rh-brain derived neurotrophic factor (bdnf)  (Promega)

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB transmembrane domain (A) Fluoxetine (10 μM/15 min) and ketamine (10 μM/15 min) increased pTRKB.Y816 in cortical neurons immunoprecipitated with anti-PLC-γ1 (F[2,45] = 11.03, p = 0.0001, n = 16/group). (B) Fluoxetine facilitates BDNF-induced activation of TRKB under high cholesterol concentrations (interaction: F[2,132] = 5.15, p = 0.0070, n = 12/group) in cultured cortical cells. (C and D) Biotinylated fluoxetine binds to TRKB in lysates of TRKB expressing HEK cells (interaction: F[7,153] = 16.18, p < 0.0001; n = 6–14), but not (C) to TRKB.Y433F mutant or (D) to TRKB carrying the TMD of TRKA (TRKB/TRKA.TM) (interaction: F[7,80] = 43.75, p < 0.0001, n = 6/group). (E and F) Binding of biotinylated R,R-HNK (interaction: F[7,160] = 14.91, p < 0.0001; n = 6–14) (E) and tritiated imipramine (interaction: F[7,16] = 106.1, p < 0.0001; n = 2) (F) to TRKB, but not to TRKB.Y433F. Data expressed mean ± SEM of percentage of binding at 100 μM for fluoxetine and R,R-HNK or at 30 μM for imipramine. (G) Esketamine displaces the interaction of biotinylated fluoxetine (1 μM) with TRKB (n = 8/group). (H and I) Cholesterol facilitates the interaction of (H) biotinylated fluoxetine (F[5,30] = 7.198, p = 0.0002, n = 6/group)and (I) R,R-HNK (F[5,30] = 4.592, p = 0.0031, n = 6/group) with TRKB. (J) In situ PLA demonstrates close proximity between biotinylated fluoxetine and TRKB on TRKB-expressing N2A cells (red dots). (K) No PLA signal is seen in cells not expressing TRKB. Blue, DAPI; scale bar, 10 μm. (L) MST demonstrated direct interaction between fluoxetine and GFP-tagged TRKB (15 min) in lysates from GFP-TRKB expressing HEK293T cells (n = 4/group). Experimental traces depicted in the inset, vertical bars: blue, fluorescence cold; red, fluorescence hot. See also , , and .

Article Snippet: biotinylated rh-BDNF , Alomone Labs , Cat#B-250.

Techniques: Immunoprecipitation, Activation Assay, Cell Culture, Expressing, Mutagenesis, Binding Assay, In Situ, Fluorescence

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB transmembrane domain, related to Figure 2 Lysate from HEK293T cells expressing TRKB were submitted to ligand binding assays. (A) Schematic representation of the biotinylated fluoxetine interaction with immobilized TRKB. (B-J) Biotinylated fluoxetine (1 μM) interaction with TRKB is reduced by non-biotinylated (B) fluoxetine (n = 6/group), (C) imipramine (n = 8/group), (D) moclobemide (n = 10/group), (E) venlafaxine (n = 6/group), (F) ketamine (n = 8/group), (G) R,R-HNK (n = 8/group), but not reduced by (H) S,S-HNK (n = 8/group), (I) chlorpromazine (n = 8/group), isoproterenol (n = 8/group) or diphenhydramine (n = 8/group), or (J) BDNF (n = 6/group). (K) Biotinylated R,R-HNK (1 μM) interaction with TRKB is not reduced by S,S-HNK (n = 12/group). (L,M) Biotinylated fluoxetine interaction with (L) TRKA (n = 7/group) from MG87 cells, or (M) lysates from non-transfected HEK cells (n = 10/group) are negligible compared to TRKB. The interaction of biotinylated fluoxetine is not altered in (N) TRKB lacking most of the intra and extracellular domains (TRKB.T1ΔEC, n = 12/group), but it is reduced by (O) V437A and Y433F mutations, and partially attenuated by S440A (n = 6/group).

Article Snippet: biotinylated rh-BDNF , Alomone Labs , Cat#B-250.

Techniques: Expressing, Ligand Binding Assay, Transfection

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB in the intact cells, related to Figure 2 (A) Schematic representation of the in situ proximity ligation assay (PLA) between TRKB and biotinylated fluoxetine. (B-G) HEK cells were transfected to express TRKB and farnesylated GFP and were exposed to biotinylated fluoxetine (10uM/15min). The cells were fixed in PFA and the PLA reaction was conducted in permeabilized cells. (B-D) No signal from TRKB-FLX interaction was observed when cells were not transfected to express TRKB. (E-G) positive signal of TRKB-FLX (PLA). Scale bar: 20 μm. Zoom in square: 2.5x.

Article Snippet: biotinylated rh-BDNF , Alomone Labs , Cat#B-250.

Techniques: In Situ, Proximity Ligation Assay, Transfection

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet:

Article Snippet: biotinylated rh-BDNF , Alomone Labs , Cat#B-250.

Techniques: Recombinant, In Situ, Stable Transfection, Expressing, Plasmid Preparation, Software

Journal: Reproduction (Cambridge, England)

Article Title: Neurotrophin NT3 promotes ovarian primordial to primary follicle transition

doi: 10.1530/REP-09-0179

Figure Lengend Snippet: Follicle development (follicle category percentage) in cultured rat ovaries treated with NT3, BDNF or NGF. Postnatal 4-day (P4) old rat ovaries were cultured for 10 days in the absence or presence of: (A) NT3 (50 ng/ml); (B) BDNF (50 or 250 ng/ml); (C) NGF (50 ng/ml); or KL (50 ng/ml). Follicles were classified as primordial or developing. KL was used as a positive control and has previously been shown to stimulate follicle development. A minimum of three different experiments in replicate were performed with the mean±S.E.M. presented and asterisks indicating a statistical difference from control with *P<0.05 and **P<0.01 by Dunnet's post-hoc test after a significant ANOVA result.

Article Snippet: Ovaries were treated with no factor (control), NT3 (rh NT3, 50 ng/ml; R&D Systems, Minneapolis, MN, USA), BDNF (rh BDNF, 50–250 ng/ml; R&D Systems), NGF (rr β-NGF, 50 ng/ml; R&D Systems) or KL/stem cell factor (rm SCF, 50 ng/ml; R&D Systems).

Techniques: Cell Culture, Positive Control, Control

Journal: Journal of drug targeting

Article Title: Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS

doi: 10.3109/10611860903318134

Figure Lengend Snippet: (A) Intranasal delivery in nM of BDNF 25 min after intranasal delivery (N = 7). (B) Intranasal delivery in nM of BDNF 60 min after intranasal delivery (N = 3).

Article Snippet: Recombinant human (rh) BDNF (molecular weight 26,984 Da), NT-4 (22,428 Da), CNTF (22,706 Da), and EPO (glycosylated form 30,400 Da) were each purchased from Cell Sciences, Inc. (Canton, MA, USA).

Techniques:

Journal: Journal of drug targeting

Article Title: Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS

doi: 10.3109/10611860903318134

Figure Lengend Snippet: Concentration of drug 25 min after intranasal delivery in nanograms/mL.

Article Snippet: Recombinant human (rh) BDNF (molecular weight 26,984 Da), NT-4 (22,428 Da), CNTF (22,706 Da), and EPO (glycosylated form 30,400 Da) were each purchased from Cell Sciences, Inc. (Canton, MA, USA).

Techniques: Concentration Assay

Journal: Journal of drug targeting

Article Title: Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS

doi: 10.3109/10611860903318134

Figure Lengend Snippet: Concentration of drug 60 min after intranasal delivery in nanograms/mL.

Article Snippet: Recombinant human (rh) BDNF (molecular weight 26,984 Da), NT-4 (22,428 Da), CNTF (22,706 Da), and EPO (glycosylated form 30,400 Da) were each purchased from Cell Sciences, Inc. (Canton, MA, USA).

Techniques: Concentration Assay

Journal: Journal of drug targeting

Article Title: Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS

doi: 10.3109/10611860903318134

Figure Lengend Snippet: Western blot examination of activation of the pAkt pathway caused by intranasal delivery of either CNTF or BDNF. (A) Representative western blot of control and CNTF-treated occipital cortex 24 and 48 h after a single intranasal application. Control pAkt protein is on the far right. HPRT was used as a loading control. (B) Densitometric examination of relative amounts of pAkt in CNTF-treated occipital cortex 24, 48, 72, and 96 h after a single intranasal application compared to saline-only control cortex (N=6 for each time point). * indicates significantly different from control. (C) Densitometric examination of relative amounts of pAkt in BDNF-treated frontal cortex 24, 48, 72, and 96 h after a single intranasal application compared to saline-only control cortex (N = 6 for each time point). * indicates significantly different from control.

Article Snippet: Recombinant human (rh) BDNF (molecular weight 26,984 Da), NT-4 (22,428 Da), CNTF (22,706 Da), and EPO (glycosylated form 30,400 Da) were each purchased from Cell Sciences, Inc. (Canton, MA, USA).

Techniques: Western Blot, Activation Assay, Control, Saline