Journal: Molecular Brain

Article Title: proBDNF is modified by advanced glycation end products in Alzheimer’s disease and causes neuronal apoptosis by inducing p75 neurotrophin receptor processing

doi: 10.1186/s13041-018-0411-6

Figure Lengend Snippet: proBDNF modified by MGO induces apoptosis in control mice differentiated hippocampal neural stem cells . a) Representative pictures of neuronal primary culture treated for 6 days (7DIV) with: untreated (control), MGO modified BSA (MBSA), mBDNF, proBDNF and two doses of MGO modified proBDNF (MproBDNF). DAPI staining (white) of nuclei shows apoptotic morphology (white arrow). Immunostaining with anti βIIItubulin (red) show differentiation degree. b) Bars represent the quantification of apoptosis (by nuclei morphology), expressed as the mean of % apoptosis ± SD of the total nuclei. c) The quantification of differentiation is expressed as the mean of % differentiation (βIIItubulin immunoreactivity quantified by ImageJ, divided by the number of β III tubulin positive cells). Values represent the mean of three independent experiments. Scale bar =25 μm . ** p < 0,01, *** p < 0,001 two tailed Student’s “t” test

Article Snippet: Recombinant human proBDNF (Alomone) was modified by the reactive carbonyl specie MGO that react with free amino groups of Lys residues on proteins, leading to the formation of CEL adducts and intermolecular crosslinks [ ].

Techniques: Modification, Staining, Immunostaining, Two Tailed Test, T-Test

Journal: Molecular Brain

Article Title: proBDNF is modified by advanced glycation end products in Alzheimer’s disease and causes neuronal apoptosis by inducing p75 neurotrophin receptor processing

doi: 10.1186/s13041-018-0411-6

Figure Lengend Snippet: Human proBDNF from CSF AD patients induces apoptosis in neuronal culture. a) Representative pictures of neuronal primary culture from control mice Dentate Gyrus. Cells were treated for 6 days (7DIV) with human CSF from controls (control CSF) and from AD-affected patients (ADCSF). DAPI staining of nuclei shows apoptotic morphology (white arrow). Immunofluorescence with anti p75 ICD domain (green) and anti Sortilin (red). Inset indicate a higher magnification image of the boxed areas. White arrow in control CSF shows neurons with peripheral p75ICD location. White arrows in AD CSF show p75ICD nuclear location. b) Representative image of western blot showing the levels of proBDNF in CSF and in CSF immunodepleted with anti proBDNF (ID), from control and AD samples. c and d) Cells are treated with human 8 control CSF and 8 ADCSF, in both cases directly (CSF) or proBDNF immunodepleted (SCFID). c) Bars represent the quantification of apoptosis (by nuclei morphology), expressed as the mean of % apoptosis ± SD of the positive cells for both p75 and Sortilin. d) Bars represent the quantification of the mean of the % ± SD of neurons with the ICDp75 translocated to the nuclei (% ICD nuclear). Scale bar =50 μm . * p < 0,05, ** p < 0,01, two tailed Student’s “t” test

Article Snippet: Recombinant human proBDNF (Alomone) was modified by the reactive carbonyl specie MGO that react with free amino groups of Lys residues on proteins, leading to the formation of CEL adducts and intermolecular crosslinks [ ].

Techniques: Staining, Immunofluorescence, Western Blot, Two Tailed Test, T-Test

Journal: Molecular Brain

Article Title: proBDNF is modified by advanced glycation end products in Alzheimer’s disease and causes neuronal apoptosis by inducing p75 neurotrophin receptor processing

doi: 10.1186/s13041-018-0411-6

Figure Lengend Snippet: Apoptosis and p75 processing are increased in neuronal cultures from APP/PS1 animals. Primary cultures of differentiated mouse hippocampal NSCs were treated for 6 days (7DIV) with MGO modified BSA (MBSA) or with MGO modified proBDNF (MproBDNF). a) Representative immunofluorescence pictures of cells from WT and APP/P1 mice labeled with ICDp75 (green), βIIItubulin (red) and DAPI (white). Inset represents a higher magnification image of the boxed areas indicating citoplasmatic (WT) and nuclear localization (APP/PS1) of ICDp75. b) Bars represent the quantification of apoptosis (by nuclei morphology), expressed as the mean of % apoptosis ± SD of the positive cells for both ICDp75 and βIIItubulin. c) Bars represent the mean of the % ± SD of neurons with the ICDp75 translocated to the nuclei (% ICD nuclear). The values represent the mean of three independent experiments Scale bar =50 μm . ** p < 0,01, two tailed Student’s “t” test

Article Snippet: Recombinant human proBDNF (Alomone) was modified by the reactive carbonyl specie MGO that react with free amino groups of Lys residues on proteins, leading to the formation of CEL adducts and intermolecular crosslinks [ ].

Techniques: Modification, Immunofluorescence, Labeling, Two Tailed Test, T-Test

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: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Stable Transfection

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 (XIC) of (A) nonglycosylated, NYLDAANMSMR and (B) deglycosylated, NYLDAADMSMR glycopeptide of proBDNF secreted by HEK293F cells confirm site occupancy higher than 99%. C, tandem mass spectrum verifies deglycosylation of the proBDNF peptide under [18O]water.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques:

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: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

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

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: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Concentration Assay, FLAG-tag

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: Mass spectra of proBDNF under the following conditions. A, precursor profile of the tryptic glycopeptide NYLDAANM(ox)SM(ox)R of proBDNF expressed in HEK293F cells; B, HCD fragmentation spectrum of the major glycoform (m/z 1132.42) at low collision energy (10% NCE) showing that characteristic LacdiNAc oxonium ion HexNAc-HexNAc (m/z 407.17) and its fucosylated form (m/z 553.22) with complementary y-ions (m/z 1373, 1381, 1454, and 1555) dominate the composition.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques:

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: Relative abundance of the glycoforms of HEK293F-produced proBDNF and prodomain expressed as % of all identified glycoforms

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Produced

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: Impact of modulators of glycosylation on the BDNF/proBDNF ratio in HEK293F cells. Western blotting using antibodies to prodomain and mature BDNF regions was analyzed by densitometry for the following: A, intracellular BDNF to proBDNF ratio; B, secreted BDNF to proBDNF ratio; C, intracellular prodomain to proBDNF ratio; and D, secreted prodomain to proBDNF ratio. The representative Western blotting is labeled as the graphs: CTRL, control; CHLOR, 50 mm sodium chlorate; KIF, 1 μg/ml kifunensine; TNMC, 5 μg/ml of tunicamycin. Results are expressed as scatter plot (mean ± S.D., n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with CTRL, one-way analysis of variance with Bonferroni adjustment.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Western Blot, Labeling

Journal: The Journal of Neuroscience

Article Title: Pro-Brain-Derived Neurotrophic Factor Inhibits GABAergic Neurotransmission by Activating Endocytosis and Repression of GABA A Receptors

doi: 10.1523/JNEUROSCI.2069-14.2014

Figure Lengend Snippet: Endogenous proBDNF decreases the colocalization of postsynaptic GABAAR β2/3 clusters with presynaptic VAMP1 puncta. A, proBDNF protein levels in hippocampal culture supernatants in control and aprotinin conditions. Treatment with aprotinin (Apro; 3 μg/ml, 12 h) increases significantly the concentration of proBDNF in the supernatant compared with control (CTR). n = 3; ***p < 0.001. B, Hippocampal cultures (14 DIV) were treated for 12 h with vehicle (CTR), aprotinin (Apro), aprotinin plus TAT-pep5 (Apro + TATpep5), or TAT-pep5 alone, and stained for GABAAR β2/3 subunit (green), VAMP1 (red), and MAP2 (blue). Scale bars: top, 20 μm; bottom, 3 μm. C, The percentage of β2/3 subunit-positive clusters that colocalized with VAMP1 puncta/intracellular MAP2 fluorescence ratio was significantly reduced in cultures treated with aprotinin (3 μg/ml, 12 h). D, E, Aprotinin (Apro) significantly decreased the average β2/3 subunit-positive area fraction/intracellular MAP2 fluorescence ratio (D) and the average perimeter of GABAAR β2/3 clusters (E) at the cell surface of MAP2-positive neurons, in comparison with the vehicle-treated control (CTR), in the presence of TAT-pep5 (Apro + TATpep5) or TAT-pep5 alone (TATpep5). Thirty-five neurons analyzed per condition, n = 4; ***p < 0.001. F, Quantification of surface VAMP1/MAP2 fluorescence ratio, in neurons treated with aprotinin alone (Apro), aprotinin plus TAT-pep5 (Apro + TATpep5), or TAT-pep5 alone (Tatpep5; 2 μm), in comparison with the vehicle-treated control (CTR). Thirty-five neurons analyzed per condition, per experiment; n = 4; ***p < 0.001, **p < 0.01. G, Cell-surface levels of GABAARs measured using ELISA assays with GABAAR β2/3 subunit-specific antibody in neurons treated with aprotinin alone (Apro; 3 μg/ml, 12 h), with Apro in the presence of TAT-pep5 (Apro + TAT-pep5; 2 μm), with Apro and nifedipine (Apro + nifedipine; 10 μm), and only in the presence TAT-pep5 (TAT-pep5; 10 μm), and expressed as a percentage of vehicle-treated controls (CTR). H, Total GABAAR β2/3 subunit expression normalized to control nontreated conditions. n = 6; ***p < 0.001. I, Relative expression of GABAAR β3 mRNA transcripts in neurons treated with aprotinin (Apro; 3 μg/ml, 12 h), with aprotinin and TAT-pep5 (Apro + TATpep5; 2 μm), with K252a alone (200 nm), or with TAT-pep5 alone (2 μm), and expressed as a percentage of vehicle-treated controls (CTR). GABAAR β3 subunit gene expression was normalized to the expression of the housekeeping gene, GAPDH, in the same RNA preparation. n = 6; ***p < 0.001.

Article Snippet: TTX was purchased from Abcam; ROCKi (Y-27632) was purchased from Millipore; tPA-Stop was purchased from American Diagnostica; k252a, TAT-pep5, and Stattic were purchased from Calbiochem; nifedipine, aprotinin, leupeptin, and AG490 were purchased from Sigma-Aldrich; CR-proBDNF recombinant ( Escherichia coli ) mutated human and mBDNF were purchased from Alomone Labs. NBQX, CNQX, and d -APV were obtained from the Molecular, Cellular, and Genomic Neuroscience Research Branch of the National Institute of Mental Health.

Techniques: Concentration Assay, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Journal of Neuroscience

Article Title: Pro-Brain-Derived Neurotrophic Factor Inhibits GABAergic Neurotransmission by Activating Endocytosis and Repression of GABA A Receptors

doi: 10.1523/JNEUROSCI.2069-14.2014

Figure Lengend Snippet: Apoptosis is not triggered by proBDNF. A, Image of hippocampal neurons transfected at 10 DIV with the CR-proBDNF Cherry (in red) and/or Cherry (in red), and costained at 14 DIV with the cleaved caspase-3 (in blue). B, Cultured hippocampal neurons (14 DIV) in control conditions (CTR), or treated with CR-proBDNF (25 ng, 30 min), or CR-proBDNF plus TAT-pep5 (CR-proBDNF + TATpep5; 2 μm), or aprotinin (Apro; 3 μg/ml, 12 h), or aprotinin plus TAT-pep5 (Apro + TATpep5; 2 μm), and immunolabeled with cleaved caspase-3 antibody (red), and costained with anti-MAP2 antibody (green). C, Ratio of cleaved caspase-3-positive (apoptotic) neuronal number to total MAP2-positive cells, and expressed as a percentage of vehicle-treated controls (CTR). Overexpression of CR-proBDNF (CR-proBDNF Cherry), or exogenous application of CR-proBDNF (CR-proBDNF) or treatment with aprotinin (Apro) did not increase the number of apoptotic neurons compared with control (CTR). Fifty neurons analyzed per condition, per experiment; n = 4; p > 0.05.

Article Snippet: TTX was purchased from Abcam; ROCKi (Y-27632) was purchased from Millipore; tPA-Stop was purchased from American Diagnostica; k252a, TAT-pep5, and Stattic were purchased from Calbiochem; nifedipine, aprotinin, leupeptin, and AG490 were purchased from Sigma-Aldrich; CR-proBDNF recombinant ( Escherichia coli ) mutated human and mBDNF were purchased from Alomone Labs. NBQX, CNQX, and d -APV were obtained from the Molecular, Cellular, and Genomic Neuroscience Research Branch of the National Institute of Mental Health.

Techniques: Transfection, Cell Culture, Immunolabeling, Over Expression

Journal: The Journal of Neuroscience

Article Title: Pro-Brain-Derived Neurotrophic Factor Inhibits GABAergic Neurotransmission by Activating Endocytosis and Repression of GABA A Receptors

doi: 10.1523/JNEUROSCI.2069-14.2014

Figure Lengend Snippet: ProBDNF activates the RhoA-ROCK-PTEN pathway and promotes GABAAR dephosphorylation and endocytosis. A, CR-proBDNF decreases the phosphorylation of Ser408/409 in the GABAAR β3 subunit (pGABAAR β3). Total cellular proteins were analyzed using Western blot with polyclonal GABAAR β3 subunit and pGABAAR β3 subunit antibodies. Hippocampal neurons were treated with vehicle (CTR), CR-proBDNF (25 ng, 30 min), CR-proBDNF plus TAT-pep5 (CR-proBDNF + TATpep5; 2 μm), CR-proBDNF plus ROCKi (CR-proBDNF + ROCKi; 50 μm), CR-proBDNF plus Ag490 (CR-proBDNF + Ag490; 50 μm), or CR-proBDNF plus Stattic (CR-proBDNF + Stattic; 10 μm). Proteins were visualized using ECL following incubation with HRP-conjugated secondary antibody. Normalized data (pGABAAR β3/GABAAR β3) are expressed as percentage change with respect to vehicle-treated cultures (defined as 100%). Levels of total GABAAR β3 subunits were used as internal control. n = 4–6 experiments; *p < 0.05, **p < 0.01. B, Cell-surface levels of GABAARs measured using ELISA assays with GABAAR β2/3 subunit-specific antibody in neurons treated with CR-proBDNF alone (25 ng, 30 min) with CR-proBDNF in the presence of ROCKi (CR-proBDNF + ROCKi; 50 μm), or with ROCKi alone (50 μm), and expressed as a percentage of vehicle-treated controls. C, CR-proBDNF decreases the phosphorylation of T366 in the PTEN (pPTEN). Total cellular proteins were analyzed using Western blot with polyclonal PTEN and pPTEN antibodies. Hippocampal neurons were treated with vehicle (CTR), CR-proBDNF (25 ng, 30 min), CR-proBDNF plus ROCKi (CR-proBDNF + ROCKi; 50 μm), or ROCKi alone (50 μm). Proteins were visualized using ECL following incubation with HRP-conjugated secondary antibody. Normalized data (pPTEN/PTEN) are expressed as percentage change with respect to vehicle-treated cultures (defined as 100%). Levels of total PTEN were used as internal control. n = 5 experiments; *p < 0.05. D, Neurons were permeabilized and immunolabeled with anti-GABAAR β2/3 subunit-specific antibody (green), anti-EEA1 rabbit polyclonal antibody (red), and anti-MAP2 chicken polyclonal antibody (blue) to reveal the intracellular distribution of these proteins. GABAAR β2/3 subunits and EEA1 colocalization appear yellow in merge panels (right). Scale bar, 20 μm. E, The percentage of β2/3 subunit-positive clusters that colocalized with EEA1 puncta/intracellular MAP2 fluorescence ratio was significantly increased in cultures treated with CR-proBDNF (25 ng, 30 min). Thirty-five neurons analyzed per condition, n = 4; ***p < 0.001. F, Western blot analysis with the antibody to EEA1. EEA1 expression was upregulated after treatment with CR-proBDNF (25 ng, 30 min). Proteins were visualized using ECL following incubation with HRP-conjugated secondary antibody. Normalized data (EEA1/β-Tubulin) are expressed as percentage change with respect to vehicle-treated cultures (defined as 100%). n = 5 experiments; *p < 0.05. G, Cell-surface levels of GABAARs measured using ELISA assays with GABAAR β2/3 subunit-specific antibody in neurons treated with aprotinin (Apro; 3 μg/ml, 12 h) in the presence or absence of leupeptine (5 μg/ml), or with leupeptine alone (5 μg/ml), and expressed as a percentage of vehicle-treated controls (CTR). H, Total GABAAR β2/3 subunit expression normalized to control nontreated conditions. n = 9; **p < 0.01, ***p < 0.001.

Article Snippet: TTX was purchased from Abcam; ROCKi (Y-27632) was purchased from Millipore; tPA-Stop was purchased from American Diagnostica; k252a, TAT-pep5, and Stattic were purchased from Calbiochem; nifedipine, aprotinin, leupeptin, and AG490 were purchased from Sigma-Aldrich; CR-proBDNF recombinant ( Escherichia coli ) mutated human and mBDNF were purchased from Alomone Labs. NBQX, CNQX, and d -APV were obtained from the Molecular, Cellular, and Genomic Neuroscience Research Branch of the National Institute of Mental Health.

Techniques: De-Phosphorylation Assay, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Immunolabeling, Fluorescence, Expressing

Journal: The Journal of Neuroscience

Article Title: Pro-Brain-Derived Neurotrophic Factor Inhibits GABAergic Neurotransmission by Activating Endocytosis and Repression of GABA A Receptors

doi: 10.1523/JNEUROSCI.2069-14.2014

Figure Lengend Snippet: ProBDNF has no effect on phospho-CREB activation but induces ICER expression via the JAK2/STAT3 signaling cascade. A, Immunofluorescence signal of CREB (red, top), pCREB (green, middle) in hippocampal neurons in control conditions (CTR), or treated with CR-proBDNF alone (25 ng; CR-proBDNF), or CR-proBDNF plus mBDNF (CR-proBDNF + mBDNF; 25 ng), or CR-proBDNF plus mBDNF in the presence of K252a (CR-proBDNF + mBDNF + K252a; 200 nm). Right, Merged images with MAP2 staining (blue). Scale bar, 40 μm. B, Average ratio of pCREB/CREB in the different conditions normalized to control. n = 15 neurons analyzed per treatment in n = 3 independent experiments; ***p < 0.001. C, Immunofluorescence signal of ICER (red; top) in hippocampal neurons in control conditions (CTR), or treated with CR-proBDNF (25 ng) for 30 min (CR-proBDNF 30 min), for 12 h (CR-proBDNF 12 h), or for 30 min in the presence of TAT-pep5 (CR-pro-BDNF 30 min + TATpep5; 2 μm), following a costaining with anti-MAP2 chicken polyclonal antibody (green; bottom) to reveal neuronal cells. Scale bar: 20 μm. D, Average fluorescence intensity ratio of ICER/MAP2 in the different conditions normalized to control. Forty-five neurons analyzed per condition, n = 4; ***p < 0.001. E, Western blot analysis with the antibody to ICER. ICER expression was upregulated after acute treatment with CR-proBDNF (25 ng, 30 min) but not with chronic application of CR-proBDNF (25 ng, 12 h). The increase of ICER was antagonized by TAT-pep5 (2 μm), AG490 (50 μm), and Stattic (10 μm). This increase was insensitive to ROCKi (50 μm). Proteins were visualized using ECL following incubation with HRP-conjugated secondary antibody. Normalized data (ICER/β-Tubulin) are expressed as percentage change with respect to vehicle-treated cultures (defined as 100%). n = 4–6 experiments; **p < 0.01, ***p < 0.001. F, Relative expression of ICER mRNA transcripts in neurons treated with CR-proBDNF for 30 min (CR-proBDNF 30 min), with CR-proBDNF for 12 h (CR-proBDNF 12 h; 25 ng), or with CR-proBDNF plus TAT-pep5 for 30 min (CR-proBDNF 30 min + TATpep5; 2 μm), and expressed as a percentage of vehicle-treated controls (CTR). ICER gene expression was normalized to the expression of the housekeeping gene, GAPDH, in the same RNA preparation. n = 6; ***p < 0.001.

Article Snippet: TTX was purchased from Abcam; ROCKi (Y-27632) was purchased from Millipore; tPA-Stop was purchased from American Diagnostica; k252a, TAT-pep5, and Stattic were purchased from Calbiochem; nifedipine, aprotinin, leupeptin, and AG490 were purchased from Sigma-Aldrich; CR-proBDNF recombinant ( Escherichia coli ) mutated human and mBDNF were purchased from Alomone Labs. NBQX, CNQX, and d -APV were obtained from the Molecular, Cellular, and Genomic Neuroscience Research Branch of the National Institute of Mental Health.

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot, Incubation

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: ProBDNF cleavage by furin Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Stable Transfection

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: ProBDNF cleavage by furin Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

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

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: ProBDNF cleavage by furin Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Concentration Assay, FLAG-tag