recombinant mouse cleavage resistant probdnf  (Alomone Labs)

Journal: bioRxiv

Article Title: CNTF specifically slows down the axonal transport of signalling endosomes

doi: 10.1101/2025.10.09.681259

Figure Lengend Snippet: A) Male and female ChAT.eGFP mice were allocated into one of five groups: i) PBS (control); ii) CNTF; iii) proBDNF; iv) HGF; and v) NTRN. B) Each mouse received intramuscular injections of H C T combined with a single NTF into the left tibialis anterior and right soleus muscles to target fast and slow motor neurons, respectively. After a 4-8 h incubation period, time-lapse microscopy was performed on both sciatic nerves. C) H C T-labelled signalling endosomes (pseudo-coloured in magenta) from single ChAT.eGFP motor axons were individually tracked to quantify retrograde transport dynamics. Three representative retrogradely transported signalling endosomes are identified by yellow, cyan, and peach arrowheads connected by dashed lines across frames. Grey arrowheads and dashed lines identify a stationary endosome. See also Video 1 . Scale bar = 5 μm, frame interval = 3 s.

Article Snippet: Mice were divided into five experimental cohorts, and received 5-7 μg of H C T mixed with: a) phosphate buffered saline (PBS) as the control; b) 50 ng recombinant human CNTF protein (Peprotech, 450-13), c) 50 ng recombinant mouse cleavage-resistant proBDNF (Alomone labs, B-243), d) 25 ng recombinant human HGF (Bio-Techne, 294-HG[CF]), or e) 50 ng recombinant human NTRN (Peprotech, 450-11), as summarised in .

Techniques: Control, Muscles, Incubation, Time-lapse Microscopy

Journal: bioRxiv

Article Title: CNTF specifically slows down the axonal transport of signalling endosomes

doi: 10.1101/2025.10.09.681259

Figure Lengend Snippet: In both fast motor neurons (FMNs) and slow motor neurons (SMNs), proBDNF did not alter A) mean endosome speed ( p = 0.06 for motor neuron type; p = 0.19 for stimulation factor; p = 0.70 for interaction), B) maximum endosome speed ( p = 0.30 for motor neuron type; p = 0.62 for stimulation factor; p = 0.25 for interaction) or C) pausing percentage ( p = 0.24 for motor neuron type; p = 0.92 for stimulation factor; p = 0.34 for interaction). D ) Violin plots of individual endosomes show comparable distributions in all conditions (FMNs: PBS mean = 2.78 µm/s ± 0.03 [n = 495], proBDNF mean = 2.67 µm/s ± 0.03 [n = 496]; SMNs: PBS mean = 2.57 µm/ s ± 0.03 [n = 495], proBDNF mean = 2.53 µm/s ± 0.03 [n = 477]). Overlapping endosome frame-to-frame and mean endosome speed distribution curves confirm that proBDNF does not modulate retrograde transport in E) FMNs or F) SMNs. Statistical analyses were performed using two-way ANOVA and Holm-Šídák 1 s multiple comparisons tests. ns, not significant. n = 7-8. Black circles = males (n = 4 PBS; n = 2 CNTF), white circles = females (n = 4 PBS; n = 5 proBDNF).

Article Snippet: Mice were divided into five experimental cohorts, and received 5-7 μg of H C T mixed with: a) phosphate buffered saline (PBS) as the control; b) 50 ng recombinant human CNTF protein (Peprotech, 450-13), c) 50 ng recombinant mouse cleavage-resistant proBDNF (Alomone labs, B-243), d) 25 ng recombinant human HGF (Bio-Techne, 294-HG[CF]), or e) 50 ng recombinant human NTRN (Peprotech, 450-11), as summarised in .

Techniques:

Journal: bioRxiv

Article Title: Matrix Metalloproteinase-9 controls structural synaptic plasticity via BDNF-dependent signaling

doi: 10.1101/2023.12.08.569797

Figure Lengend Snippet: TrkB activation depends on MMP-9 activity. A TrkB FRET sensor schematic. B Two-photon FLIM images of TrkB activation averaged at indicated time points. Warmer colors represent shorter lifetimes, increased binding fraction and higher TrkB activity. White cross indicate uncaging spot, scale bar: 1 μm. C Averaged time courses of TrkB activation (measured as a change of the sensor binding fraction) for spines stimulated by uncaging. Data are means ± SEM. Grey box indicates duration of sLTP protocol. D Statistical analysis of (C). Averaged TrkB activation (measured as a Δ Binding Fraction) for transient phase (1-3 min after sLTP induction) and sustained phase (9-11 min. after sLTP induction) in spines incubated with DMSO (n = 70 spines, 29 cells, 16 animals) or Inhibitor I (n = 49 spines, 21 cells, 10 animals). Gray dots represent individual values for spines, bars (black – DMSO, red – Inhibitor I) are means ± SEM. Repeated measures ANOVA (Time (F (1, 117) = 11.46, p = 0.0010); Inhibitor (F (1, 117) = 10.80, p = 0.0013); Time=Inhibitor (F (1, 117) = 0.1103, p = 0.7403)) followed by Šidák’s multiple comparison test (DMSO vs Inhibitor I at 1-3 min (p = 0.0113, 95% C.I. = [0.005425 to 0.05049]) and at 9-11 min (p = 0.0045, 95% C.I. = [0.008394 to 0.05346])). E Same as in (C). Averaged time courses of TrkB activation observed in spines obtained from WT, and MMP-9 KO mice. F same as in (D). WT (black bars, n = 66 spines; 22 cells, 10 animals), MMP-9 KO (blue bars, n = 73 spines; 25 cells, 11 animals). Repeated measures ANOVA (Time F (1, 137) = 5.084, p = 0.0257; MMP-9 KO F (1, 137) = 7.982, p = 0.0054; Time=MMP-9 KO F (1, 137) = 0.1253, p = 0.7238)) followed by Šidák’s multiple comparison test (WT vs MMP-9 KO at 1-3 min (p = 0.0351, 95% C.I.=[0.001241 to 0.04204] and at 9-11 min (p = 0.0149, 95% C.I. = [0.004051 to 0.04485])). G Representative immunoblot of digestion reaction of proBDNF incubated with active MMP- 9, inactive MMP-9 (E402A), or only in the reaction buffer. Arrows indicate proBDNF (∼26 kDa) and mBDNF (∼14 kDa). H Quantification of three separate Western-blots of proBDNF digestion. Gray dots represent individual values of mBDNF band intensity in separate experiments and Western-blots for each experimental condition. Bars are means ± SEM. One-way ANOVA (F (2, 6) = 20.38, p = 0.0021) followed by Tukey’s multiple comparisons test (Buffer vs MMP-9 p = 0.0034, 95% C.I. = [-213821761 to -62305372] and E402A vs MMP-9 p = 0.0038, 95% C.I. = [59136932 to 210653321]).

Article Snippet: Twenty ng of recombinant proBDNF (Alomone Labs) was incubated with 50 ng of recombinant MMP-9 (Calbiochem) or 50 ng of recombinant, human, inactive MMP- 9(E402A) in total volume of 20 μl.

Techniques: Activation Assay, Activity Assay, Binding Assay, Incubation, Comparison, Western Blot

Journal: eNeuro

Article Title: Cortical Brain Injury Causes Retrograde Degeneration of Afferent Basal Forebrain Cholinergic Neurons via the p75NTR

doi: 10.1523/ENEURO.0067-23.2023

Figure Lengend Snippet: Proneurotrophins are induced in the ipsilateral cortex but not the basal forebrain after cortical FPI. a–c , Brain tissue lysates from naive, sham, and injured (2 atm) wild-type adult mice were obtained 1DPI, 3DPI, and 7DPI to determine levels of proBDNF ( a , b ) and proNGF ( c ) in the injured versus uninjured side. Cortical tissue lysate ( a ) harvested for Western blot was probed for proBDNF (32 kDa) in the ipsilateral and contralateral cortex at 1DPI, 3DPI, and 7DPI in naive, sham, and injured mice. Basal forebrain tissue lysate ( b ) harvested for Western blot was probed for proBDNF (32 kDa) in the ipsilateral versus contralateral basal forebrain at 1DPI, 3DPI, and 7DPI in naive, sham, and injured mice. Cortex and basal forebrain tissue lysates ( c ) harvested for Western blot were probed for proNGF (37 kDa) at 3DPI after FPI in the ipsilateral versus contralateral side of the cortex and the basal forebrain; n = 4 (naive), n = 4 (sham 1DPI), n = 4 (injured 1DPI), n = 4 (sham 3DPI), n = 4 (injured 3DPI), n = 3 (sham 7DPI), n = 3 (injured 7DPI; a , b ); n = 3 (naive), n = 4 (sham 3DPI), n = 4 (injured 3DPI; c ). The established size of proBDNF is 32 kDa; however, a prominent band of 25 kDa was also recognized by the BDNF antibody that appeared to be regulated by injury, but the identity of that band is unclear.

Article Snippet: Recombinant human proNGF (cleavage resistant) protein (catalog #N-285) and recombinant mouse proBDNF (cleavage resistant) protein (catalog #B-243) were purchased from Alomone Labs. Poly- d -lysine, glucose, transferrin, insulin, putrescine, selenium, progesterone, penicillin, and streptomycin were purchased from Sigma-Aldrich.

Techniques: Western Blot