Journal: PLoS ONE

Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

doi: 10.1371/journal.pone.0025097

Figure Lengend Snippet: (A): RT-PCR of BDNF, its high (TrkB) and low (p75 NTR ) affinity receptors, TrkA, and TrkC from cells cultured in basal (10% FCS medium), WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). GAPDH mRNA levels was used as an internal control. Positive control (lane 5) was the neuroblastoma cell line (IMR32) for BDNF, TrkB and p75 NTR ; and the erythromyeloblastoid leukemia cells (K562) for TrkA and C. A representative result of at least three to five independent experiments. (B) Comparison of TrkB95 and p75 NTR on total RNA extracted from WiDr cells cultured in 10% FCS or after 24 to 72 h serum deprivation (0% FCS). TrkB95 and p75 NTR mRNA/GAPDH mRNA quantification of band intensities evaluated by densitometry are shown above lanes and expressed in arbitrary units (mean of three independent experiments). (C) Same experiment: RT-PCR of TrkA and TrkC on total RNA extracted from WiDr cells cultured under basal culture conditions (10% FCS) and after 24–72 h of serum starvation in comparison to positive control (K562) (D) Expression of pro-BDNF and BDNF, full length TrkB 145 and truncated TrkB 95 and p75 NTR proteins in CRC cell lines cultured in 10% FCS. TrkA and TrkC were not detected. Actin was used as loading protein control. WiDr (lane: 1), SW480 (lane: 2), SW620 (lane: 3), COLO 205 (lane: 4). Positive control (lane: 5) were IMR32 cells for BDNF, pro-BDNF, TrkB and p75 NTR and K562 cells or TrkA and TrkC. A representative result of at least three independent experiments.

Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Positive Control, Expressing

Journal: PLoS ONE

Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

doi: 10.1371/journal.pone.0025097

Figure Lengend Snippet: BDNF release and effect of exogenous BDNF, pro-BDNF, TrkB inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in primary CRC cell lines.

Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

Techniques:

Journal: PLoS ONE

Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

doi: 10.1371/journal.pone.0025097

Figure Lengend Snippet: BDNF release and effect of exogenous BDNF, pro-BDNF, TrkB inhibitor (K252a) and neutralizing anti-BDNF on apoptosis and proliferation in metastatic CRC cell lines.

Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

Techniques:

Journal: PLoS ONE

Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

doi: 10.1371/journal.pone.0025097

Figure Lengend Snippet: Confocal microscopy of WiDr (A, B) and COLO 205 (C, D) cells, stained with an anti-BDNF Ab (red), anti-TrkB mAb (green) or both (overlay) cultured with 10% FCS (A, C) or after 24-h serum deprivation (B, D). Under basal culture conditions (10% FCS), TrkB and BDNF were sequestered in the cytoplasm (arrows) in WiDr (A) and COLO 205 (C) cells. The same staining patterns were obtained with the two other cell lines (data not shown). After serum starvation relocation to the cell membrane and colocalization of TrkB and BDNF (yellow in merged, arrows) were detected in WiDr (B) and COLO 205 (D). Images were representative for at least three to five independent experiments.

Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

Techniques: Confocal Microscopy, Staining, Cell Culture

Journal: PLoS ONE

Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

doi: 10.1371/journal.pone.0025097

Figure Lengend Snippet: (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

Article Snippet: The following Abs were used: rabbit anti-BDNF Ab (1 µg/ml; Santa Cruz Biotechnology), rabbit anti-pro-BDNF Ab (8 µg/ml; Alomone Labs), mouse anti-TrkB Ab (2.5 µg/ml; R&D Systems), rabbit anti-p75 NTR (2 µg/ml; Santa Cruz Biotechnology) and goat anti-sortilin Ab (1 µg/ml; Santa Cruz Biotechnology).

Techniques: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: A Novel Peptide Interfering with proBDNF-Sortilin Interaction Alleviates Chronic Inflammatory Pain

doi: 10.7150/thno.29703

Figure Lengend Snippet: Mapping of proBDNF-sortilin interaction interface. (A) Schematic diagram illustrating the design of human BDNF prodomain variants. Six variants with a series of amino acids deletions were used in this study. V66M, Val66Met; S, Signal peptide (amino acid 1-18); BDNF, mature BDNF (amino acid 129-247); HA, C-terminal HA epitope. (B) Immunoblots of co-immunoprecipitation (Left) or inputs (Right) of sortilin and proBDNF variants obtained with antibody against HA or Myc epitope. IP, Immunoprecipitation; IB, Immunoblot. (C) Left , live fluorescence micrograph of BiFC mapping assay (Yellow) using HEK293T cells with VC155-sortilin (Sort1) and VN155(I152L)-BDNF prodomain variants. Scale bar represents 50 µm. Right , quantification of fluorescence intensity of BiFC assay. P <0.0001, one-way ANOVA. ** P <0.01 versus Fl group; *** P <0.0001 versus Fl group; ## P <0.001 versus variant I; ### P <0.0001 versus variant I group; n.s., non-significant. Values are means ± SEM. n = 100 individual cells from 4 images. (D) Left , representative immunoblots of supernatant and cell lysates of HEK293 cells overexpressing sortilin and BDNF prodomain variants obtained with antibodies against HA. Right , densitometry analyses of secreted BDNF in supernatants, normalized to input in lysates. P <0.001, one-way ANOVA. * P <0.05, versus Fl group; *** P <0.001, versus Fl group; ## P <0.05, versus I group. Values are means ± SEM. n = 4. IB, Immunoblot. (E) BDNF ELISA quantification of supernatant collected from HEK293 cells overexpressing sortilin and BDNF prodomain variants. P <0.0001, one-way ANOVA. * P <0.05; ** P <0.01; *** P <0.001, versus Sort1/Fl group; ## P <0.05, versus Sort1/variant I group. Values are means ± SEM. Each data point represents the average of 3 independent experiments.

Article Snippet: The primary antibodies used included anti-proBDNF antibody (#ANT-006-AG, Alomone Labs Ltd, Jerusalem, Israel), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell signalling Technology, Danvers, MA, USA), anti-NeuN antibody (MAB377, EMD Millipore Corporation, Billerica, MA, USA) and anti-sortilin antibody (Abcam, Cambridge, UK).

Techniques: Western Blot, Immunoprecipitation, Fluorescence, Mapping Assay, Bimolecular Fluorescence Complementation Assay, Variant Assay, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: A Novel Peptide Interfering with proBDNF-Sortilin Interaction Alleviates Chronic Inflammatory Pain

doi: 10.7150/thno.29703

Figure Lengend Snippet: Construction of a novel peptide targeting proBDNF-sortilin Interaction. (A) Schematic diagram illustrating the design of a series of blocking peptides targeting proBDNF-sortilin interaction interface. Scr-bdnf84-83 (scr), scrambled peptide with permutation of peptide sequence at amino acid 84-93. (B) Immunoblots of co-immunoprecipitation (upper) or reverse co-immunoprecipitation (lower) of sortilin and proBDNF variants in cell lysates overexpressing full-length proBDNF and sortilin followed by peptide incubation obtained with antibodies against HA or Myc. IP, Immunoprecipitation; IB, Immunoblot. (C) Schematic diagram of the design of the Tat-tagged interfering peptide. Scr-bdnf89-98-Tat, Tat-tagged scrambled peptide with permutation of peptide sequence at amino acid 89-98. (D) Live fluorescence micrographs of 5FAM (green) or bright field in rat DRG neuronal cultures before (0 min) and incubated with 5 µM 5FAM-tagged bdnf89-98-Tat (bdnf-Tat) for 2 h. Scale bar represents 150 µm. (E) Fluorescence micrograph of 5FAM (green), sortilin (red) or DAPI (blue) in rat DRG neuronal cultures with peptides pre-treatment for 30 min. Scale bars represent 25 µm. (F) Left , fluorescence micrograph of BiFC assay (yellow) of HEK293T cells overexpressing full-length BDNF and sortilin with vehicle, 10 µM scr-Tat or bdnf-Tat pre-treatment for 30 min. Scale bars represent 25 µm. Right , Determination of the corrected total cell fluorescence (CTCF) of the BiFC signal. P <0.0001 , one-way ANOVA. *** P <0.0001, scr-Tat treatment versus bdnf-Tat treatment. Mean values ± SEM. n = 100 individual cells from 5 images. (G) Effect of 30 min peptide pre-treatment on activity-dependent secretion of BDNF in rat DRG neuronal cultures with high-potassium stimulus using ELISA. P <0.05, one-way ANOVA. * P <0.05, vehicle versus scr-Tat treatment or scr-Tat treatment versus bdnf-Tat treatment. Values are means ± SEM. Each data point represents the average of 3 independent experiments.

Article Snippet: The primary antibodies used included anti-proBDNF antibody (#ANT-006-AG, Alomone Labs Ltd, Jerusalem, Israel), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell signalling Technology, Danvers, MA, USA), anti-NeuN antibody (MAB377, EMD Millipore Corporation, Billerica, MA, USA) and anti-sortilin antibody (Abcam, Cambridge, UK).

Techniques: Blocking Assay, Sequencing, Western Blot, Immunoprecipitation, Incubation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: Dexmedetomidine Attenuates Neurotoxicity in Developing Rats Induced by Sevoflurane through Upregulating BDNF-TrkB-CREB and Downregulating ProBDNF-P75NRT-RhoA Signaling Pathway

doi: 10.1155/2020/5458061

Figure Lengend Snippet: Dexmedetomidine could decrease the level of proBDNF and restore the ratio of proBDNF/mBDNF and alleviates activation of the proBDNF-P75NRT-RHOA pathway after sevoflurane. (a) Western blot band. (b) ProBDNF/mBDNF. (c–e) Bar graph of Western blot. (f) Immunofluorescence of proBDNF (scale bar = 50 μ m). ∗ Compare with the control group, P < 0.05; # Compared with the Sev group, P < 0.05.

Article Snippet: The primary antibodies were added: anti-BDNF (1 : 2000, Abcam, Cambridge, UK), anti-proBDNF (1 : 500, Alomone Labs, Israel), P75NRT (1 : 2000, Cell Signaling Technology, USA), TrkB (1 : 1000, ABclonal, Woburn, MA, USA), p-TrkB (1 : 1000, ABclonal, Woburn, MA, USA), CREB (1 : 1000, Cell Signaling Technology, USA), anti-RhoA (1 : 500, Proteintech, China), PSD95 (1 : 500, Proteintech Biotechnology, Chicago, IL, USA), SYP (1 : 1000, Abcam, USA), anti- β -actin (1 : 500, Proteintech, China), and GAPDH (1 : 1000, Cell Signaling Technology, Boston, MA, USA).

Techniques: Activation Assay, Western Blot, Immunofluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pro-BDNF Contributes to Hypoxia/Reoxygenation Injury in Myocardial Microvascular Endothelial Cells: Roles of Receptors p75 NTR and Sortilin and Activation of JNK and Caspase 3

doi: 10.1155/2018/3091424

Figure Lengend Snippet: Effects of the anti-pro-BDNF antibody on apoptosis, migration, and capillary-like-structure formation among MMECs after exposure to HG and H/R. (a) Effects of pro-BDNF on apoptosis were analyzed by flow cytometry of MMECs after different treatments: control, H/R, H/R + anti-pro-BDNF, and H/R + vehicle. (b, c) The functional effects of pro-BDNF on MMECs were assessed by capillary-like-structure formation and cell scratch assays. (d) Relative apoptosis levels and fold changes are expressed in relation to the control group. The H/R group showed markedly increased relative apoptosis levels, decreased capillary-like-structure formation, and reduced cell migration when compared with the control group. These effects were reversed by treatment with the anti-pro-BDNF antibody to the levels similar to those in the control group. The data were subjected to one-way ANOVA. The error bars represent SEM. ∗ P < 0.05 as compared with the control group; # P < 0.05 as compared with the H/R group or the H/R + vehicle group.

Article Snippet: After blocking in 5% fat-free dry milk, antibodies against pro-BDNF (Alomone, 1 : 400), BDNF (Abcam, 1 : 500), P75 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 100), sortilin (Abcam, 1 : 500), JNK (Santa Cruz Biotechnology, 1 : 100), cleaved caspase 3 (Asp175, Cell Signaling Technology, 1 : 1000), caspase 3, and β -actin (Santa Cruz Biotechnology) were employed.

Techniques: Migration, Flow Cytometry, Functional Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pro-BDNF Contributes to Hypoxia/Reoxygenation Injury in Myocardial Microvascular Endothelial Cells: Roles of Receptors p75 NTR and Sortilin and Activation of JNK and Caspase 3

doi: 10.1155/2018/3091424

Figure Lengend Snippet: Effects of the anti-pro-BDNF antibody on the expression of p75 NTR and sortilin and apoptosis-related proteins. (a) Representative immunofluorescent images of pro-BDNF (red, first column), p-JNK (green, second column), BDNF (red, fourth column), JNK (green, fifth column), sortilin (red, seventh column), and p75 NTR (green, eighth column) in groups control, H/R, H/R + anti-pro-BDNF, and H/R + vehicle. (b–e) Representative Western blots and quantitative analysis of pro-BDNF, BDNF (b), p75 NTR , sortilin (c), JNK, p-JNK (d), caspase 3 and cleaved-caspase 3 expression (e) in response to different treatments. All the data were normalized to β -actin, and fold changes are expressed in relation to the control group. Exposure of MMECs to H/R resulted in significantly higher expression levels of pro-BDNF, p75 NTR , and sortilin and in activation of JNK and caspase 3 as compared with MMECs maintained under normal conditions (control). Nonetheless, there were no significant differences in BDNF, JNK, and caspase 3 expression levels after H/R. Treatment with the anti-pro-BDNF antibody significantly reversed the increase in the protein expression of pro-BDNF, p75 NTR , sortilin, p-JNK, and cleaved caspase 3 in MMECs after exposure to HG and H/R (H/R + anti-pro-BDNF). The data were subjected to one-way ANOVA. The error bars represent SEM. ∗ P < 0.05 as compared with the control group; # P < 0.05 as compared with the H/R or H/R + vehicle group.

Article Snippet: After blocking in 5% fat-free dry milk, antibodies against pro-BDNF (Alomone, 1 : 400), BDNF (Abcam, 1 : 500), P75 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 100), sortilin (Abcam, 1 : 500), JNK (Santa Cruz Biotechnology, 1 : 100), cleaved caspase 3 (Asp175, Cell Signaling Technology, 1 : 1000), caspase 3, and β -actin (Santa Cruz Biotechnology) were employed.

Techniques: Expressing, Western Blot, Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pro-BDNF Contributes to Hypoxia/Reoxygenation Injury in Myocardial Microvascular Endothelial Cells: Roles of Receptors p75 NTR and Sortilin and Activation of JNK and Caspase 3

doi: 10.1155/2018/3091424

Figure Lengend Snippet: Effects of the anti-pro-BDNF antibody on apoptosis, migration, and capillary-like-structure formation among MMECs after exposure to HG and H/R. (a) Effects of pro-BDNF on apoptosis were analyzed by flow cytometry of MMECs after different treatments: control, H/R, H/R + anti-pro-BDNF, and H/R + vehicle. (b, c) The functional effects of pro-BDNF on MMECs were assessed by capillary-like-structure formation and cell scratch assays. (d) Relative apoptosis levels and fold changes are expressed in relation to the control group. The H/R group showed markedly increased relative apoptosis levels, decreased capillary-like-structure formation, and reduced cell migration when compared with the control group. These effects were reversed by treatment with the anti-pro-BDNF antibody to the levels similar to those in the control group. The data were subjected to one-way ANOVA. The error bars represent SEM. ∗ P < 0.05 as compared with the control group; # P < 0.05 as compared with the H/R group or the H/R + vehicle group.

Article Snippet: Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 30 min and permeabilized or not permeabilized with 0.5% Triton X-100 (Sigma-Aldrich), and nonspecific binding was blocked by incubation with 5% donkey serum (Jackson ImmunoResearch Laboratories) at RT for 30 min. Coverslips were incubated overnight at 4°C with the following primary antibodies: rabbit anti-BDNF (Abcam, 1 : 500), rabbit anti-pro-BDNF (Alomone Labs, 1 : 400), anti-JNK (Santa Cruz Biotechnology, 1 : 100), anti-phosphorylated-JNK (p-JNK) (Santa Cruz Biotechnology, 1 : 100), rabbit anti-p75 NTR (Santa Cruz Biotechnology, 1 : 100), and goat anti-sortilin (Abcam, 1 : 500) antibodies.

Techniques: Migration, Flow Cytometry, Functional Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pro-BDNF Contributes to Hypoxia/Reoxygenation Injury in Myocardial Microvascular Endothelial Cells: Roles of Receptors p75 NTR and Sortilin and Activation of JNK and Caspase 3

doi: 10.1155/2018/3091424

Figure Lengend Snippet: Effects of the anti-pro-BDNF antibody on the expression of p75 NTR and sortilin and apoptosis-related proteins. (a) Representative immunofluorescent images of pro-BDNF (red, first column), p-JNK (green, second column), BDNF (red, fourth column), JNK (green, fifth column), sortilin (red, seventh column), and p75 NTR (green, eighth column) in groups control, H/R, H/R + anti-pro-BDNF, and H/R + vehicle. (b–e) Representative Western blots and quantitative analysis of pro-BDNF, BDNF (b), p75 NTR , sortilin (c), JNK, p-JNK (d), caspase 3 and cleaved-caspase 3 expression (e) in response to different treatments. All the data were normalized to β -actin, and fold changes are expressed in relation to the control group. Exposure of MMECs to H/R resulted in significantly higher expression levels of pro-BDNF, p75 NTR , and sortilin and in activation of JNK and caspase 3 as compared with MMECs maintained under normal conditions (control). Nonetheless, there were no significant differences in BDNF, JNK, and caspase 3 expression levels after H/R. Treatment with the anti-pro-BDNF antibody significantly reversed the increase in the protein expression of pro-BDNF, p75 NTR , sortilin, p-JNK, and cleaved caspase 3 in MMECs after exposure to HG and H/R (H/R + anti-pro-BDNF). The data were subjected to one-way ANOVA. The error bars represent SEM. ∗ P < 0.05 as compared with the control group; # P < 0.05 as compared with the H/R or H/R + vehicle group.

Article Snippet: Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 30 min and permeabilized or not permeabilized with 0.5% Triton X-100 (Sigma-Aldrich), and nonspecific binding was blocked by incubation with 5% donkey serum (Jackson ImmunoResearch Laboratories) at RT for 30 min. Coverslips were incubated overnight at 4°C with the following primary antibodies: rabbit anti-BDNF (Abcam, 1 : 500), rabbit anti-pro-BDNF (Alomone Labs, 1 : 400), anti-JNK (Santa Cruz Biotechnology, 1 : 100), anti-phosphorylated-JNK (p-JNK) (Santa Cruz Biotechnology, 1 : 100), rabbit anti-p75 NTR (Santa Cruz Biotechnology, 1 : 100), and goat anti-sortilin (Abcam, 1 : 500) antibodies.

Techniques: Expressing, Western Blot, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Plasmalogens, the Vinyl Ether-Linked Glycerophospholipids, Enhance Learning and Memory by Regulating Brain-Derived Neurotrophic Factor

doi: 10.3389/fcell.2022.828282

Figure Lengend Snippet: Alteration of memory-related gene expression and cellular signaling in the hippocampus following the Gnpat knockdown. (A,B) Real-time PCR data show the changes of Gnpat (A) and Bdnf (B) mRNA expressions in the hippocampus following 1 and 3 weeks after microinjection of sh- Gnpat lentiviruses. (C) Real-time PCR data show the changes in memory-related gene expressions in the hippocampus 3 weeks after the injections of lentiviruses. (D) Western blotting assays show the representative protein expressions in the hippocampus 3 weeks after the sh-control and sh- Gnpat injection. (E) Quantification data of panel (D) show the relative change of GNPAT, p-Akt, p-CREB, p-ERK, pro-BDNF, Synapsin-1, SYT-1, and PSD-95 in the hippocampus. The protein expression was normalized with the endogenous expression of β-Actin. The data represent the mean ± S.E.M. Each experiment included more than five mice. *, p < 0.05 and **, p < 0.01. The p values were obtained by Student’s t-tests.

Article Snippet: Western blotting assays were carried out by standard protocols ( ) using antibodies against GNPAT (ab75060, Abcam, Cambridge, MA), CREB (9,197, Cell signaling), p-CREB (9,191, cell signaling), Akt (610,860, BD Biosciences), p-Akt (s-473, 4,060, Cell signaling), ERK (4,695, Cell signaling), p-ERK (4,370, Cell signaling), pro-BDNF (ANT-006, alomone labs), Synapsin-1 (AB1543, Millipore), SYT-1 (AB5600, Millipore), PSD-95 (MAB1598, Millipore), Flotillin (610,820, BD Biosciences), TrkB (610,102, BD Biosciences) and β-Actin (sc-47778, Cell Signaling).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Plasmalogens, the Vinyl Ether-Linked Glycerophospholipids, Enhance Learning and Memory by Regulating Brain-Derived Neurotrophic Factor

doi: 10.3389/fcell.2022.828282

Figure Lengend Snippet: Impact of Pls diet in spatial memory and memory-related protein expression in the hippocampus. (A) Mass spectroscopic data show the expression of hippocampal Pls in the mice fed with the diet containing 0.01% Pls for 6 weeks (0.2–0.25 mg Pls/day/mouse, n = 5). (B) Memory tasks show the change in escape latency by Pls diet. (C) The probe test following the memory task shows the changes of time spent in the target quadrant. The right panel shows examples of traces for control (upper) and Pls diet (lower) mice. The red circle indicates the place of the missing platform. (D,E) The swimming speed of the mice in both groups during the memory tasks (D) and the probe tests (E) . (F) Immunohistochemistry data show the expression of p-Akt and p-ERK in the control and Pls diet mice hippocampus. Scale bar, 300 μm. The right panel shows the image intensities in relative pixel values. (G) Representative Western blotting assays show the protein expression in the control and Pls diet mice hippocampus tissue. (H) Quantification data of the panel (G) show the relative change in the expression of p-Akt, p-ERK, p-CREB, synapsin-1, PSD-95, and SYT-1 by the Pls diet in the mice hippocampus ( n = 5). (I) Real-time PCR data show the mRNA expression of Bdnf , Synapsin-1 , Syt-1 , and Psd-95 in the mice hippocampus ( n = 5). The data represent mean ± S.E.M. *, p < 0.05 and **, p < 0.01. The p values of panel B were obtained from ANOVA analysis followed by post hoc Bonferroni’s tests. Student’s t-tests were performed in panels (D,E,F,H,I) .

Article Snippet: Western blotting assays were carried out by standard protocols ( ) using antibodies against GNPAT (ab75060, Abcam, Cambridge, MA), CREB (9,197, Cell signaling), p-CREB (9,191, cell signaling), Akt (610,860, BD Biosciences), p-Akt (s-473, 4,060, Cell signaling), ERK (4,695, Cell signaling), p-ERK (4,370, Cell signaling), pro-BDNF (ANT-006, alomone labs), Synapsin-1 (AB1543, Millipore), SYT-1 (AB5600, Millipore), PSD-95 (MAB1598, Millipore), Flotillin (610,820, BD Biosciences), TrkB (610,102, BD Biosciences) and β-Actin (sc-47778, Cell Signaling).

Techniques: Expressing, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Plasmalogens, the Vinyl Ether-Linked Glycerophospholipids, Enhance Learning and Memory by Regulating Brain-Derived Neurotrophic Factor

doi: 10.3389/fcell.2022.828282

Figure Lengend Snippet: Attenuation of Pls-mediated memory enhancement by knockdown of hippocampal TrkB or Bdnf expression. (A) Mice were subjected to the Pls feeding for 4 weeks followed by the sh- Bdnf or sh- TrkB injection bilaterally into the hippocampus. The water maze task was performed 6 weeks after Pls feeding. (B) Water maze data show the difference in escape latency between the experimental group mice. The data represent the mean values of the swimming time to reach the goal on the fourth-day trails (3 trails per day). (C) Real-time PCR data show the expression of Bdnf and synapsin-1 in the hippocampus tissues of the mice. The data represent the mean ± S.E.M. Each experiment included more than five mice. *, p < 0.05 and **, p < 0.01 (ANOVA followed by Bonferroni’s post hoc tests).

Article Snippet: Western blotting assays were carried out by standard protocols ( ) using antibodies against GNPAT (ab75060, Abcam, Cambridge, MA), CREB (9,197, Cell signaling), p-CREB (9,191, cell signaling), Akt (610,860, BD Biosciences), p-Akt (s-473, 4,060, Cell signaling), ERK (4,695, Cell signaling), p-ERK (4,370, Cell signaling), pro-BDNF (ANT-006, alomone labs), Synapsin-1 (AB1543, Millipore), SYT-1 (AB5600, Millipore), PSD-95 (MAB1598, Millipore), Flotillin (610,820, BD Biosciences), TrkB (610,102, BD Biosciences) and β-Actin (sc-47778, Cell Signaling).

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of proBDNF precursor and cleaved BDNFpro domain. αBDNFpro antibody recognizes the furin cleavage site of the prodomain. Western blotting probing recombinant mBDNF, BDNFpro, and proBDNF CR with αBDNFpro and αmBDNF antibodies. b Cortical slices from control mice injected with AAV-GFAP-GFP virus were recorded and fixed 10 min after TBS for immunostaining. z-stack reconstruction shows astrocytes labeled by GFP. Magnification of a single stack from a region of interest (ROI) shows BDNFpro immunoreactivity and BDNFpro/GFP colocalization signal of one GFP-astrocyte delimited by an approximate territory (white dashed). Scale bars: 10 µm. c z-stack reconstruction of BDNFpro/GFP colocalization signals in astrocytes from baseline- and TBS-slices from control mice. The insets show GFP signal. BDNFpro/GFP colocalization was quantified in the whole cell and branches using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 9 cells, 4 slices, 3 mice for baseline; n = 12 cells, 4 slices, 3 mice for TBS). Scale bars: 10 µm. d mBDNF/GFP colocalization was quantified in the whole cell and branches using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 10 cells, 3 slices, 3 mice for baseline; n = 10 cells, 4 slices, 3 mice for TBS). e BDNFpro/GFP and mBDNF/GFP colocalizations in baseline and TBS-slices treated with plasmin were quantified in branches using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 20 cells, 3 slices, 3 mice for baseline BDNFpro/GFP; n = 11 cells, 3 slices, 3 mice for TBS BDNFpro/GFP; n = 21 cells, 3 slices, 3 mice for baseline mBDNF/GFP; n = 13 cells, 3 slices, 3 mice for TBS mBDNF/GFP). f z-stack reconstruction of BDNFpro/GFP colocalization signals in astrocytes from basal- and TBS-slices from tamoxifen-treated p75-flox mice. Insets show GFP signal. BDNFpro/GFP colocalization was quantified using Mander’s overlap ( n = 11 cells, 5 slices, 4 mice for baseline; n = 16 cells, 5 slices, 4 mice for TBS). Scale bars: 10 µm. Data are normalized to baseline and presented as mean ± SEM.

Article Snippet: The following antibodies were used: rabbit α-GFP (Thermo Fisher Scientific Cat#A-6455; RRID:AB#2536208; IHC 1:1000 ICC 1:1000), chicken α-GFP (Thermo Fisher Scientific Cat#A10262; RRID:AB#2534023, IHC 1:1000), chicken α-BDNF (Promega Cat#G1641; RRID:AB#430850, IHC 1:300; WB 1:500), rabbit α-BDNF (Alomone Labs Cat# ANT-010; RRID:AB_2039756, EM 1:20), chicken α-proBDNF (Millipore Cat#AB9042; RID:AB#2274709, IHC 1:300), rabbit α-proBDNF (Alomone Labs Cat#ANT-006; RRID:AB_2039758, EM 1:20), rabbit α-BDNFpro (Laboratory of Bai Lu, Govern Institute for Brain Research, Tsinghua University, Beijing, IHC 1:300, EM 1:20, WB 1:500), mouse α-NeuN (Abcam Cat#ab77315; RRID:AB#1566475, IHC 1:1000), guinea pig α-NeuN (Millipore Cat#ABN90; RRID:AB#11205592, IHC 1:1000), rabbit α-pTrkB (Tyr 816) (Laboratory of Moses Chao, Skirball Intitute of Biomolecular Medicine, New York, USA, IHC 1.25 mg/ml), goat α-TrkB (Santa Cruz Biotechnology Cat#sc-12-G; RRID:AB#632558, IHC 1:300), mouse α-PSD95 (Merck-Millipore Cat#MAB1596; RRID:AB_2092365, IHC 1:500), sheep α-SorCS2 (R&D System Cat#AF4238; RRID:AB_10645642), rabbit α-SorCS2 (MyBioSource Cat# MBS5302436), rabbit α-p75 NTR (Promega Cat#G3231; RRID:AB_430853, IHC 1:1000), chicken α-GFAP (Abcam Cat#ab134436; RRID:AB_2818977, IHC 1:1000), mouse α-GFAP (Abcam Cat#ab10062; RRID:AB_296804, IHC 1:1000), mouse α-synaptobrevin2\VAMP2 (Synaptic System Cat#104 211; RRID:AB_2619758, IHC 1:300), rabbit α-beta galactosidase (Proteintech Cat#15518-1-AP; RRID:AB_2263448, IHC 1:500), rabbit α-RFP (Rockland Antibodies Cat#600-401-379; RRID:AB_2209751 IHC 1:1000), mouse α DsRed (Santa Cruz Biotechnology Cat# sc-390909; RRID:AB_2801575 IHC 1:1000).

Techniques: Western Blot, Recombinant, Injection, Immunostaining, Labeling

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of the experimental design. Step I, deletion of p75 NTR in astrocytes from tamoxifen-treated p75-flox mice precludes proBDNF transfer from neurons to astrocyte following TBS. Step II, LV-BDNFpro stop transduction replaces BDNFpro in astrocytes. Schematic representation of the experimental paradigms (right); mice were treated with tamoxifen (−5 to 0), injected with lentiviruses the last day of tamoxifen treatment (0 dptm) and finally recorded (14 dptm). LTP evoked in slices from p75-flox mice and control littermates injected with LV-GFP stop or LV-BDNFpro stop is shown. *** p < 0.001 (unpaired t -test) (p75-flox/LV-GFP stop 102.00 ± 1.85%, p75-flox/LV-BDNFpro stop 177.41 ± 10.74% and control littermates/LV-GFP stop 165.27 ± 2.24% fEPSP 180 min from TBS; n = 10 slices, 6 mice for p75-flox/LV-GFP stop ; n = 9 slices, 6 mice for p75-flox/LV-BDNFpro stop ; n = 6 slices, 4 mice for control littermates/LV-GFP stop ). b Schematic representation of the experimental design. Step II, and I as in ( a ). Step III, LV-TeTN stop transduction in astrocytes prevents from BDNFpro release. Schematic representation of the experimental paradigm as in ( a ). LTP evoked in slices from p75-flox mice and control littermates injected with LV-GFP stop or co-injected with LV-GFP stop /LV-BDNFpro stop and LV-TeTN stop /LV-BDNFpro stop is shown. *** p < 0.001 (unpaired t -test) (p75-flox/LV-BDNFpro stop /LV-GFP stop 167.50 ± 8.78%, p75-flox/LV-BDNFpro stop /LV-TeTN stop 106.26 ± 2.47% and control littermates/LV-GFP stop 151.33 ± 7.39% fEPSP 180 min from TBS; n = 8 slices, 6 mice for p75-flox/LV-BDNFpro stop /LV-GFP stop ; n = 9 slices, 7 mice for p75-flox/LV-TeTN stop /LV-BDNFpro stop ; n = 7 slices, 4 mice control littermates/LV-GFP stop ). c LTP evoked in slices from p75-flox mice and control littermates. Mice were treated with tamoxifen (−5 to 0) and recorded 14 dptm. Slices were perfused (18–28 min) with vehicle or exogenous BDNFpro. *** p < 0.001 (unpaired t -test) (p75-flox/vehicle 103.54 ± 3.43%, p75-flox/BDNFpro 171.09 ± 10.17% and control littermates/vehicle 162.81 ± 9.87% fEPSP 180 min from TBS; n = 8 slices, 4 mice for p75-flox/vehicle; n = 7 slices, 5 mice for p75-flox/BDNFpro; n = 7 slices, 5 mice for control littermates/vehicle). d LTP evoked as in ( c ). Slices were perfused (18–28 min) with vehicle or BDNFpro Val/Met . *** p < 0.001 (unpaired t -test) (p75-flox/vehicle 105.26 ± 1.59%, p75-flox/BDNFpro Val/Met 148.76 ± 8.69% and control littermates/vehicle 147.55 ± 3.55% fEPSP 180 min from TBS; n = 8 slices, 5 mice for p75-flox/vehicle; n = 6 slices, 4 mice for p75-flox/BDNFpro Val/Met ; n = 7 slices, 5 mice for control littermates/vehicle). Data are presented as mean ± SEM.

Article Snippet: The following antibodies were used: rabbit α-GFP (Thermo Fisher Scientific Cat#A-6455; RRID:AB#2536208; IHC 1:1000 ICC 1:1000), chicken α-GFP (Thermo Fisher Scientific Cat#A10262; RRID:AB#2534023, IHC 1:1000), chicken α-BDNF (Promega Cat#G1641; RRID:AB#430850, IHC 1:300; WB 1:500), rabbit α-BDNF (Alomone Labs Cat# ANT-010; RRID:AB_2039756, EM 1:20), chicken α-proBDNF (Millipore Cat#AB9042; RID:AB#2274709, IHC 1:300), rabbit α-proBDNF (Alomone Labs Cat#ANT-006; RRID:AB_2039758, EM 1:20), rabbit α-BDNFpro (Laboratory of Bai Lu, Govern Institute for Brain Research, Tsinghua University, Beijing, IHC 1:300, EM 1:20, WB 1:500), mouse α-NeuN (Abcam Cat#ab77315; RRID:AB#1566475, IHC 1:1000), guinea pig α-NeuN (Millipore Cat#ABN90; RRID:AB#11205592, IHC 1:1000), rabbit α-pTrkB (Tyr 816) (Laboratory of Moses Chao, Skirball Intitute of Biomolecular Medicine, New York, USA, IHC 1.25 mg/ml), goat α-TrkB (Santa Cruz Biotechnology Cat#sc-12-G; RRID:AB#632558, IHC 1:300), mouse α-PSD95 (Merck-Millipore Cat#MAB1596; RRID:AB_2092365, IHC 1:500), sheep α-SorCS2 (R&D System Cat#AF4238; RRID:AB_10645642), rabbit α-SorCS2 (MyBioSource Cat# MBS5302436), rabbit α-p75 NTR (Promega Cat#G3231; RRID:AB_430853, IHC 1:1000), chicken α-GFAP (Abcam Cat#ab134436; RRID:AB_2818977, IHC 1:1000), mouse α-GFAP (Abcam Cat#ab10062; RRID:AB_296804, IHC 1:1000), mouse α-synaptobrevin2\VAMP2 (Synaptic System Cat#104 211; RRID:AB_2619758, IHC 1:300), rabbit α-beta galactosidase (Proteintech Cat#15518-1-AP; RRID:AB_2263448, IHC 1:500), rabbit α-RFP (Rockland Antibodies Cat#600-401-379; RRID:AB_2209751 IHC 1:1000), mouse α DsRed (Santa Cruz Biotechnology Cat# sc-390909; RRID:AB_2801575 IHC 1:1000).

Techniques: Transduction, Injection

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of the experimental design. Circular DNA probes (−) and (+) are coupled to II° antibody targeting αSorCS2 and αTrkB I° antibody. BDNFpro induces TrkB/SorCS2 complex formation (PLA TrkB/SorCS2 ) that is prevented in the presence of αSorCS2 (blocking) antibody. b Panels show PLA TrkB/SorCS2 signals in primary culture of cortical neurons treated with vehicle or BDNFpro. The insets show reference GFP-neurons. Scale bars: 5 μm. c Panels show a GFP-neuron treated with BDNFpro. Scale bar: 5 μm. Magnification of regions of interest 1 and 2 shows dendritic PLA TrkB/SorCS2 localization (red arrowheads). Scale bar: 1 μm. d Quantification of PLA TrkB/SorCS2 signal in cultured neurons treated with vehicle, BDNFpro (in presence or absence of αSorCS2), mBDNF or proBDNF CR . Data are presented as mean ± SEM; ** p < 0.01 (unpaired t -test) ( n = 111 cells, 3 cultures for vehicle; n = 152 cells, 4 cultures for BDNFpro; n = 98 cells, 3 cultures for BDNFpro/aSorCS2; n = 89 cells, 3 cultures for mBDNF; n = 79 cells, 3 cultures for proBDNF CR ). e z-stack reconstruction showing NeuN, PSD95 and PLA TrkB/SorCS2 signals in baseline and TBS slices. NeuN/PLA TrkB/SorCS2 and PSD95/PLA TrkB/SorCS2 colocalization signals are shown. Scale bars: 40 μm. NeuN/PLA TrkB/SorCS2 and PSD95/PLA TrkB/SorCS2 colocalization was quantified using Mander’s overlap. Data are normalized to baseline and presented as mean ± SEM; * p < 0.05 (unpaired t -test) ( n = 4 slices, 3 mice for each experimental condition).

Article Snippet: The following antibodies were used: rabbit α-GFP (Thermo Fisher Scientific Cat#A-6455; RRID:AB#2536208; IHC 1:1000 ICC 1:1000), chicken α-GFP (Thermo Fisher Scientific Cat#A10262; RRID:AB#2534023, IHC 1:1000), chicken α-BDNF (Promega Cat#G1641; RRID:AB#430850, IHC 1:300; WB 1:500), rabbit α-BDNF (Alomone Labs Cat# ANT-010; RRID:AB_2039756, EM 1:20), chicken α-proBDNF (Millipore Cat#AB9042; RID:AB#2274709, IHC 1:300), rabbit α-proBDNF (Alomone Labs Cat#ANT-006; RRID:AB_2039758, EM 1:20), rabbit α-BDNFpro (Laboratory of Bai Lu, Govern Institute for Brain Research, Tsinghua University, Beijing, IHC 1:300, EM 1:20, WB 1:500), mouse α-NeuN (Abcam Cat#ab77315; RRID:AB#1566475, IHC 1:1000), guinea pig α-NeuN (Millipore Cat#ABN90; RRID:AB#11205592, IHC 1:1000), rabbit α-pTrkB (Tyr 816) (Laboratory of Moses Chao, Skirball Intitute of Biomolecular Medicine, New York, USA, IHC 1.25 mg/ml), goat α-TrkB (Santa Cruz Biotechnology Cat#sc-12-G; RRID:AB#632558, IHC 1:300), mouse α-PSD95 (Merck-Millipore Cat#MAB1596; RRID:AB_2092365, IHC 1:500), sheep α-SorCS2 (R&D System Cat#AF4238; RRID:AB_10645642), rabbit α-SorCS2 (MyBioSource Cat# MBS5302436), rabbit α-p75 NTR (Promega Cat#G3231; RRID:AB_430853, IHC 1:1000), chicken α-GFAP (Abcam Cat#ab134436; RRID:AB_2818977, IHC 1:1000), mouse α-GFAP (Abcam Cat#ab10062; RRID:AB_296804, IHC 1:1000), mouse α-synaptobrevin2\VAMP2 (Synaptic System Cat#104 211; RRID:AB_2619758, IHC 1:300), rabbit α-beta galactosidase (Proteintech Cat#15518-1-AP; RRID:AB_2263448, IHC 1:500), rabbit α-RFP (Rockland Antibodies Cat#600-401-379; RRID:AB_2209751 IHC 1:1000), mouse α DsRed (Santa Cruz Biotechnology Cat# sc-390909; RRID:AB_2801575 IHC 1:1000).

Techniques: Blocking Assay, Cell Culture

Journal: Communications Biology

Article Title: Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention

doi: 10.1038/s42003-021-02678-x

Figure Lengend Snippet: a Schematic representation of the experimental design. Step I, deletion of p75 NTR in astrocytes from tamoxifen-treated p75-flox mice precludes proBDNF transfer from neurons to astrocyte following TBS. Step II, LV-BDNFpro stop transduction replaces BDNFpro in astrocytes. Step III, astrocytic BDNFpro provides final increase of TrkB/SorCS2 complexes in dendritic spines and LTP maintenance. b z-stack reconstruction showing NeuN/PLA TrkB/SorCS2 colocalization signal in TBS-slices from p75-flox mice transduced with LV-GFP stop or LV-BDNFpro stop . Scale bars: 40 μm. The insets show the field of analysis. Scale bars: 15 μm. NeuN/PLA TrkB/SorCS2 colocalization was quantified using Mander’s overlap. ** p < 0.01 (unpaired t -test) ( n = 4 slices, 3 mice for each experimental condition). c Dot plot shows quantification of pTrkB/TrkB colocalization in baseline- and TBS-slices from p75-flox mice transduced with LV-GFP stop or LV-BDNFpro stop and control littermates using Mander’s overlap. *** p < 0.001 (unpaired t -test) ( n = 4 slices, 3 mice for p75-flox mice/LV-GFP stop /TBS; n = 4 slices, 3 mice for p75-flox mice/LV-GFP stop /baseline; n = 6 slices, 4 mice for p75-flox mice/LV-BDNFpro stop /TBS; n = 5 slices, 4 mice for p75-flox mice/LV-BDNFpro stop /baseline; n = 5 slices, 3 mice for control littermates/TBS; n = 6 slices, 3 mice for control littermates/baseline). Data are normalized to baseline and presented as mean ± SEM.

Article Snippet: The following antibodies were used: rabbit α-GFP (Thermo Fisher Scientific Cat#A-6455; RRID:AB#2536208; IHC 1:1000 ICC 1:1000), chicken α-GFP (Thermo Fisher Scientific Cat#A10262; RRID:AB#2534023, IHC 1:1000), chicken α-BDNF (Promega Cat#G1641; RRID:AB#430850, IHC 1:300; WB 1:500), rabbit α-BDNF (Alomone Labs Cat# ANT-010; RRID:AB_2039756, EM 1:20), chicken α-proBDNF (Millipore Cat#AB9042; RID:AB#2274709, IHC 1:300), rabbit α-proBDNF (Alomone Labs Cat#ANT-006; RRID:AB_2039758, EM 1:20), rabbit α-BDNFpro (Laboratory of Bai Lu, Govern Institute for Brain Research, Tsinghua University, Beijing, IHC 1:300, EM 1:20, WB 1:500), mouse α-NeuN (Abcam Cat#ab77315; RRID:AB#1566475, IHC 1:1000), guinea pig α-NeuN (Millipore Cat#ABN90; RRID:AB#11205592, IHC 1:1000), rabbit α-pTrkB (Tyr 816) (Laboratory of Moses Chao, Skirball Intitute of Biomolecular Medicine, New York, USA, IHC 1.25 mg/ml), goat α-TrkB (Santa Cruz Biotechnology Cat#sc-12-G; RRID:AB#632558, IHC 1:300), mouse α-PSD95 (Merck-Millipore Cat#MAB1596; RRID:AB_2092365, IHC 1:500), sheep α-SorCS2 (R&D System Cat#AF4238; RRID:AB_10645642), rabbit α-SorCS2 (MyBioSource Cat# MBS5302436), rabbit α-p75 NTR (Promega Cat#G3231; RRID:AB_430853, IHC 1:1000), chicken α-GFAP (Abcam Cat#ab134436; RRID:AB_2818977, IHC 1:1000), mouse α-GFAP (Abcam Cat#ab10062; RRID:AB_296804, IHC 1:1000), mouse α-synaptobrevin2\VAMP2 (Synaptic System Cat#104 211; RRID:AB_2619758, IHC 1:300), rabbit α-beta galactosidase (Proteintech Cat#15518-1-AP; RRID:AB_2263448, IHC 1:500), rabbit α-RFP (Rockland Antibodies Cat#600-401-379; RRID:AB_2209751 IHC 1:1000), mouse α DsRed (Santa Cruz Biotechnology Cat# sc-390909; RRID:AB_2801575 IHC 1:1000).

Techniques: Transduction