Journal: Cell death & disease

Article Title: Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

doi: 10.1038/cddis.2012.80

Figure Lengend Snippet: Figure 2 MT2 interactions with TrkA. (a) Displacement of 125I-hrNGF bound to PC12 cells by MT2. PC12 cells were incubated with 0.1 nM 125I-hrNGF in the presence or absence of different concentrations of MT2. Specific cell bound radioactivity was calculated and the results analyzed by Origin software. Results of one representative experiment out of three performed are shown. (b and c) Binding of 3H-MT2 to TrkA NIH-3T3. NIH-3T3, stably transfected with full-length human TrkA, were incubated in triplicate with different concentrations of 3H-MT2, in the presence or absence of excess cold MT2 (b) or 4 nM cold hrNGF (c). Specific cell bound radioactivity was calculated and the results analyzed by the Origin software (one-site binding assay). No specific binding was recorded on mock-transfected NIH-3T3 cells (not shown). (d) Internalization of 3H-MT2. TrkA-NIH-3T3 or mock-transfected cells were incubated for 1 h at 41 C with 3H-MT2 in the presence or absence of excess cold MT2 or hrNGF. Then, cells were washed and brought at 371 C for 1 h. Membrane radioactivity was eluted with 0.1 M glycine buffer, pH 2.8. RBC were incubated for 1 h at 371 C with 3H-MT2 in the presence or absence of excess cold MT2 or hrNGF. Cells were lysed, and cell-bound radioactivity recorded. Data are expressed as mean bound radioactivity±S.E. of triplicate cultures. Results of one representative experiments out of three performed are shown. (e) MT2 interaction with the ECD fraction of human recombinant TrkA. One microgram purified TrkA ECD was incubated in triplicate with different concentrations of 3H-MT2, in the presence or absence of excess cold MT2. The mixture was absorbed on filter papers and, after washing, the radioactivity recorded. Data were analyzed by Origin software. Results from one representative experiments out of three performed are shown

Article Snippet: Membranes were stained with rabbit antibodies anti-P-TrkA-Y490, anti-PTrkA-Y674/675, anti-P-TrkA-Y 785, anti-P-Akt, anti-P-ERK 1/2, and anti-P-SAPK/ JNK (Cell Signaling Technology, Danvers, MA, USA), anti-MKP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VGF (OriGene, Rockville, MD, USA) and mouse anti b-actin (Santa Cruz Biotecnology); all the antibodies were used at 1 : 1000, final dilution.

Techniques: Incubation, Radioactivity, Software, Binding Assay, Stable Transfection, Transfection, Membrane, Recombinant

Journal: Cell death & disease

Article Title: Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

doi: 10.1038/cddis.2012.80

Figure Lengend Snippet: Figure 3 Binding sites of MT2 in TrkA molecule. In silico prediction of amino- acid residues involved. The predicted bound conformation of MT2 at the binding site of TrkA is shown. The surface of the pocket is reported explicitly with the structure of TrkA in gray ribbons as background. The amino acids of TrkA that establish direct interaction with MT2 are displayed in ball and stick representation. MT2 is displayed in ball and stick with carbon atoms colored dark yellow. As a term of comparison, Arg103 and Ile31 of NGF are reported in ball and stick with the carbon atoms colored orange. The H-bond between Thr325 of TrkA and MT2 is highlighted with green dots. The van der Waals volume of part of Thr352 and Val354 side chains is highlighted with a black dotted line

Article Snippet: Membranes were stained with rabbit antibodies anti-P-TrkA-Y490, anti-PTrkA-Y674/675, anti-P-TrkA-Y 785, anti-P-Akt, anti-P-ERK 1/2, and anti-P-SAPK/ JNK (Cell Signaling Technology, Danvers, MA, USA), anti-MKP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VGF (OriGene, Rockville, MD, USA) and mouse anti b-actin (Santa Cruz Biotecnology); all the antibodies were used at 1 : 1000, final dilution.

Techniques: Binding Assay, In Silico, Comparison

Journal: Cell death & disease

Article Title: Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

doi: 10.1038/cddis.2012.80

Figure Lengend Snippet: Figure 5 Biochemical pathways induced by MT2 in PC12 cells. (a) TrkA phosphorylation. Serum-starved PC12 cells were stimulated with 10 mM MT2, or 4 nM hrNGF as positive control, for 15 min. Cells lysates were blotted with antibodies to P-Y490, P-Y674/675, P-Y785 and with anti-actin as loading control. Membranes were stripped and stained with anti-total TrkA IgG. The relative histograms represent the data of densitometric analysis and are expressed as the ratio between phosphoprotein (P) and total protein (T) of five different experiments (mean±S.E.). Statistical analysis was performed by paired Student’s t-test. (b) Kinases phosphorylation. Serum-starved PC12 cells were stimulated with 10 mM MT2, or 4 nM hrNGF, for 30 min. Cell lysates were blotted with rabbit anti-P-ERK, anti-P-Akt, anti-P-p38 MAPK, anti-P-JNK. Then, membranes were stripped and stained with antibodies to the respective total protein and with anti-actin antibodies as loading control. The relative histograms represent the data of densitometric analysis and are expressed as the ratio between phosphoprotein (P) and total protein (T) of five different experiments (mean±S.E.). Statistical analysis was performed by paired Student’s t-test. (c) Phosphatase. Serum-starved PC12 cells were stimulated with 10 mM MT2, or 4 nM hrNGF as positive control, for 30 min. Cell lysates were blotted with rabbit anti-MKP1 IgG and anti-actin antibodies as loading control. The relative histogram represents the data of densitometric analysis and is expressed as the ratio between MKP1 expression and actin of five different experiments (mean±S.E.). Statistical analysis was performed by paired Student’s t-test

Article Snippet: Membranes were stained with rabbit antibodies anti-P-TrkA-Y490, anti-PTrkA-Y674/675, anti-P-TrkA-Y 785, anti-P-Akt, anti-P-ERK 1/2, and anti-P-SAPK/ JNK (Cell Signaling Technology, Danvers, MA, USA), anti-MKP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VGF (OriGene, Rockville, MD, USA) and mouse anti b-actin (Santa Cruz Biotecnology); all the antibodies were used at 1 : 1000, final dilution.

Techniques: Phospho-proteomics, Positive Control, Control, Staining, Expressing

Journal: Cell death & disease

Article Title: Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

doi: 10.1038/cddis.2012.80

Figure Lengend Snippet: Figure 6 Biochemical pathways induced by MT2 in WT TrkA-NIH-3T3 or mutants. NIH-3T3 cells, stably transfected with full-length WT human TrkA, T352A, or F327A TrkA mutants, were cultured in serum-free medium and incubated with 10 mM MT2 or 4 nM hrNGF, in the presence or absence of K252a. Cell lysates were blotted with rabbit anti-P-ERK (a) and anti-P-p38 MAPK (b). Then, membranes were stripped and stained with antibodies to the respective total protein. The relative histograms represent the densitometric analysis. Data are expressed as ratio between phosphoprotein (P) and total protein (T) of three different experiments (mean±S.E.). Statistical analysis was performed by paired Student’s t-test

Article Snippet: Membranes were stained with rabbit antibodies anti-P-TrkA-Y490, anti-PTrkA-Y674/675, anti-P-TrkA-Y 785, anti-P-Akt, anti-P-ERK 1/2, and anti-P-SAPK/ JNK (Cell Signaling Technology, Danvers, MA, USA), anti-MKP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VGF (OriGene, Rockville, MD, USA) and mouse anti b-actin (Santa Cruz Biotecnology); all the antibodies were used at 1 : 1000, final dilution.

Techniques: Stable Transfection, Transfection, Cell Culture, Incubation, Staining

Journal: Cell death & disease

Article Title: Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

doi: 10.1038/cddis.2012.80

Figure Lengend Snippet: Figure 7 MT2 protects neurons from Ab amyloid-mediated death in NGF-deficient neurons. (a and b) 3–4 days cultured hippocampal neurons were exposed to 2 nM NGF or to MT2 (5–30 mM) and the highest active concentration of MT2 evaluated as induction of TrkA phosphorylation by western blot analysis with anti-phosphorylated (P) and anti total (T) Trk-A antibodies or by counting the number of NeuN stained nuclei (b). (c and d) hippocampal neurons were deprived of NGF by exposure to anti-NGF antibodies (30 mg/ml) for 24 h and incubated in the presence or absence of 10 mM MT2 or 2 nM hrNGF. Cells were lysed and western blot analysis performed with antibodies against APP full-length (c) and PS1 28 kDa N-terminal fragment (d). (e and f) Hippocampal neurons were deprived of NGF by exposure to anti-NGF antibodies (30 mg/ml) for 24 h and then treated with concentrations of MT2 ranging from 5 to 30 mM or 2 nM hrNGF. The percentage of alive neurons was obtained by counting the number of intact nuclei (d) or alternatively by counting the number of condensed nuclei (e). Ctrl, neurons not exposed to NGF or MT2; NGF, neurons exposed for 48 h to NGF; MT2, neurons exposed for 48 h to MT2 (10 mM); Dep, neurons deprived of NGF for 24 hours; Dep þ NGF, neurons washed with NGF-free media and immediately exposed to NGF containing media; Dep þ MT2 neurons washed with NGF-free media and immediately exposed to MT2-containing media. Statistical analysis was performed by Newman–Keuls test (N ¼ 4), Po0.05. Consistent with the inhibition of the amyloidogenic pathway, neurons were completely protected from death

Article Snippet: Membranes were stained with rabbit antibodies anti-P-TrkA-Y490, anti-PTrkA-Y674/675, anti-P-TrkA-Y 785, anti-P-Akt, anti-P-ERK 1/2, and anti-P-SAPK/ JNK (Cell Signaling Technology, Danvers, MA, USA), anti-MKP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VGF (OriGene, Rockville, MD, USA) and mouse anti b-actin (Santa Cruz Biotecnology); all the antibodies were used at 1 : 1000, final dilution.

Techniques: Cell Culture, Concentration Assay, Phospho-proteomics, Western Blot, Staining, Incubation, Inhibition

Journal: British journal of pharmacology

Article Title: Chondroitin sulfate proteoglycan is a potential target of memantine to improve cognitive function via the promotion of adult neurogenesis.

doi: 10.1111/bph.15920

Figure Lengend Snippet: FIGURE 5 MEM affects the expression levels of genes related to the BDNF signalling in the hippocampus. (a–f) The expression levels of mRNA encoding BDNF and BDNF-related molecules in the dentate gyrus (DG) and CA1 region (CA1): BDNF (a), TrkB (b), Akt (c), ERK (d), CaMKII (e), and CREB (f). (g, h) The expression levels of mRNA encoding NGF (g) and its receptor TrkA (h) in the dentate gyrus and CA1 region. The graphs show the values relative to the mean of the DG of VEH-treated mice. Statistical differences were analysed by two-way ANOVA with post hoc Tukey's HSD test. Data are presented as mean ± SD (n = 7 for VEH-treated MA mice and n = 7 for MEM-treated MA mice). Each circle represents one animal. One outlier in panel (d) (CA1 region of VEH-treated MA mouse) was excluded based on the Grubbs test. Asterisks indicate statistical significance: *P < 0.05.

Article Snippet: Transferred polyvinylidene difluoride membranes were blocked with 5.0% skim milk in PBS containing 0.1% Tween 20 (PBS-T) for 30 min at room temperature and incubated overnight at 4 C with the following antibodies; rabbit monoclonal anti-chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT1) antibody (1:1000, Proteintech, Rosemont, IL, USA; Cat# 26913-1-AP, Lot# 0049252; RRID:AB_2880682), rabbit monoclonal anti-CREB antibody (1:2000, Cell Signaling Technology; Cat# 9197, Lot# 17; RRID:AB_331277), rabbit monoclonal anti-p-CREB antibody (1:1000, Cell Signaling Technology; Cat# 9198, Lot# 18; RRID:AB_ 2561044), rabbit monoclonal anti-TrkB antibody (1:2000, Cell Signaling Technology; Cat# 92991, Lot# 10; RRID:AB_2800196), rabbit monoclonal anti-p-TrkB antibody (1:1000, Cell Signaling Technology; Cat# 4619, Lot# 4; RRID:AB_10235585), and mouse monoclonal anti-β-actin antibody (1:20000; Sigma, Cat# A5441, Lot# 014M4759; RRID:AB_476744).

Techniques: Expressing

Journal: Translational Psychiatry

Article Title: Carboxypeptidase E conditional knockout mice exhibit learning and memory deficits and neurodegeneration

doi: 10.1038/s41398-023-02429-y

Figure Lengend Snippet: a – e Western blot analysis of p -TrkB, BDNF, p -mTOR, mTOR, p -AKT, and AKT levels in the hippocampus. f Immunofluorescence of CPE, MAP2, DCX, and GFAP; and the relative fluorescent intensities of g CPE in Sub, h MAP2 in Sub, i MAP2 in hilius, j DCX in DG, k GFAP in DG of WT, CPE flox/− , and CPE flox/flox mice at 100× and 400× (square in the panel). n = 6; * P < 0.05 and ** P < 0.01 compared with WT; values are mean ± SEM.

Article Snippet: After blocking with 5% nonfat milk, the membrane was blotted with antibodies against mouse anti-β-actin antibody (1:1000, CST, 4967S), mouse anti-CPE antibody (1:1000; BD bioscience, 610758), mouse anti-BDNF antibody (1:600, Abcam, UK, ab108319), rabbit anti-p-TrkB antibody (1:1000; Boster, Wuhan, China, BM4437), rabbit anti-AKT (1:2000, Cell Signaling Technology, 4691S), and anti-p-AKT (1:2000, Cell Signaling Technology, 23430S) overnight at 4 °C.

Techniques: Western Blot, Immunofluorescence