an antibody recognizing the prodomain region. The gel images were spliced as indicated by space to exclude samples not related to the study.
    Figure Legend Snippet: ProBDNF stably expressed in HEK293F cells is glycosylated. N -Glycosylation of proBDNF and the prodomain is documented by mass shift following deglycosylation with PNGase F: A, detection of proBDNF and BDNF by an antibody recognizing the BDNF region; B, detection of proBDNF and the prodomain by an antibody recognizing the prodomain region. The gel images were spliced as indicated by space to exclude samples not related to the study.

    Techniques Used: Stable Transfection

    A, schematic illustration of proBDNF with zoom into the region containing the N -glycosylation and proteolytic cleavage sites involved in the generation of mature BDNF; B, ) (please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site) (multiple alignments were performed with Align tool of the Clustal Omega 1.2.4 engine). Letters in red represent amino acids that differ from the human sequence. Underlined text highlights the NX(S/T) N -glycosylation sequon.
    Figure Legend Snippet: A, schematic illustration of proBDNF with zoom into the region containing the N -glycosylation and proteolytic cleavage sites involved in the generation of mature BDNF; B, ) (please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site) (multiple alignments were performed with Align tool of the Clustal Omega 1.2.4 engine). Letters in red represent amino acids that differ from the human sequence. Underlined text highlights the NX(S/T) N -glycosylation sequon.

    Techniques Used: Sequencing

    Extracted ion chromatograms of four of the most abundant glycopeptides of proBDNF expressed in PC12 cells. A , C , and D, LacdiNAc containing structures; B, LacNAc containing structure. The arrow points to the peaks of glycopeptides aligned with slight retention time shifts as expected based on the hydrophilicity of the attached glycan. Numbers represent intensity of the extracted peaks (×10 5 ).
    Figure Legend Snippet: Extracted ion chromatograms of four of the most abundant glycopeptides of proBDNF expressed in PC12 cells. A , C , and D, LacdiNAc containing structures; B, LacNAc containing structure. The arrow points to the peaks of glycopeptides aligned with slight retention time shifts as expected based on the hydrophilicity of the attached glycan. Numbers represent intensity of the extracted peaks (×10 5 ).

    Techniques Used:

    4) Product Images from "Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator"

    Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    Journal: Scientific Reports

    doi: 10.1038/srep27171

    Exogenous proBDNF induces pain hypersensitivity and spinal cord activation in mice. ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P
    Figure Legend Snippet: Exogenous proBDNF induces pain hypersensitivity and spinal cord activation in mice. ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P

    Techniques Used: Activation Assay, Mouse Assay, Injection

    Polyclonal Ab-proBDNF pretreatment attenuates inflammatory pain in mice. ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P
    Figure Legend Snippet: Polyclonal Ab-proBDNF pretreatment attenuates inflammatory pain in mice. ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P

    Techniques Used: Mouse Assay, Injection

    Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P
    Figure Legend Snippet: Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Western Blot, Migration

    Upregulation of p75NTR and effect of local proBDNF injection on inflammatory reaction in mice. ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p
    Figure Legend Snippet: Upregulation of p75NTR and effect of local proBDNF injection on inflammatory reaction in mice. ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p

    Techniques Used: Injection, Mouse Assay, Western Blot, Expressing

    Upregulation of proBDNF in the local tissue in acute and persistent inflammatory pain in mice. ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p
    Figure Legend Snippet: Upregulation of proBDNF in the local tissue in acute and persistent inflammatory pain in mice. ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p

    Techniques Used: Mouse Assay, Western Blot, Injection

    5) Product Images from "A method for reproducible measurements of serum BDNF: comparison of the performance of six commercial assays"

    Article Title: A method for reproducible measurements of serum BDNF: comparison of the performance of six commercial assays

    Journal: Scientific Reports

    doi: 10.1038/srep17989

    Line-blot for qualitative analysis of anti-BDNF antibodies specificity. ( A ) The antibodies from each ELISA kit were tested for specificity against pro-BDNF or mature BDNF. The BDNF standards blotted were commercial pro-BDNF (Alomone; 10 pg/lane), mature BDNF (1 and 2 from Alomone and Sigma, respectively; both 1000 pg/lane) and the standard BDNF protein included in each kit (Aviscera-Bioscience and Biosensis: 10 pg/lane; Millipore-ChemiKine TM , Millipore-Milliplex ® - and R D System-Quantikine ® : 100 pg/lane; Promega-Emax ® : 1000 pg/lane). BSA (1000 pg/lane) was used as a negative control. The mouse monoclonal anti-BDNF antibody, (1:1000; Sigma) was tested as a control. ( B ) Central region of the same blot shown in A, from an overexposed film to better visualize the reactivity against pro-BDNF. ( C ) Reactivity of antibodies from Biosensis, Promega-Emax ® pAb and Sigma on a dot blot in which the same quantity of pro-BNDF and mature BDNF were spotted (100 pg each). Each antibody from the ELISA kits was used at the dilution suggested by the manufacturer’s instructions. mAb: Promega-Emax ® monoclonal capture antibody for plate coating. pAb: Promega-Emax ® polyclonal detection antibody.
    Figure Legend Snippet: Line-blot for qualitative analysis of anti-BDNF antibodies specificity. ( A ) The antibodies from each ELISA kit were tested for specificity against pro-BDNF or mature BDNF. The BDNF standards blotted were commercial pro-BDNF (Alomone; 10 pg/lane), mature BDNF (1 and 2 from Alomone and Sigma, respectively; both 1000 pg/lane) and the standard BDNF protein included in each kit (Aviscera-Bioscience and Biosensis: 10 pg/lane; Millipore-ChemiKine TM , Millipore-Milliplex ® - and R D System-Quantikine ® : 100 pg/lane; Promega-Emax ® : 1000 pg/lane). BSA (1000 pg/lane) was used as a negative control. The mouse monoclonal anti-BDNF antibody, (1:1000; Sigma) was tested as a control. ( B ) Central region of the same blot shown in A, from an overexposed film to better visualize the reactivity against pro-BDNF. ( C ) Reactivity of antibodies from Biosensis, Promega-Emax ® pAb and Sigma on a dot blot in which the same quantity of pro-BNDF and mature BDNF were spotted (100 pg each). Each antibody from the ELISA kits was used at the dilution suggested by the manufacturer’s instructions. mAb: Promega-Emax ® monoclonal capture antibody for plate coating. pAb: Promega-Emax ® polyclonal detection antibody.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Negative Control, Dot Blot

    6) Product Images from "NMDA-Dependent Switch of proBDNF Actions on Developing GABAergic Synapses"

    Article Title: NMDA-Dependent Switch of proBDNF Actions on Developing GABAergic Synapses

    Journal: Cerebral Cortex (New York, NY)

    doi: 10.1093/cercor/bhs071

    Paracrine actions of proBDNF on GABAergic synaptic activity. Schematic representation of the steps leading to the p75 NTR -dependent LLP GABA-A and LLD GABA-A . CA3 pyramidal cells release proBDNF in response to the activation of L-type Ca 2+ channel during
    Figure Legend Snippet: Paracrine actions of proBDNF on GABAergic synaptic activity. Schematic representation of the steps leading to the p75 NTR -dependent LLP GABA-A and LLD GABA-A . CA3 pyramidal cells release proBDNF in response to the activation of L-type Ca 2+ channel during

    Techniques Used: Activity Assay, Activation Assay

    Endogenous activation of p75 NTR is required for LLD GABA-A induction. ( A ) Immunofluorescence detection of proBDNF and p75 NTR in a 4-days-old rat hippocampi (red signal, upper images), superimposed on neuro-tracer immunofluorescence (Nt, green signal, lower
    Figure Legend Snippet: Endogenous activation of p75 NTR is required for LLD GABA-A induction. ( A ) Immunofluorescence detection of proBDNF and p75 NTR in a 4-days-old rat hippocampi (red signal, upper images), superimposed on neuro-tracer immunofluorescence (Nt, green signal, lower

    Techniques Used: Activation Assay, Immunofluorescence

    Regulated secretion of proBDNF is required for LLD GABA-A induction. ( A ) Average time course of sGABA A -PSCs frequency before and after activity recovery with aprotinin and nifedipine (10 μM) in the absence (control, open symbol, n = 6) or the presence
    Figure Legend Snippet: Regulated secretion of proBDNF is required for LLD GABA-A induction. ( A ) Average time course of sGABA A -PSCs frequency before and after activity recovery with aprotinin and nifedipine (10 μM) in the absence (control, open symbol, n = 6) or the presence

    Techniques Used: Activity Assay

    7) Product Images from "Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF"

    Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.575607

    Molar concentrations of proBDNF are lower in platelets and higher in plasma than those of BDNF. ELISA quantification of proBDNF and BDNF levels in the intraplatelet (A, B) and plasma (C, D) compartments. Concentrations are normalized for 250 x 10 6 platelets. Horizontal bar represents median, *p
    Figure Legend Snippet: Molar concentrations of proBDNF are lower in platelets and higher in plasma than those of BDNF. ELISA quantification of proBDNF and BDNF levels in the intraplatelet (A, B) and plasma (C, D) compartments. Concentrations are normalized for 250 x 10 6 platelets. Horizontal bar represents median, *p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Human platelets contain proBDNF. (A) ProBDNF immunoblotting of human washed platelet lysates (15 µg) from six different healthy volunteers. Recombinant proBDNF (3 ng) and human cortex lysate (3 µg) were used as positive controls. Molecular weight is indicated on the left (kDa) and primary antibody on the right. Experiments representative of n=9 for R-176 and n=10 for mab31751 antibody. IB, immunoblotting. (B) Immunoblotting of proBDNF and BDNF in different fractions of washed human platelets. P-Selectin was used as control protein in the membrane fraction, p65 NF-ĸB was used as control protein in the cytosol, and α-tubulin was used as control protein in the cytoskeleton. The equivalent of the protein content of 3 x 10 7 platelets was loaded for each fraction on the gel. Representative experiment of n=4 different volunteers. (C) ProBDNF treatment with PNGase F in washed human platelet lysates. U87-MG glioblastoma cells were used as a control. rProBDNF, recombinant proBDNF (3 ng); cortex, human cortex lysate (3 µg); platelets, whole human platelet lysate (representative experiment of n=4 different volunteers, 7.5 x 10 8 platelets per well); −PNGF, platelets treated with GlycoBuffer, and incubated at 37°C for 60 min without PNGase F; +PNGF, platelets treated with GlycoBuffer and incubated at 37°C for 60 min with PNGase F; PNGF alone, PNGase F incubated at 37°C for 60 min without platelet lysate. CD42b and sortilin were used as controls of protein deglycosylation in platelets and in U87-MG cells, respectively. n=3 different volunteers for PNGase treatments in human platelets and n=4 independent experiments for U87-MG cells. (D) Representative flow cytometry experiment showing surface and intracellular proBDNF in human washed platelets and in U87-MG and U251-MG glioblastoma cell lines. Mouse IgG 2b was used as isotype control. Percentage of expression are indicated on the figure. n=10 different healthy volunteers for human platelets; n=3 independent experiments for each glioblastoma cell line. (E) Confocal microscopy imaging of proBDNF in human permeabilized washed platelets (top) and in permeabilized U-251 MG cells (bottom). Mouse IgG 2b was used as isotype control. ProBDNF was labelled using Alexa488 fluorochrome (in green). Nuclei were stained with DAPI (in blue). Scale bar = 2 µm and 200 µm for washed platelets and U-251 MG cells images, respectively. (F) Immunoblotting of α 2 -macroglobulin at increasing quantities of loaded proteins (1–20 µg) obtained from a washed platelet lysate or platelet-poor plasma (PPP) from the same individual (#23). α-tubulin was used as loading control. Molecular weight is indicated on the left (kDa) and primary antibody on the right. PPP, platelet poor plasma; PLTs, platelets; IB, immunoblotting.
    Figure Legend Snippet: Human platelets contain proBDNF. (A) ProBDNF immunoblotting of human washed platelet lysates (15 µg) from six different healthy volunteers. Recombinant proBDNF (3 ng) and human cortex lysate (3 µg) were used as positive controls. Molecular weight is indicated on the left (kDa) and primary antibody on the right. Experiments representative of n=9 for R-176 and n=10 for mab31751 antibody. IB, immunoblotting. (B) Immunoblotting of proBDNF and BDNF in different fractions of washed human platelets. P-Selectin was used as control protein in the membrane fraction, p65 NF-ĸB was used as control protein in the cytosol, and α-tubulin was used as control protein in the cytoskeleton. The equivalent of the protein content of 3 x 10 7 platelets was loaded for each fraction on the gel. Representative experiment of n=4 different volunteers. (C) ProBDNF treatment with PNGase F in washed human platelet lysates. U87-MG glioblastoma cells were used as a control. rProBDNF, recombinant proBDNF (3 ng); cortex, human cortex lysate (3 µg); platelets, whole human platelet lysate (representative experiment of n=4 different volunteers, 7.5 x 10 8 platelets per well); −PNGF, platelets treated with GlycoBuffer, and incubated at 37°C for 60 min without PNGase F; +PNGF, platelets treated with GlycoBuffer and incubated at 37°C for 60 min with PNGase F; PNGF alone, PNGase F incubated at 37°C for 60 min without platelet lysate. CD42b and sortilin were used as controls of protein deglycosylation in platelets and in U87-MG cells, respectively. n=3 different volunteers for PNGase treatments in human platelets and n=4 independent experiments for U87-MG cells. (D) Representative flow cytometry experiment showing surface and intracellular proBDNF in human washed platelets and in U87-MG and U251-MG glioblastoma cell lines. Mouse IgG 2b was used as isotype control. Percentage of expression are indicated on the figure. n=10 different healthy volunteers for human platelets; n=3 independent experiments for each glioblastoma cell line. (E) Confocal microscopy imaging of proBDNF in human permeabilized washed platelets (top) and in permeabilized U-251 MG cells (bottom). Mouse IgG 2b was used as isotype control. ProBDNF was labelled using Alexa488 fluorochrome (in green). Nuclei were stained with DAPI (in blue). Scale bar = 2 µm and 200 µm for washed platelets and U-251 MG cells images, respectively. (F) Immunoblotting of α 2 -macroglobulin at increasing quantities of loaded proteins (1–20 µg) obtained from a washed platelet lysate or platelet-poor plasma (PPP) from the same individual (#23). α-tubulin was used as loading control. Molecular weight is indicated on the left (kDa) and primary antibody on the right. PPP, platelet poor plasma; PLTs, platelets; IB, immunoblotting.

    Techniques Used: Recombinant, Molecular Weight, Incubation, Flow Cytometry, Expressing, Confocal Microscopy, Imaging, Staining

    Unlike BDNF, intraplatelet proBDNF is not released during platelet activation. Intraplatelet (A, D) and plasma (B, E) concentrations of BDNF and proBDNF following platelet activation by different agonists. Intraplatelet concentrations are normalized for 250 x 10 6 platelets. Proportion of BDNF (C) and proBDNF (F) in plasma vs . in platelets are expressed in percentage. Error bar represents IQR, *p
    Figure Legend Snippet: Unlike BDNF, intraplatelet proBDNF is not released during platelet activation. Intraplatelet (A, D) and plasma (B, E) concentrations of BDNF and proBDNF following platelet activation by different agonists. Intraplatelet concentrations are normalized for 250 x 10 6 platelets. Proportion of BDNF (C) and proBDNF (F) in plasma vs . in platelets are expressed in percentage. Error bar represents IQR, *p

    Techniques Used: Activation Assay

    8) Product Images from "ProNGF promotes neurite growth from a subset of NGF-dependent neurons by a p75NTR-dependent mechanism"

    Article Title: ProNGF promotes neurite growth from a subset of NGF-dependent neurons by a p75NTR-dependent mechanism

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.085266

    Specificity of neurite growth-promoting effects of proNGF, proBDNF and proNT3. ( A , B ) Neurite length and branching of E17 to P5 trigeminal neurons cultured for 24 hours without factors or with 10 ng/ml cleavage-resistant murine proNGF or 10 ng/ml mNGF. ( C , D ) Neurite length and branching of P5 trigeminal neurons cultured with a range of concentrations of mNGF and cleavage-resistant murine proNGF. ( E-G ) YFP-transfected SCG neurons (green) and pDsRed-transfected trigeminal neurons (red) co-cultured for 24 hours either without factors (E), or with 10 ng/ml cleavage-resistant murine proNGF (F) or 10 ng/ml mNGF (G). Scale bars: 100 μm. ( H ) Sholl plot of P3 trigeminal neurons incubated with mNGF, cleavage-resistant murine proNGF or cleavage-resistant human proNGF. ( I , J ) Neurite length and branching of P3 SCG, trigeminal and nodose neurons cultured for 24 hours with 10 ng/ml cleavage-resistant murine proNGF, 10 ng/ml cleavage-resistant murine proBDNF or 10 ng/ml native human proNT3. All cultures were supplemented with 25 μM Boc-D-FMK and the cultures with native proNT3 additionally received 1 μM batimastat. The mean±s.e.m. for at least three individual datasets are shown.
    Figure Legend Snippet: Specificity of neurite growth-promoting effects of proNGF, proBDNF and proNT3. ( A , B ) Neurite length and branching of E17 to P5 trigeminal neurons cultured for 24 hours without factors or with 10 ng/ml cleavage-resistant murine proNGF or 10 ng/ml mNGF. ( C , D ) Neurite length and branching of P5 trigeminal neurons cultured with a range of concentrations of mNGF and cleavage-resistant murine proNGF. ( E-G ) YFP-transfected SCG neurons (green) and pDsRed-transfected trigeminal neurons (red) co-cultured for 24 hours either without factors (E), or with 10 ng/ml cleavage-resistant murine proNGF (F) or 10 ng/ml mNGF (G). Scale bars: 100 μm. ( H ) Sholl plot of P3 trigeminal neurons incubated with mNGF, cleavage-resistant murine proNGF or cleavage-resistant human proNGF. ( I , J ) Neurite length and branching of P3 SCG, trigeminal and nodose neurons cultured for 24 hours with 10 ng/ml cleavage-resistant murine proNGF, 10 ng/ml cleavage-resistant murine proBDNF or 10 ng/ml native human proNT3. All cultures were supplemented with 25 μM Boc-D-FMK and the cultures with native proNT3 additionally received 1 μM batimastat. The mean±s.e.m. for at least three individual datasets are shown.

    Techniques Used: Cell Culture, Transfection, Incubation

    9) Product Images from "Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator"

    Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    Journal: Scientific Reports

    doi: 10.1038/srep27171

    Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P
    Figure Legend Snippet: Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Western Blot, Migration

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    Alomone Labs wild type probdnf
    Proneurotrophins abolish retinal axon branching via p75 NTR . The outgrowth/branching assay was performed as described [ 6 ]. Cells from E8 nasal retina were electroporated with eGFP and plated on a merosin/laminin substrate. (A) Cultures were treated at 1 day in vitro with 5 ng/ml BDNF, or 5 ng/ml proneurotrophins in the presence of 5 ng/ml BDNF as indicated, and fixed and analysed for branch number per axon after 3 days in vitro . The basal level of branching was not affected by treatment of retinal cultures with proneurotrophins alone. However, both <t>proBDNF</t> and proNGF led to a downregulation of the BDNF-induced branching to basal levels. (B) The length of outgrowth of retinal axons is not affected by treatment with neurotrophins and/or proneurotrophins. Axon length is given in arbitrary units. (C) To show that the proBDNF effect is mediated via p75 NTR , retinal cultures were electroporated either with an RNAi vector resulting in the knockdown of p75 NTR , with an RNAi vector not affecting p75 NTR protein levels or with empty vector. After plating, the cultures were treated with pro/neurotrophins as described in (A). p75 NTR knockdown obliterates the branch-suppressing effect of proBDNF. Three independent experiments were performed. The statistical analysis was done using Kruskal-Wallis test and Dunn's multiple comparison test post hoc . *** P
    Wild Type Probdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wild type probdnf/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
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    wild type probdnf - by Bioz Stars, 2022-05
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    94
    Alomone Labs cleavage resistant probdnf
    The increase <t>proBDNF</t> alters synaptic currents, promotes LFS-induced synaptic depression and strengthens the theta phase-gamma amplitude coupling during the PR-LTM test. (A) Schematic describing the timeline for morphological analysis (Top-left). Illustration of the region of interest in prelimbic images (Top-middle), and dendritic segment analysis for spine quantification (Top-right). Red circles indicated the mushroom type spine, yellow circles indicated thin type spine and blue circles indicated stubby type spine. Sample images were projected at minimal intensity and inverted, background was then subtracted, followed by brightness/contrast adjustment. Scale bars, 5 μm. Quantification of spine density (Bottom-left) and the proportion of spine (Bottom-right). No statistical difference in spine density was found between juvenile and adult groups. However, a significant higher proportion of thin type spine but a lower mushroom type spine was observed in juveniles compared with adults. (B) Schematic describing the timeline for EPSCs recordings (Top-left). Representative continuous traces (Top-middle) and average waveform (Top-right) of the pharmacologically isolated NMDA EPSCs in the prelimbic neurons of adult, juvenile and juvenile+anti groups. No change in the amplitude of EPSCs (Bottom-left) was found but the frequency (Bottom-middle) and decay time (Bottom-right) were significantly increased in juvenile group. The enhanced frequency and decay time of NMDA currents in juvenile group were inhibited after infusions of anti-proBDNF antibody. (* P
    Cleavage Resistant Probdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proneurotrophins abolish retinal axon branching via p75 NTR . The outgrowth/branching assay was performed as described [ 6 ]. Cells from E8 nasal retina were electroporated with eGFP and plated on a merosin/laminin substrate. (A) Cultures were treated at 1 day in vitro with 5 ng/ml BDNF, or 5 ng/ml proneurotrophins in the presence of 5 ng/ml BDNF as indicated, and fixed and analysed for branch number per axon after 3 days in vitro . The basal level of branching was not affected by treatment of retinal cultures with proneurotrophins alone. However, both proBDNF and proNGF led to a downregulation of the BDNF-induced branching to basal levels. (B) The length of outgrowth of retinal axons is not affected by treatment with neurotrophins and/or proneurotrophins. Axon length is given in arbitrary units. (C) To show that the proBDNF effect is mediated via p75 NTR , retinal cultures were electroporated either with an RNAi vector resulting in the knockdown of p75 NTR , with an RNAi vector not affecting p75 NTR protein levels or with empty vector. After plating, the cultures were treated with pro/neurotrophins as described in (A). p75 NTR knockdown obliterates the branch-suppressing effect of proBDNF. Three independent experiments were performed. The statistical analysis was done using Kruskal-Wallis test and Dunn's multiple comparison test post hoc . *** P

    Journal: Neural Development

    Article Title: Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance

    doi: 10.1186/1749-8104-5-30

    Figure Lengend Snippet: Proneurotrophins abolish retinal axon branching via p75 NTR . The outgrowth/branching assay was performed as described [ 6 ]. Cells from E8 nasal retina were electroporated with eGFP and plated on a merosin/laminin substrate. (A) Cultures were treated at 1 day in vitro with 5 ng/ml BDNF, or 5 ng/ml proneurotrophins in the presence of 5 ng/ml BDNF as indicated, and fixed and analysed for branch number per axon after 3 days in vitro . The basal level of branching was not affected by treatment of retinal cultures with proneurotrophins alone. However, both proBDNF and proNGF led to a downregulation of the BDNF-induced branching to basal levels. (B) The length of outgrowth of retinal axons is not affected by treatment with neurotrophins and/or proneurotrophins. Axon length is given in arbitrary units. (C) To show that the proBDNF effect is mediated via p75 NTR , retinal cultures were electroporated either with an RNAi vector resulting in the knockdown of p75 NTR , with an RNAi vector not affecting p75 NTR protein levels or with empty vector. After plating, the cultures were treated with pro/neurotrophins as described in (A). p75 NTR knockdown obliterates the branch-suppressing effect of proBDNF. Three independent experiments were performed. The statistical analysis was done using Kruskal-Wallis test and Dunn's multiple comparison test post hoc . *** P

    Article Snippet: Experimental reagents EphA7-Fc was from R & D Systems, Fc control from Calbiochem, BDNF from Promega, wild-type proBDNF and proNGF from Alomone Lab (Jerusalem, Israel).

    Techniques: In Vitro, Plasmid Preparation

    Expression of proBDNF in chick RGC axons . Retinal single cell cultures derived from E6 chick retina were stained after 2 days in vitro using a monoclonal antibody against the pro-domain of proBDNF [ 14 ] according to protocols given in Yang et al . [ 14 ]. (A-C) An RGC growth cone stained with control antibody (B), and phalloidin (C) to show the location of actin. (A) The composite of (B) and (C). In all composites phalloidin is shown in green and proBDNF/control antibody in red. (D-F) An RGC axon stained with control antibody (E), and phalloidin (F). (D) The composite of (E) and (F). (G-I) An RGC axon stained with a proBDNF antibody (H) [ 14 ], and phalloidin (I). (G) The composite of (H) and (I). (J-L) . An RGC growth cone stained with proBDNF antibody (K), and phalloidin (L). (J) The composite of (K) and (L).

    Journal: Neural Development

    Article Title: Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance

    doi: 10.1186/1749-8104-5-30

    Figure Lengend Snippet: Expression of proBDNF in chick RGC axons . Retinal single cell cultures derived from E6 chick retina were stained after 2 days in vitro using a monoclonal antibody against the pro-domain of proBDNF [ 14 ] according to protocols given in Yang et al . [ 14 ]. (A-C) An RGC growth cone stained with control antibody (B), and phalloidin (C) to show the location of actin. (A) The composite of (B) and (C). In all composites phalloidin is shown in green and proBDNF/control antibody in red. (D-F) An RGC axon stained with control antibody (E), and phalloidin (F). (D) The composite of (E) and (F). (G-I) An RGC axon stained with a proBDNF antibody (H) [ 14 ], and phalloidin (I). (G) The composite of (H) and (I). (J-L) . An RGC growth cone stained with proBDNF antibody (K), and phalloidin (L). (J) The composite of (K) and (L).

    Article Snippet: Experimental reagents EphA7-Fc was from R & D Systems, Fc control from Calbiochem, BDNF from Promega, wild-type proBDNF and proNGF from Alomone Lab (Jerusalem, Israel).

    Techniques: Expressing, Derivative Assay, Staining, In Vitro

    Repellent axon guidance is disrupted in the presence of an anti-proBDNF antibody or a soluble extracellular domain of p75 NTR . (A) Quantification of axon growth preferences in the presence of an anti-proBDNF antibody [ 14 ] or a control antibody. Stripe assay experiments were performed in the presence of a proBDNF antibody (1:200) [ 14 ] or a control antibody (mouse monoclonal antibody for placental alkaline phosphatase; 1:200). The quantification of axon growth preferences shows an abolishment of repellent guidance in the presence of the proBDNF antibody (see also Additional file 4 ). Error bars represent the standard error of the mean. Statistics were performed using Kruskal-Wallis test and Dunn's multiple comparison test with *** P

    Journal: Neural Development

    Article Title: Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance

    doi: 10.1186/1749-8104-5-30

    Figure Lengend Snippet: Repellent axon guidance is disrupted in the presence of an anti-proBDNF antibody or a soluble extracellular domain of p75 NTR . (A) Quantification of axon growth preferences in the presence of an anti-proBDNF antibody [ 14 ] or a control antibody. Stripe assay experiments were performed in the presence of a proBDNF antibody (1:200) [ 14 ] or a control antibody (mouse monoclonal antibody for placental alkaline phosphatase; 1:200). The quantification of axon growth preferences shows an abolishment of repellent guidance in the presence of the proBDNF antibody (see also Additional file 4 ). Error bars represent the standard error of the mean. Statistics were performed using Kruskal-Wallis test and Dunn's multiple comparison test with *** P

    Article Snippet: Experimental reagents EphA7-Fc was from R & D Systems, Fc control from Calbiochem, BDNF from Promega, wild-type proBDNF and proNGF from Alomone Lab (Jerusalem, Israel).

    Techniques: Stripping Membranes

    Exogenous proBDNF induces pain hypersensitivity and spinal cord activation in mice. ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P

    Journal: Scientific Reports

    Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    doi: 10.1038/srep27171

    Figure Lengend Snippet: Exogenous proBDNF induces pain hypersensitivity and spinal cord activation in mice. ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P

    Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

    Techniques: Activation Assay, Mouse Assay, Injection

    Polyclonal Ab-proBDNF pretreatment attenuates inflammatory pain in mice. ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P

    Journal: Scientific Reports

    Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    doi: 10.1038/srep27171

    Figure Lengend Snippet: Polyclonal Ab-proBDNF pretreatment attenuates inflammatory pain in mice. ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P

    Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

    Techniques: Mouse Assay, Injection

    Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P

    Journal: Scientific Reports

    Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    doi: 10.1038/srep27171

    Figure Lengend Snippet: Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P

    Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

    Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay, Western Blot, Migration

    Upregulation of p75NTR and effect of local proBDNF injection on inflammatory reaction in mice. ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p

    Journal: Scientific Reports

    Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    doi: 10.1038/srep27171

    Figure Lengend Snippet: Upregulation of p75NTR and effect of local proBDNF injection on inflammatory reaction in mice. ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p

    Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

    Techniques: Injection, Mouse Assay, Western Blot, Expressing

    Upregulation of proBDNF in the local tissue in acute and persistent inflammatory pain in mice. ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p

    Journal: Scientific Reports

    Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    doi: 10.1038/srep27171

    Figure Lengend Snippet: Upregulation of proBDNF in the local tissue in acute and persistent inflammatory pain in mice. ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p

    Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

    Techniques: Mouse Assay, Western Blot, Injection

    Expression of wildtype ( WT ) proBDNF and its N121Q mutant ( NQ ) in stably transfected HEK293F cells. Protein expression was determined by Western blotting using an antibody, recognizing both BDNF and proBDNF, in the conditioned media ( A ) and cell lysates ( B ). Arrows point to (pro)BDNF forms: glycosylated proBDNF ( black arrow ), nonglycosylated proBDNF ( white arrow ), and mature BDNF ( gray arrow ). M indicates MagicMark molecular weight marker. Expression of mRNA was determined in WT and N121Q mutant by RT-PCR ( C ). ProBDNF/BDNF expression in cell lysates ( D ) or conditioned media ( E ) of HEK293F cells expressing N121Q mutant treated with proteasome inhibitor MG132 ( MG ), lysosomal inhibitor chloroquine ( CQ ), or chemical chaperone 4-phenylbutyric acid ( PBA ). The black arrow points to the position of nonglycosylated proBDNF.

    Journal: The Journal of Biological Chemistry

    Article Title: N-Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

    doi: 10.1074/jbc.RA119.009989

    Figure Lengend Snippet: Expression of wildtype ( WT ) proBDNF and its N121Q mutant ( NQ ) in stably transfected HEK293F cells. Protein expression was determined by Western blotting using an antibody, recognizing both BDNF and proBDNF, in the conditioned media ( A ) and cell lysates ( B ). Arrows point to (pro)BDNF forms: glycosylated proBDNF ( black arrow ), nonglycosylated proBDNF ( white arrow ), and mature BDNF ( gray arrow ). M indicates MagicMark molecular weight marker. Expression of mRNA was determined in WT and N121Q mutant by RT-PCR ( C ). ProBDNF/BDNF expression in cell lysates ( D ) or conditioned media ( E ) of HEK293F cells expressing N121Q mutant treated with proteasome inhibitor MG132 ( MG ), lysosomal inhibitor chloroquine ( CQ ), or chemical chaperone 4-phenylbutyric acid ( PBA ). The black arrow points to the position of nonglycosylated proBDNF.

    Article Snippet: The concentration of proBDNF in individual fractions was routinely quantified by commercial sandwich proBDNF Duoset ELISA (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions and confirmed by Coomassie Blue staining compared with proBDNF standards (Alomone Labs, Jerusalem, Israel) of known concentrations.

    Techniques: Expressing, Mutagenesis, Stable Transfection, Transfection, Western Blot, Molecular Weight, Marker, Reverse Transcription Polymerase Chain Reaction

    Kinetics of cleavage of glycosylated and nonglycosylated proBDNF by furin. Mass difference between glycosylated and nonglycosylated proBDNF enabled densitometric quantification of each proBDNF form: A, representative blot of the cleavage kinetic at the indicated time intervals; B, densitometric analysis of proBDNF intensities at the indicated time intervals. Results are expressed as percent of the proBDNF concentration (mean ± S.D., n = 3) at the beginning of the reaction. Note that proBDNF without C-terminal Myc-FLAG tag was used in the reaction as described under “Experimental procedures.”

    Journal: The Journal of Biological Chemistry

    Article Title: N-Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

    doi: 10.1074/jbc.RA119.009989

    Figure Lengend Snippet: Kinetics of cleavage of glycosylated and nonglycosylated proBDNF by furin. Mass difference between glycosylated and nonglycosylated proBDNF enabled densitometric quantification of each proBDNF form: A, representative blot of the cleavage kinetic at the indicated time intervals; B, densitometric analysis of proBDNF intensities at the indicated time intervals. Results are expressed as percent of the proBDNF concentration (mean ± S.D., n = 3) at the beginning of the reaction. Note that proBDNF without C-terminal Myc-FLAG tag was used in the reaction as described under “Experimental procedures.”

    Article Snippet: The concentration of proBDNF in individual fractions was routinely quantified by commercial sandwich proBDNF Duoset ELISA (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions and confirmed by Coomassie Blue staining compared with proBDNF standards (Alomone Labs, Jerusalem, Israel) of known concentrations.

    Techniques: Concentration Assay, FLAG-tag

    ProBDNF stably expressed in HEK293F cells is glycosylated. N -Glycosylation of proBDNF and the prodomain is documented by mass shift following deglycosylation with PNGase F: A, detection of proBDNF and BDNF by an antibody recognizing the BDNF region; B, detection of proBDNF and the prodomain by an antibody recognizing the prodomain region. The gel images were spliced as indicated by space to exclude samples not related to the study.

    Journal: The Journal of Biological Chemistry

    Article Title: N-Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

    doi: 10.1074/jbc.RA119.009989

    Figure Lengend Snippet: ProBDNF stably expressed in HEK293F cells is glycosylated. N -Glycosylation of proBDNF and the prodomain is documented by mass shift following deglycosylation with PNGase F: A, detection of proBDNF and BDNF by an antibody recognizing the BDNF region; B, detection of proBDNF and the prodomain by an antibody recognizing the prodomain region. The gel images were spliced as indicated by space to exclude samples not related to the study.

    Article Snippet: The concentration of proBDNF in individual fractions was routinely quantified by commercial sandwich proBDNF Duoset ELISA (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions and confirmed by Coomassie Blue staining compared with proBDNF standards (Alomone Labs, Jerusalem, Israel) of known concentrations.

    Techniques: Stable Transfection

    A, schematic illustration of proBDNF with zoom into the region containing the N -glycosylation and proteolytic cleavage sites involved in the generation of mature BDNF; B, ) (please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site) (multiple alignments were performed with Align tool of the Clustal Omega 1.2.4 engine). Letters in red represent amino acids that differ from the human sequence. Underlined text highlights the NX(S/T) N -glycosylation sequon.

    Journal: The Journal of Biological Chemistry

    Article Title: N-Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

    doi: 10.1074/jbc.RA119.009989

    Figure Lengend Snippet: A, schematic illustration of proBDNF with zoom into the region containing the N -glycosylation and proteolytic cleavage sites involved in the generation of mature BDNF; B, ) (please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site) (multiple alignments were performed with Align tool of the Clustal Omega 1.2.4 engine). Letters in red represent amino acids that differ from the human sequence. Underlined text highlights the NX(S/T) N -glycosylation sequon.

    Article Snippet: The concentration of proBDNF in individual fractions was routinely quantified by commercial sandwich proBDNF Duoset ELISA (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions and confirmed by Coomassie Blue staining compared with proBDNF standards (Alomone Labs, Jerusalem, Israel) of known concentrations.

    Techniques: Sequencing

    Extracted ion chromatograms of four of the most abundant glycopeptides of proBDNF expressed in PC12 cells. A , C , and D, LacdiNAc containing structures; B, LacNAc containing structure. The arrow points to the peaks of glycopeptides aligned with slight retention time shifts as expected based on the hydrophilicity of the attached glycan. Numbers represent intensity of the extracted peaks (×10 5 ).

    Journal: The Journal of Biological Chemistry

    Article Title: N-Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

    doi: 10.1074/jbc.RA119.009989

    Figure Lengend Snippet: Extracted ion chromatograms of four of the most abundant glycopeptides of proBDNF expressed in PC12 cells. A , C , and D, LacdiNAc containing structures; B, LacNAc containing structure. The arrow points to the peaks of glycopeptides aligned with slight retention time shifts as expected based on the hydrophilicity of the attached glycan. Numbers represent intensity of the extracted peaks (×10 5 ).

    Article Snippet: The concentration of proBDNF in individual fractions was routinely quantified by commercial sandwich proBDNF Duoset ELISA (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions and confirmed by Coomassie Blue staining compared with proBDNF standards (Alomone Labs, Jerusalem, Israel) of known concentrations.

    Techniques:

    The increase proBDNF alters synaptic currents, promotes LFS-induced synaptic depression and strengthens the theta phase-gamma amplitude coupling during the PR-LTM test. (A) Schematic describing the timeline for morphological analysis (Top-left). Illustration of the region of interest in prelimbic images (Top-middle), and dendritic segment analysis for spine quantification (Top-right). Red circles indicated the mushroom type spine, yellow circles indicated thin type spine and blue circles indicated stubby type spine. Sample images were projected at minimal intensity and inverted, background was then subtracted, followed by brightness/contrast adjustment. Scale bars, 5 μm. Quantification of spine density (Bottom-left) and the proportion of spine (Bottom-right). No statistical difference in spine density was found between juvenile and adult groups. However, a significant higher proportion of thin type spine but a lower mushroom type spine was observed in juveniles compared with adults. (B) Schematic describing the timeline for EPSCs recordings (Top-left). Representative continuous traces (Top-middle) and average waveform (Top-right) of the pharmacologically isolated NMDA EPSCs in the prelimbic neurons of adult, juvenile and juvenile+anti groups. No change in the amplitude of EPSCs (Bottom-left) was found but the frequency (Bottom-middle) and decay time (Bottom-right) were significantly increased in juvenile group. The enhanced frequency and decay time of NMDA currents in juvenile group were inhibited after infusions of anti-proBDNF antibody. (* P

    Journal: bioRxiv

    Article Title: Prelimbic proBDNF facilitates memory destabilization by regulation of neuronal function in juveniles

    doi: 10.1101/2021.12.30.474526

    Figure Lengend Snippet: The increase proBDNF alters synaptic currents, promotes LFS-induced synaptic depression and strengthens the theta phase-gamma amplitude coupling during the PR-LTM test. (A) Schematic describing the timeline for morphological analysis (Top-left). Illustration of the region of interest in prelimbic images (Top-middle), and dendritic segment analysis for spine quantification (Top-right). Red circles indicated the mushroom type spine, yellow circles indicated thin type spine and blue circles indicated stubby type spine. Sample images were projected at minimal intensity and inverted, background was then subtracted, followed by brightness/contrast adjustment. Scale bars, 5 μm. Quantification of spine density (Bottom-left) and the proportion of spine (Bottom-right). No statistical difference in spine density was found between juvenile and adult groups. However, a significant higher proportion of thin type spine but a lower mushroom type spine was observed in juveniles compared with adults. (B) Schematic describing the timeline for EPSCs recordings (Top-left). Representative continuous traces (Top-middle) and average waveform (Top-right) of the pharmacologically isolated NMDA EPSCs in the prelimbic neurons of adult, juvenile and juvenile+anti groups. No change in the amplitude of EPSCs (Bottom-left) was found but the frequency (Bottom-middle) and decay time (Bottom-right) were significantly increased in juvenile group. The enhanced frequency and decay time of NMDA currents in juvenile group were inhibited after infusions of anti-proBDNF antibody. (* P

    Article Snippet: Needles were inserted into bilateral cannulae and then cleavage-resistant proBDNF (2 ng/ml; Cat#B257 Alomone Labs), anti-proBDNF antibody (10 μg/μL; Cat#ANT-006, Alomone Labs), TAT-Pep5 (4 ng/μL; Cat#506181, EMD Millipore), K252a (25 μg/μL; Cat#82497; Sigma-Aldrich), 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP; 32 ng/μL; Cat#01773, Tocris Bioscience), NVP-AAM077 (0.8 ng/μL; Cat#P1999, Sigma-Aldrich), Ro25-6981 (2.0 ng/μL; Cat#1594, Tocris Bioscience), mature BDNF (1.5 μg/mL; Cat#B250; Alomone Labs) or artificial CSF (ACSF, Cat#3525, Tocris Bioscience) into prelimbic area (at a rate of 0.5 μL/min/side for 2 min) was infused immediately or one day following memory retrieval.

    Techniques: Isolation

    The higher prelimbic proBDNF expression during the juvenile period facilitates retrieval-dependent memory destabilization. Quantification of the proBDNF (A) and its receptor p75 NTR (B) levels in prelimbic cortex of juvenile and adult rats. Representative immunoblots the expression of proBDNF and p75 NTR (Top). A significant increase in the proBDNF levels was detected in juvenile group, as well the p75 NTR levels (Bottom). (* P

    Journal: bioRxiv

    Article Title: Prelimbic proBDNF facilitates memory destabilization by regulation of neuronal function in juveniles

    doi: 10.1101/2021.12.30.474526

    Figure Lengend Snippet: The higher prelimbic proBDNF expression during the juvenile period facilitates retrieval-dependent memory destabilization. Quantification of the proBDNF (A) and its receptor p75 NTR (B) levels in prelimbic cortex of juvenile and adult rats. Representative immunoblots the expression of proBDNF and p75 NTR (Top). A significant increase in the proBDNF levels was detected in juvenile group, as well the p75 NTR levels (Bottom). (* P

    Article Snippet: Needles were inserted into bilateral cannulae and then cleavage-resistant proBDNF (2 ng/ml; Cat#B257 Alomone Labs), anti-proBDNF antibody (10 μg/μL; Cat#ANT-006, Alomone Labs), TAT-Pep5 (4 ng/μL; Cat#506181, EMD Millipore), K252a (25 μg/μL; Cat#82497; Sigma-Aldrich), 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP; 32 ng/μL; Cat#01773, Tocris Bioscience), NVP-AAM077 (0.8 ng/μL; Cat#P1999, Sigma-Aldrich), Ro25-6981 (2.0 ng/μL; Cat#1594, Tocris Bioscience), mature BDNF (1.5 μg/mL; Cat#B250; Alomone Labs) or artificial CSF (ACSF, Cat#3525, Tocris Bioscience) into prelimbic area (at a rate of 0.5 μL/min/side for 2 min) was infused immediately or one day following memory retrieval.

    Techniques: Expressing, Western Blot

    Up-regulation of proBDNF-p75NTR signaling mediated by NMDA-GluN2B contributes to enhance the modulation of existing fear memory traces in juvenile rats. (A) Schematic describing the behavioral timeline for the retrieval-dependent memory destabilization experiment using rats conditioned with four tones (Top). Immediately following the memory retrieval, the rats infused with TAT-Pep5, K252a or vehicle into the prelimbic cortex 15 min prior to the mBDNF, proBDNF or vehicle infusion. Two days later, PR-LTM was assessed by exposed the rats to the novel context. Similar, no significant difference in the percentage freezing during the memory retrieval but the percentage freezing level during the PR-LTM test was significant lower in juvenile group than adult group (Bottom). No obvious effect of mBDNF on freeze behavior was found. Infusions of p75 NTR blocker TAT-Pep5 could significantly enhance the percentage of freeze behavior. Meanwhile, infusions of TAT-Pep5, but not K252a, markedly blocked the effects of proBDNF treatment. (* P

    Journal: bioRxiv

    Article Title: Prelimbic proBDNF facilitates memory destabilization by regulation of neuronal function in juveniles

    doi: 10.1101/2021.12.30.474526

    Figure Lengend Snippet: Up-regulation of proBDNF-p75NTR signaling mediated by NMDA-GluN2B contributes to enhance the modulation of existing fear memory traces in juvenile rats. (A) Schematic describing the behavioral timeline for the retrieval-dependent memory destabilization experiment using rats conditioned with four tones (Top). Immediately following the memory retrieval, the rats infused with TAT-Pep5, K252a or vehicle into the prelimbic cortex 15 min prior to the mBDNF, proBDNF or vehicle infusion. Two days later, PR-LTM was assessed by exposed the rats to the novel context. Similar, no significant difference in the percentage freezing during the memory retrieval but the percentage freezing level during the PR-LTM test was significant lower in juvenile group than adult group (Bottom). No obvious effect of mBDNF on freeze behavior was found. Infusions of p75 NTR blocker TAT-Pep5 could significantly enhance the percentage of freeze behavior. Meanwhile, infusions of TAT-Pep5, but not K252a, markedly blocked the effects of proBDNF treatment. (* P

    Article Snippet: Needles were inserted into bilateral cannulae and then cleavage-resistant proBDNF (2 ng/ml; Cat#B257 Alomone Labs), anti-proBDNF antibody (10 μg/μL; Cat#ANT-006, Alomone Labs), TAT-Pep5 (4 ng/μL; Cat#506181, EMD Millipore), K252a (25 μg/μL; Cat#82497; Sigma-Aldrich), 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP; 32 ng/μL; Cat#01773, Tocris Bioscience), NVP-AAM077 (0.8 ng/μL; Cat#P1999, Sigma-Aldrich), Ro25-6981 (2.0 ng/μL; Cat#1594, Tocris Bioscience), mature BDNF (1.5 μg/mL; Cat#B250; Alomone Labs) or artificial CSF (ACSF, Cat#3525, Tocris Bioscience) into prelimbic area (at a rate of 0.5 μL/min/side for 2 min) was infused immediately or one day following memory retrieval.

    Techniques: