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A-D Kinetics of TrkB-FL downregulation. Primary cortical cultures were treated with NMDA (100 μM) and its coagonist glycine (10 μM), denoted as “NMDA”. Immunofluorescence (A) and immunoblot (B) were performed with a C-ter isoform-specific antibody (TrkB-FL Ct) recognizing full-length protein (FL) and intracellular fragment (f32). A TrkB-FL (green) and nuclei (blue), detected with DAPI. Arrowheads indicate varicosities in neuronal projections. Scale bar: 10 μm. Cell body details are shown for untreated cells or those with NMDA (120 min). B Decrease of TrkB-FL and f32 formation compared to PSD-95 downregulation, detected by a C-ter antibody (PSD-95 Ct). Calpain activation was established by accumulation of characteristic spectrin breakdown products (BDPs; 150 and 145 kDa). Neuron-specific enolase (NSE) was used as a loading control and for protein normalization. C, D Quantification of normalized TrkB-FL and PSD-95 levels relative to values in the absence of NMDA. Means ± SEM are represented. Statistical analysis was performed by one-way analysis of variance (ANOVA) test followed by a Bonferroni post hoc test (* *P < 0.01, *** P < 0.001; n = 5). E, F Cultures were preincubated or not with dynasore (80 μM, 30 min) and treated with NMDA (2 h). Means ± SEM ( n = 4), statistical analysis as above ( *P < 0.05). G Sequences of cell-penetrating neuroprotective (MTFL 457 ) and control (MTMyc) peptides, which contain Tat aa 47–57 (italic) followed by, respectively, rat TrkB-FL (aa 457-471, light blue) or c-Myc (aa 408-421, dark blue) sequences. H , I Cultures were preincubated with peptides (25 μM, 30 min) followed by NMDA treatment. Culture media was analysed with antibodies directed to TrkB-FL extracellular domain (panTrkB), recognizing all isoform ECDs (TrkB-ECD), to investigate receptor shedding by MP activation. Analysis of total lysates with TrkB- FL Ct investigated TrkB-FL calpain-processing through f32 production. Means ± SEM ( n = 7) of TrkB- ECD values are represented, and statistically analysed by ANOVA followed by a Bonferroni’s honestly significant difference test ( *P < 0.05, *** P < 0.001, MTMyc + NMDA vs MTMyc; # # P < 0.01, MTMyc vs MTFL 457 at each time). J, K NMDA effect on total and cell-surface <t>pY816-TrkB-FL</t> levels. Cultures were preincubated with peptides as above, before a brief NMDA treatment (1 h) to avoid extensive receptor degradation, followed by biotin-labelling and sedimentation of membrane proteins, and comparison to corresponding total lysates. Means ± SEM ( n = 4) are represented and analysed by unpaired Student’s t-test (n.s. = not significant; * * P < 0.01).
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A-D Kinetics of TrkB-FL downregulation. Primary cortical cultures were treated with NMDA (100 μM) and its coagonist glycine (10 μM), denoted as “NMDA”. Immunofluorescence (A) and immunoblot (B) were performed with a C-ter isoform-specific antibody (TrkB-FL Ct) recognizing full-length protein (FL) and intracellular fragment (f32). A TrkB-FL (green) and nuclei (blue), detected with DAPI. Arrowheads indicate varicosities in neuronal projections. Scale bar: 10 μm. Cell body details are shown for untreated cells or those with NMDA (120 min). B Decrease of TrkB-FL and f32 formation compared to PSD-95 downregulation, detected by a C-ter antibody (PSD-95 Ct). Calpain activation was established by accumulation of characteristic spectrin breakdown products (BDPs; 150 and 145 kDa). Neuron-specific enolase (NSE) was used as a loading control and for protein normalization. C, D Quantification of normalized TrkB-FL and PSD-95 levels relative to values in the absence of NMDA. Means ± SEM are represented. Statistical analysis was performed by one-way analysis of variance (ANOVA) test followed by a Bonferroni post hoc test (* *P < 0.01, *** P < 0.001; n = 5). E, F Cultures were preincubated or not with dynasore (80 μM, 30 min) and treated with NMDA (2 h). Means ± SEM ( n = 4), statistical analysis as above ( *P < 0.05). G Sequences of cell-penetrating neuroprotective (MTFL 457 ) and control (MTMyc) peptides, which contain Tat aa 47–57 (italic) followed by, respectively, rat TrkB-FL (aa 457-471, light blue) or c-Myc (aa 408-421, dark blue) sequences. H , I Cultures were preincubated with peptides (25 μM, 30 min) followed by NMDA treatment. Culture media was analysed with antibodies directed to TrkB-FL extracellular domain (panTrkB), recognizing all isoform ECDs (TrkB-ECD), to investigate receptor shedding by MP activation. Analysis of total lysates with TrkB- FL Ct investigated TrkB-FL calpain-processing through f32 production. Means ± SEM ( n = 7) of TrkB- ECD values are represented, and statistically analysed by ANOVA followed by a Bonferroni’s honestly significant difference test ( *P < 0.05, *** P < 0.001, MTMyc + NMDA vs MTMyc; # # P < 0.01, MTMyc vs MTFL 457 at each time). J, K NMDA effect on total and cell-surface pY816-TrkB-FL levels. Cultures were preincubated with peptides as above, before a brief NMDA treatment (1 h) to avoid extensive receptor degradation, followed by biotin-labelling and sedimentation of membrane proteins, and comparison to corresponding total lysates. Means ± SEM ( n = 4) are represented and analysed by unpaired Student’s t-test (n.s. = not significant; * * P < 0.01).

Journal: bioRxiv

Article Title: Retrograde transport of neurotrophin receptor TrkB-FL induced by excitotoxicity regulates Golgi stability and is a target for stroke neuroprotection

doi: 10.1101/2024.10.29.620835

Figure Lengend Snippet: A-D Kinetics of TrkB-FL downregulation. Primary cortical cultures were treated with NMDA (100 μM) and its coagonist glycine (10 μM), denoted as “NMDA”. Immunofluorescence (A) and immunoblot (B) were performed with a C-ter isoform-specific antibody (TrkB-FL Ct) recognizing full-length protein (FL) and intracellular fragment (f32). A TrkB-FL (green) and nuclei (blue), detected with DAPI. Arrowheads indicate varicosities in neuronal projections. Scale bar: 10 μm. Cell body details are shown for untreated cells or those with NMDA (120 min). B Decrease of TrkB-FL and f32 formation compared to PSD-95 downregulation, detected by a C-ter antibody (PSD-95 Ct). Calpain activation was established by accumulation of characteristic spectrin breakdown products (BDPs; 150 and 145 kDa). Neuron-specific enolase (NSE) was used as a loading control and for protein normalization. C, D Quantification of normalized TrkB-FL and PSD-95 levels relative to values in the absence of NMDA. Means ± SEM are represented. Statistical analysis was performed by one-way analysis of variance (ANOVA) test followed by a Bonferroni post hoc test (* *P < 0.01, *** P < 0.001; n = 5). E, F Cultures were preincubated or not with dynasore (80 μM, 30 min) and treated with NMDA (2 h). Means ± SEM ( n = 4), statistical analysis as above ( *P < 0.05). G Sequences of cell-penetrating neuroprotective (MTFL 457 ) and control (MTMyc) peptides, which contain Tat aa 47–57 (italic) followed by, respectively, rat TrkB-FL (aa 457-471, light blue) or c-Myc (aa 408-421, dark blue) sequences. H , I Cultures were preincubated with peptides (25 μM, 30 min) followed by NMDA treatment. Culture media was analysed with antibodies directed to TrkB-FL extracellular domain (panTrkB), recognizing all isoform ECDs (TrkB-ECD), to investigate receptor shedding by MP activation. Analysis of total lysates with TrkB- FL Ct investigated TrkB-FL calpain-processing through f32 production. Means ± SEM ( n = 7) of TrkB- ECD values are represented, and statistically analysed by ANOVA followed by a Bonferroni’s honestly significant difference test ( *P < 0.05, *** P < 0.001, MTMyc + NMDA vs MTMyc; # # P < 0.01, MTMyc vs MTFL 457 at each time). J, K NMDA effect on total and cell-surface pY816-TrkB-FL levels. Cultures were preincubated with peptides as above, before a brief NMDA treatment (1 h) to avoid extensive receptor degradation, followed by biotin-labelling and sedimentation of membrane proteins, and comparison to corresponding total lysates. Means ± SEM ( n = 4) are represented and analysed by unpaired Student’s t-test (n.s. = not significant; * * P < 0.01).

Article Snippet: Primary antibodies against the following proteins were used: Hrs (Cat#sc-271455, RRID:AB_10648901, Santa Cruz Biotechnology), NSE (Cat#AB951, RRID:AB_92390, Millipore), spectrin alpha chain (Cat#MAB1622, RRID:AB_94295, Millipore), TrkB-FL C-ter (sc-11, RRID:AB_632554, Santa Cruz Biotechnology), TrkB extracellular sequences or panTrkB (sc-136990, RRID:AB_2155262, Santa Cruz Biotechnology), PSD-95 C-ter (Cat#610496, RRID:AB_2315218, Transduction Laboratories), pY816-TrkB-FL (Cat#P01388, Boster).

Techniques: Immunofluorescence, Western Blot, Activation Assay, Control, Sedimentation, Membrane, Comparison