Journal: PLoS ONE

Article Title: A Cross-Talk between TrkB and Ret Tyrosine Kinases Receptors Mediates Neuroblastoma Cells Differentiation

doi: 10.1371/journal.pone.0001643

Figure Lengend Snippet: (A) Left , RNA isolated from RA-treated SH-SY5Y, LAN-5 and SK-N-BE cells was reverse transcribed and amplified as described in . Samples loaded on the gel are as follows: lanes 1, 2 and 3 amplification of TrkB in SH-SY5Y, LAN-5 and SK-N-BE, respectively; lanes 4, 5 and 6 amplification of BDNF in SH-SY5Y, LAN-5 and SK-N-BE, respectively; lane 7 markers IX (Roche); lanes 8 to 13, RNA samples as in lanes 1 to 6 but in the absence of RT as negative controls. Right , Lysates from SH-SY5Y, LAN-5 and SK-N-BE, left untreated or RA-treated, were immunoblotted with anti-TrkB antibodies against the extracellular domain of the receptor. The molecular weights of full-length TrkB and hypothetical TrkB.T1 isoform are indicated. The membranes were stripped and re-probed with anti-αtubulin? antibodies to control equal loading. Blots shown are representative of at least five independent experiments. (B) SH-SY5Y ( left panels ), LAN-5 ( middle panels ) and SK-N-BE ( right panels ) cells were left untreated or treated with RA in the absence or in the presence of TrkB siRNA (siRNATrkB) or the negative control siRNAns, as indicated. Cell lysates were immunoprecipitated with anti-panTrk antibodies and analysed by Western blotting with anti-pTyr or anti-TrkB antibodies. Blots shown are representative of at least four independent experiments. (C) RA-treated SH-SY5Y, LAN-5 and SK-N-BE cells were left un-stimulated (lanes 1, 3 and 5) or stimulated with BDNF (lanes 2, 4 and 6). Cell extracts were immunoprecipitated with anti-panTrk antibodies and immonoblotted with anti-pTyr or anti-TrkB antibodies. Blots shown are representative of at least three independent experiments. In (A), (B) and (C), quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see legend to ).

Article Snippet: For BDNF acute stimulations, SH-SY5Y, LAN-5 and SK-N-BE (900.000 cells/10 cm-plate) were RA-treated for 72 h, serum starved for 4 h and then incubated for 10 min in the absence or in the presence of BDNF (Alomone Labs, Jerusalem, Israel) at a final concentration of 50 ng/ml.

Techniques: Isolation, Reverse Transcription, Amplification, Control, Negative Control, Immunoprecipitation, Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: A Cross-Talk between TrkB and Ret Tyrosine Kinases Receptors Mediates Neuroblastoma Cells Differentiation

doi: 10.1371/journal.pone.0001643

Figure Lengend Snippet: (A) RA-treated SH-SY5Y ( left panels ) and LAN-5 ( right panels ) cells were transfected with siRNATrkB (lane 4), siRNARet (lane 5) or negative control siRNAns (lane 3). C is referred to untreated cells (lane 1). Cell lysates were immunoblotted with anti-TrkB, anti-pRet, anti-Ret, or anti-GAP-43 antibodies, as indicated. The equal protein loading has been confirmed by immunoblotting with anti-atubulin? antibodies. (B) RA-treated LAN5, SH-SY5Y and SK-N-BE cells were left un-treated (lanes 2, 5 and 8, respectively) or treated with BDNF (lanes 3, 6 and 9, respectively). Lanes 1, 4 and 7 are mock-treated cells. Cell lysates were immunoblotted with anti-pRet or anti-Ret antibodies. To confirm equal loading the filters were stripped and reprobed with anti-αtubulin? antibodies. In (A) and (B), quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see legend to ). Blots shown in (A) and (B) are representative of at least three independent experiments.

Article Snippet: For BDNF acute stimulations, SH-SY5Y, LAN-5 and SK-N-BE (900.000 cells/10 cm-plate) were RA-treated for 72 h, serum starved for 4 h and then incubated for 10 min in the absence or in the presence of BDNF (Alomone Labs, Jerusalem, Israel) at a final concentration of 50 ng/ml.

Techniques: Transfection, Negative Control, Western Blot, Quantitation Assay

Journal: Scientific Reports

Article Title: Novel plasma cytokines identified and validated in children during lead exposure according to the new updated BLRV

doi: 10.1038/s41598-024-81215-2

Figure Lengend Snippet: Identification of abnormal expression of cytokines (A) Volcano plot analysis was performed to identify significant differentially expressed proteins (DEPs), with the criteria: P <0.05 and log2 fold change >1. The volcano plot analysis showed that 3 significantly up-regulated cytokines including IL-6, IL-8 and IL-17, and 6 significantly down-regulated cytokines including Brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 4 (BMP4), insulin-like growth factor 1 (IGF-1), IL-7, IL-10 and Leptin. (B) Classification of children with elevated blood lead reference value (BLRV) (No.1–4) and children with low BLRV (No.5–8). The DEPs data of Human Cytokine Antibody Array (RayBiotech) was shown by the method of hierarchical clustering analysis. Samples and proteins were shown in columns and rows, respectively. Red, increased expression; blue, reduced expression; and white, median expression.

Article Snippet: These 9 selected cytokines (BDNF, BMP-4, IGF-1, IL-6, IL-7, IL-8, IL-10, IL-17 and Leptin) were tested by specific ELISA kit (WuhanBoster Company, China).

Techniques: Expressing, Derivative Assay, Ab Array

Journal: Scientific Reports

Article Title: Novel plasma cytokines identified and validated in children during lead exposure according to the new updated BLRV

doi: 10.1038/s41598-024-81215-2

Figure Lengend Snippet: Validation of 9 significantly identified cytokines by ELISA. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: These 9 selected cytokines (BDNF, BMP-4, IGF-1, IL-6, IL-7, IL-8, IL-10, IL-17 and Leptin) were tested by specific ELISA kit (WuhanBoster Company, China).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Novel plasma cytokines identified and validated in children during lead exposure according to the new updated BLRV

doi: 10.1038/s41598-024-81215-2

Figure Lengend Snippet: Association between nine cytokines and characteristics.

Article Snippet: These 9 selected cytokines (BDNF, BMP-4, IGF-1, IL-6, IL-7, IL-8, IL-10, IL-17 and Leptin) were tested by specific ELISA kit (WuhanBoster Company, China).

Techniques:

Journal: Scientific Reports

Article Title: Novel plasma cytokines identified and validated in children during lead exposure according to the new updated BLRV

doi: 10.1038/s41598-024-81215-2

Figure Lengend Snippet: Multivariate Logistic regressive analysis of characteristics and nine cytokines.

Article Snippet: These 9 selected cytokines (BDNF, BMP-4, IGF-1, IL-6, IL-7, IL-8, IL-10, IL-17 and Leptin) were tested by specific ELISA kit (WuhanBoster Company, China).

Techniques:

Journal: bioRxiv

Article Title: The Rab5-Rab11 endosomal pathway is required for BDNF-induced CREB transcriptional regulation in neurons

doi: 10.1101/844720

Figure Lengend Snippet: Hippocampal neurons expressing control EGFP or dominant negative (DN) mutants of Rab5 and Rab11 were stimulated with BDNF for 0, 5 or 120 minutes, and lysates were probed for total and phosphorylated TrkB (Y515), Akt (S473) and Erk1/2 (T202/Y204). Representative western blots ( A ) and quantification of 5–7 independent experiments ( B ) are shown. Differences between 5 and 120 minutes after BDNF stimulation are indicated in brackets. One-way ANOVA followed by Brown-Forsythe and Welch post-test for multiple comparison. *, p < 0.04; ****, p < 0.0001; and NS, non-significant.

Article Snippet: When indicated, neurons were treated from 30 minutes before BDNF stimulation with 30 μM PD98059 (Promega), 10 μM LY294002 (Calbiochem) or 200 nM K252a (Santa Cruz Biotechnologies).

Techniques: Expressing, Control, Dominant Negative Mutation, Western Blot, Comparison

Journal: bioRxiv

Article Title: The Rab5-Rab11 endosomal pathway is required for BDNF-induced CREB transcriptional regulation in neurons

doi: 10.1101/844720

Figure Lengend Snippet: The ability of PD98059 to inhibit BDNF-induced phosphorylation of Erk1/2 was confirmed by stimulating hippocampal neurons with BDNF for 15 minutes and performing double immunofluorescence for MAP2 and pErk1/2 ( A ). The ability of PD98059 to reduce pErk1/2 staining was evident in somas and nuclei, and the quantifications are shown in ( B - C) . Scale bar: 10 µm. Hippocampal neurons expressing control EGFP or dominant negative (DN) mutants of Rab5 and Rab11 were stimulated with BDNF for 0 o 60 minutes, and sustained activation of Erk1/2 was analyzed in different neuronal compartments (nuclei, somas, dendrites) by immunofluorescence ( D ). Nuclei were traced using Hoechst staining as a reference and are indicated here with a dashed line. Scale bar: 10 µm. Quantification of the normalized intensity from 30 nuclei and somas and 60 dendrites from 3 independent experiments is shown ( E ). In D, differences between time=0 and 60 minutes of BDNF stimulation are indicated at the bottom of the bar. The effects of the expression of the Rab5 and Rab11 mutants compared with EGFP after 60 minutes of BDNF stimulation are indicated in brackets. Statistical differences were tested by using one-way ANOVA, followed by Dunnett’s post-test for multiple comparison. *, p < 0.05; **, p < 0.01; ****, p < 0.0005; and NS, non-significant.

Article Snippet: When indicated, neurons were treated from 30 minutes before BDNF stimulation with 30 μM PD98059 (Promega), 10 μM LY294002 (Calbiochem) or 200 nM K252a (Santa Cruz Biotechnologies).

Techniques: Phospho-proteomics, Immunofluorescence, Staining, Expressing, Control, Dominant Negative Mutation, Activation Assay, Comparison

Journal: bioRxiv

Article Title: The Rab5-Rab11 endosomal pathway is required for BDNF-induced CREB transcriptional regulation in neurons

doi: 10.1101/844720

Figure Lengend Snippet: Hippocampal neurons expressing control EGFP or dominant negative (DN) mutants of Rab5 and Rab11 were stimulated with BDNF for 15 minutes, and lysates were probed for total and phosphorylated CREB. Representative western blots ( A ) and quantification of 4 independent experiments ( B ) are shown. Statistically significant differences between vehicle and BDNF treatments analyzed by using t-tests are indicated. To specifically study the amount of phosphorylated CREB in the nucleus, we performed stimulation with BDNF for 0, 15 or 60 minutes, and we detected phosphorylated CREB by immunofluorescence ( C-E ). Quantification of the normalized intensity from 42–112 nuclei from 4 independent experiments is shown ( D-F ). Datasets were tested by using one-way ANOVA, followed by Bonferroni’s post-test for multiple comparison. Statistically significant differences in pCREB at different time points compared to time = 0 minutes are indicated within the bar. Differences between EGFP controls and mutant-expressing neurons at specific time points are indicated in brackets. Significance levels are labelled as follows: **, p < 0.01; ***, p < 0.005; and NS, non-significant. Scale bars: 10 µm.

Article Snippet: When indicated, neurons were treated from 30 minutes before BDNF stimulation with 30 μM PD98059 (Promega), 10 μM LY294002 (Calbiochem) or 200 nM K252a (Santa Cruz Biotechnologies).

Techniques: Expressing, Control, Dominant Negative Mutation, Western Blot, Immunofluorescence, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: The Rab5-Rab11 endosomal pathway is required for BDNF-induced CREB transcriptional regulation in neurons

doi: 10.1101/844720

Figure Lengend Snippet: Hippocampal neurons were stimulated with BDNF for 15 minutes in the presence of the MEK1/2 inhibitor PD98059, the PI3K inhibitor LY294002 or the Trk kinase activity inhibitor K252a and then probed for phosphorylated CREB and MAP2 by immunofluorescence ( A ). Quantification of 30 nuclei per condition from 3 independent experiments showed a complete block of BDNF-induced CREB phosphorylation in the presence of PD89059 ( B ). Significant differences compared to non-stimulated neurons were analyzed by using one-way ANOVA, followed by Bonferroni’s test, and are indicated within the bars. The dependence on TrkB was confirmed by the robust inhibition of BDNF-induced CREB phosphorylation by K252a. By contrast, LY294002 did not have any effect on pCREB, despite its significant inhibition of Akt phosphorylation (pS473), as shown in C . Null effect of LY294002 on TrkB phosphorylation (pY515) is also shown. Quantification of the pCREB levels confirmed the inhibition in the nuclei ( E ) and somas ( F ). Statistically significant differences were analyzed by using one-way ANOVA and Bonferroni’s post-test for multiple comparison and are indicated over the respective bars (n= 3 independent experiments; 20 neurons per condition). To confirm whether Erk1/2-mediated phosphorylation of CREB at S133 was critical for a cellular response, we compared BDNF-induced dendritic branching in neurons expressing either EGFP or a non-phosphorylatable mutant of CREB (S133A). Immunostaining for MAP2 was used to analyze the morphology of the dendritic arbor ( G ). Sholl’s analysis ( H ) and direct counting of the number of branching points ( I ) from 18 neurons per condition in 3 independent experiments showed that phosphorylation of CREB is required for BDNF-induced dendritic branching. Sholl’s curves were compared using two-way repeated-measures ANOVA, followed by Bonferroni’s post-test for multiple comparison. Branching point data were analyzed by using Student’s t test. Significance levels for the different statistical tests are labelled as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.005; and NS, non-significant. Scale bars: 10 µm.

Article Snippet: When indicated, neurons were treated from 30 minutes before BDNF stimulation with 30 μM PD98059 (Promega), 10 μM LY294002 (Calbiochem) or 200 nM K252a (Santa Cruz Biotechnologies).

Techniques: Activity Assay, Immunofluorescence, Blocking Assay, Phospho-proteomics, Inhibition, Comparison, Expressing, Mutagenesis, Immunostaining

Journal: bioRxiv

Article Title: The Rab5-Rab11 endosomal pathway is required for BDNF-induced CREB transcriptional regulation in neurons

doi: 10.1101/844720

Figure Lengend Snippet: Changes in the expression of the early-response gene Arc following 4 hours of BDNF stimulation were studied in neurons expressing control EGFP or dominant negative (DN) mutant of Rab11 and analyzed by using Student’s t test ( A ). Abundance of transcripts was expressed as the fold-change compared to neurons treated with BSA, showing a significant decrease in neurons expressing Rab11DN. To explore BDNF-induced changes in the expression of multiple CREB-regulated genes, we used a PCR array including 84 genes with cAMP, calcium and serum response elements. Reliable data obtained from 76 of the genes in 5 independent experiments were categorized by their annotated functions and plotted as a fold-change in BDNF-stimulated neurons compared to BSA-treated neurons, resulting in significant differences in 19 of these genes ( B ). Although 2 of the studied genes significantly decreased their expression levels upon BDNF stimulation, the remaining 17 were increased. Names and levels of significance are shown for the 19 significantly regulated genes. We performed the same analysis in neurons expressing Rab11DN in parallel to compare the expression levels of these 19 genes. We plotted EGFP and Rab11DN data together for these genes ( C ), finding overall attenuation of the BDNF effect. Moreover, 9 of these genes were significantly different. Names are color-coded following the functional classification. Statistical significance of gene expression data was analyzed by using Student’s t test. Level of significance for the different tests is indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.005; and NS, non-significant.

Article Snippet: When indicated, neurons were treated from 30 minutes before BDNF stimulation with 30 μM PD98059 (Promega), 10 μM LY294002 (Calbiochem) or 200 nM K252a (Santa Cruz Biotechnologies).

Techniques: Expressing, Control, Dominant Negative Mutation, Mutagenesis, Functional Assay, Gene Expression

Journal: The Journal of Neuroscience

Article Title: TrkB Modulates Fear Learning and Amygdalar Synaptic Plasticity by Specific Docking Sites

doi: 10.1523/JNEUROSCI.1707-09.2009

Figure Lengend Snippet: Decreased pAKT but not pMAPKs in trkBSHC/+ point mutants. A, Top, Immunoblot showing the time course of MAPK phosphorylation after BDNF stimulation of control and trkBSHC/+ mutant cortical primary neurons using Abs against the phosphorylated forms of ERK1/2, p42/44 (pMAPKs). To control for protein loading, the blots were reprobed with anti-ERK1 Abs. Bottom, Quantification of pMAPK levels. Densitometric analysis was performed on two different blots for each antibody (n = 3 cortices per time point). B–E, Immunofluorescence analysis of amygdala tissues from fear-conditioned (n = 3) versus untrained (n = 2) trkB+/+ and trkBSHC/+ mice by use of pMAPKs Abs. F, Top, Immunoblot showing the time course of AKT phosphorylation after BDNF stimulation of control and trkBSHC/+ mutant cortical primary neurons by using pAKT antibodies. To control for protein loading, blots were reprobed with anti-AKT Abs. Bottom, Quantification of pAKT levels. *, pAKT in trkBSHC/+ versus trkB+/+, p = 0.02, ANOVA. Densitometric analysis was performed on two different blots for each antibody (n = 3 cortices per time point). G, Top, Immunoblot analysis of pAKT in amygdala lysates from trkB+/+ untrained (control) (n = 2) versus trained (fear-conditioned) (n = 3) amygdala lysate and from trkBSHC/+ mice (control) (n = 2) versus trained (fear-conditioned) (n = 3) amygdala lysate. To control for protein loading, blots were reprobed with anti-AKT Abs. Bottom, Quantification of pAKT levels. *p = 0.01. C, Control (untrained); T, trained.

Article Snippet: Cells were stimulated with 5 or 50 ng/ml of purified recombinant BDNF (Genentech and Regeneron Pharmaceuticals) as described previously ( Minichiello et al., 2002 ).

Techniques: Western Blot, Phospho-proteomics, Control, Mutagenesis, Immunofluorescence