mouse anti trka  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/ijms25105475

Figure Lengend Snippet: ( a ) Indirect IFs demonstrating increased overlapping (yellow/orange) immunoreactivity for TrkA (green) and MitoTracker-labeled mitochondria (red) (upper panels) and Y490 phosphorylated TrkAIII (pTrkAIII, green) and MitoTracker-labeled mitochondria (red) (lower panels) in DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cells compared to untreated controls (control). DAPI stained nuclei are blue. (bar = 50 μm). ( b ) Western blots demonstrating TrkAIII cleavage and Y674/5 phosphorylation in mitochondria (50 mg) from DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cells compared to untreated TrkAIII SH-SY5Y controls. ( c ) Western blots demonstrating increased PTPase oxidation (arrows) in mitochondria (50 mg) from DTT-treated TrkAIII SH-SY5Y cells (5 mM for 6 h) compared to untreated controls (Con). ( d ) Phase contrast images merged with green fluorescence and histogram demonstrating significant differences (* p < 0.0001) in pcDNA-SH-SY5Y and TrkAIII SH-SY5Y percentage cell death at 24 h and 48 h, induced by 5 mM DTT.

Article Snippet: Mouse monoclonal anti-human TrkA (B3, sc-7268), rabbit polyclonal anti-human XIAP (H-202), rabbit polyclonal anti-human α-tubulin (H-3000) and monoclonal mouse anti-human calmodulin (sc-137079) antibodies were from SantaCruz (Santa Cruz, CA, USA).

Techniques: Labeling, Control, Staining, Western Blot, Phospho-proteomics, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/ijms25105475

Figure Lengend Snippet: ( a ) Western blots demonstrating differences in TrkAIII immunoreactivity in untreated (CON) and DTT-treated (5 mM DTT for 1, 2 and 3 h) TrkAIII SH-SY5Y cell extracts (30 mg) under non-reducing and reducing conditions. ( b ) Co-immunoprecipitation Western blots demonstrating increased TrkAIII pulldown of Grp78 by anti-TrkA antibody in DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cell extracts compared to untreated control extracts (CON) and similar levels of TrkAIII immunoprecipitated by anti-TrkA antibody but not by pre-immune mouse IgG in untreated (Con) and DTT-treated (5 mM for 6 h) TrkAIII SH-SY5Y cell extracts (500 μg), plus the absence of GRP78 or TrkA isoform pulldown by anti-TrkA antibody in pcDNA-SH-SY5Y cell extracts (500 μg). ( c ) Western blots of TrkAIII pulldown by calmodulin-conjugated Sepharose (CaM-Seph) in TrkAIII SH-SY5Y but not pcDNA-SH-SY5Y cell extracts (500 μg) in the presence of 150 mM CaCl 2 (left panel) plus increased TrkAIII pulldown by calmodulin-conjugated Sepharose (CaM-Seph) in TrkAIII SH-SY5Y cell extracts (500 μg) in the presence of 150 μM CaCl 2 compared to 5 mM EGTA (right panel), plus no TrkAIII pulldown by unconjugated Sepharose in TrkAIII SH-SY5Y cell extracts in the presence of 150 μM CaCl 2 or 5 mM EGTA (right panel). ( d ) Co-immunoprecipitation Western blots demonstrating enhanced pulldown of TrkAIII by anti-calmodulin antibody (anti-CaM) but not by pre-immune mouse IgG in DTT-treated (5 mM for 3 and 6 h) but not untreated (Con) TrkAIII SH-SY5Y extracts (500 μg) (upper left panels) and calmodulin pulldown by anti-calmodulin antibody (anti-CaM) but not pre-immune IgG in untreated (Con) and DTT-treated (DTT) TrkAIII SH-SY5Y cell extracts (500 μg) (upper right panels), plus no pulldown of TrkAIII by anti-calmodulin antibody in pcDNA-SH-SY5Y extracts (500 μg).

Article Snippet: Mouse monoclonal anti-human TrkA (B3, sc-7268), rabbit polyclonal anti-human XIAP (H-202), rabbit polyclonal anti-human α-tubulin (H-3000) and monoclonal mouse anti-human calmodulin (sc-137079) antibodies were from SantaCruz (Santa Cruz, CA, USA).

Techniques: Western Blot, Immunoprecipitation, Control

Journal: International Journal of Molecular Sciences

Article Title: Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/ijms25105475

Figure Lengend Snippet: ( a ) Western blots demonstrating TrkAIII cleavage and phosphorylation in mitochondria (50 μg) from TrkAIII SH-SY5Y cells treated with 5 mM DTT alone for 6 h and in mitochondria from TrkAIII SH-SY5Y cells co-treated with 5 mM DTT and either HA-15 (20 μM) (DTT/HA); brefeldin A (5 mg/mL) (DTT/BfA) or W7 (60 μM) (DTT/W7) plus non-phosphorylated TrkAIII in mitochondria from untreated TrkAIII SH-SY5Y cells (Con) and lack of TrkA or phosphorylated TrkA immunoreactivity in mitochondria (50 μg) from untreated (Con) and DTT-treated (5 mm for 6 h) pcDNA-SH-SY5Y cells. ( b ) Line graphs demonstrating significant inhibition (*) of pcDNA-SH-SY5Y and TrkAIII SH-SY5Y proliferation by BfA (5 μg/mL) and W7 (60 μM) but not by HA-15 (20 μM) at 24 and 48 h (* p < 0.0001). ( c ) Phase contrast images merged with green fluorescence plus histograms demonstrating percentage pcDNA-SH-SY5Y and TrkAIII SH-SY5Y cell death induced by 5 mM DTT alone (DTT), DTT and either HA-15 (20 μM) (DTT/HA), BfA (5 μg/mL) (DTT/BfA) or W7 (60 μM) (DTT/W7), plus pcDNA-SH-SY5Y and TrkAIII SH-SY5Y cell death induced by W7 (60 μM) alone (W7) at 24 and 48 h (* p < 0.006).

Article Snippet: Mouse monoclonal anti-human TrkA (B3, sc-7268), rabbit polyclonal anti-human XIAP (H-202), rabbit polyclonal anti-human α-tubulin (H-3000) and monoclonal mouse anti-human calmodulin (sc-137079) antibodies were from SantaCruz (Santa Cruz, CA, USA).

Techniques: Western Blot, Phospho-proteomics, Inhibition, Fluorescence

Journal: Biomedicines

Article Title: A Possible Role for Nerve Growth Factor and Its Receptors in Human Sperm Pathology.

doi: 10.3390/biomedicines11123345

Figure Lengend Snippet: Figure 2. Nerve growth factor (NGF) receptors in the ejaculated sperm, epididymis and testis of fertile men. (A) The bar graph shows the mean and standard deviation of the fold increase in TrKA (slanted-line bar graph) and p75NTR (checkerboard bar graph) messenger RNA (mRNA) levels in fertile men (n = 7). (B,C) The plots (blue gate) display the flow cytometric analysis (representative of

Article Snippet: After washing three times in PBS supplemented with BSA, sperm were incubated at 4 ◦C for 1 h in PBS/BSA containing FITC-labelled rabbit anti-human p75NTR extracellular domain (ANT-007-F, Alomone Labs, Jerusalem, Israel) (10 mL/sample) and PE-labelled mouse anti-human TrKA (FAB1751P, R&D Systems, Minneapolis, MN, USA) (10 mL/sample).

Techniques: Standard Deviation

Journal: Biomedicines

Article Title: A Possible Role for Nerve Growth Factor and Its Receptors in Human Sperm Pathology.

doi: 10.3390/biomedicines11123345

Figure Lengend Snippet: Figure 3. Nerve growth factor (NGF) receptors in altered sperm from men with infertility. (A–D) Fluorescence immunostaining of human sperm from men with infertility (varicocele (V) or uro- genital infections (UGIs)) incubated with the anti-TrKA (A,B) and anti-p75NTR (C,D) antibodies. In (A)

Article Snippet: After washing three times in PBS supplemented with BSA, sperm were incubated at 4 ◦C for 1 h in PBS/BSA containing FITC-labelled rabbit anti-human p75NTR extracellular domain (ANT-007-F, Alomone Labs, Jerusalem, Israel) (10 mL/sample) and PE-labelled mouse anti-human TrKA (FAB1751P, R&D Systems, Minneapolis, MN, USA) (10 mL/sample).

Techniques: Fluorescence, Immunostaining, Incubation