Immunofluorescence of U373 cells was executed as previously described [60 (link)]. U373 were fixed in paraformaldehyde (4% in PBS) and probed overnight with primary antibodies: anti-p75NTR (Santa Cruz Biotechnology, Dallas, TX, USA, sc-271708, dilution 1:100), and anti-TrkA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-118, dilution 1:100). Subsequently, cells were incubated for 1 h with goat anti-mouse secondary antibody Alexa Fluor 555 (ThermoFisher Scientific, Waltham, MA, USA, A28180) and with goat anti-rabbit secondary antibody Alexa Fluor 488 (ThermoFisher Scientific, Waltham, MA, USA, A27034). DAPI staining was performed to visualize nuclei, and coverslips were finally mounted with Fluoroshield mounting medium. Samples were analyzed via confocal microscopy, as described above.
Anti trka
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-TrkA is a laboratory product designed to detect the TrkA receptor in various biological samples. It is a specific antibody that recognizes the TrkA protein, which is a member of the Trk family of receptor tyrosine kinases. The primary function of Anti-TrkA is to enable the identification and quantification of TrkA in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti p75ntr, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminescence kit, by Santa Cruz Biotechnology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti ptrka, by Santa Cruz Biotechnology (2 mentions)
Dulbecco s modified eagle s medium, by Cytiva (2 mentions)
Poly d lysine, by BD (2 mentions)
Anti β actin antibody, by Abcam (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Anti akt1 2 3, by Santa Cruz Biotechnology (2 mentions)
Anti ngf, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti gst antibody, by Santa Cruz Biotechnology (1 mentions)
Nerve growth factor (ngf), by R&D Systems (1 mentions)
Anti fgfr1, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Santa Cruz Biotechnology (1 mentions)
Anti erk1 2, by Santa Cruz Biotechnology (1 mentions)
Anti p erk1 2, by Santa Cruz Biotechnology (1 mentions)
Anti p akt1 2 3, by Santa Cruz Biotechnology (1 mentions)
Tetrazolium dye 3 4 5 di methylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
7 protocols using anti trka
1
Immunofluorescence Profiling of U373 Cells
2
Antibody Sources for Cellular Signaling
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Anti-Flag (M2), anti β-actin, and anti-acetylated tubulin monoclonal antibodies were purchased from Sigma (St. Louis, MO, USA). Anti-NEAP/DUSP26 antibody was purchased from GeneTex (Hsinchu, Taiwan). Anti-phospho-Akt (S473), pTrkA (Y490 and Y674/675), anti-pFGFR1(Y653/654), anti-pFRS2 (Y196 and Y436), anti-pIGFR1(Y), and anti-IGFR1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-Akt, anti-TrkA, anti-FGFR1, anti-GST antibodies and peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NGF was purchased from R & D System (Minneapolis, MN, USA) and heregulin was purchased from Calbiochem (San Diego, CA, USA).
3
Synthesis and Evaluation of NGF-Based Dipeptides
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The dimeric dipeptides GK-6 and GK-2 were synthesized on the base of murine NGF at the Zakusov Institute of Pharmacology (Moscow, Russia).
Inhibitors of PI3K (LY294002) and MAPK (PD98059) were purchased from Tocris Bioscience (Bristol, UK). The tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT),was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals. Inc. (Costa Mesa, CA, USA). Poly-D-lysine was purchased from BD Biosciences (San Jose, CA, USA). DC protein assay was purchased from Biorad (Hercules, CA, USA). Anti-TrkA, anti-pTrkA, anti-AKT1/2/3, anti-pAKT1/2/3, anti-ERK1/2, anti-pERK1/2 antibodies and enhanced chemiluminescence kits were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin antibodies and horseradish peroxidase conjugated antibodies were purchased from Abcam (Cambridge, MA, USA).
Inhibitors of PI3K (LY294002) and MAPK (PD98059) were purchased from Tocris Bioscience (Bristol, UK). The tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT),was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals. Inc. (Costa Mesa, CA, USA). Poly-D-lysine was purchased from BD Biosciences (San Jose, CA, USA). DC protein assay was purchased from Biorad (Hercules, CA, USA). Anti-TrkA, anti-pTrkA, anti-AKT1/2/3, anti-pAKT1/2/3, anti-ERK1/2, anti-pERK1/2 antibodies and enhanced chemiluminescence kits were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin antibodies and horseradish peroxidase conjugated antibodies were purchased from Abcam (Cambridge, MA, USA).
4
Neurotrophic Signaling Pathway Analysis
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Dimethyl sulfoxide, absolute ethanol and methanol were obtained from Merck (Darmstadt, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), rat nerve growth factor (NGF-β) and all other reagents needed for chemical synthesis were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and horse serum were acquired from Invitrogen (San Diego, CA, USA), while penicillin/streptomycin, RPMI 1640, sterile phosphate-buffered saline (PBS), and trypsin EDTA 0.25% were purchased from Biosera (Ringmer, UK). Anti-phospho-TrkA, anti-MAP kinase ERK1/2, anti-phospho-MAP kinase ERK1/2 (pThr202/Tyr204), and anti-phospho-Akt antibodies were obtained from Cell Signaling (Danvers, MA, USA). Anti-TrkA, anti-Akt antibodies, and K252a were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Homo sapiens TrkA/NTRK1 cDNA clone-containing plasmid was purchased from Sino Biologicals (Beijing, China). A plasmid extraction Miniprep kit was obtained from Invitek Inc. (Berlin, Germany). Deionized water was used in all experiments.
5
Synthesis and Characterization of Dimeric Dipeptides
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The dimeric dipeptides GSB-106 ((bis-(N-monosuccinyl-l -seryl-l -lysine)hexamethylenediamide (Tm =143°C–145°C, [α] 20 (link) D =−24.7° (c=0.4%; dimethylformamide)) and GSB-214((bis-(N-monosuccinyl-l -methionyl-l -serine) heptamethylenediamide (Tm =160°C–162°C, [α] 20 (link) D =−21.75° (c=0.4%; MeOH)) were synthesized at the Zakusov Institute of Pharmacology (Moscow, Russia), as described previously.13 (link)
2,3,5-triphenyltetrazolium chloride (TTC) and Nembutal were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA, USA). Poly-d -lysine was purchased from BD Biosciences (San Jose, CA, USA). DC protein assay was purchased from Bio-Rad (Hercules, CA, USA). Anti-TrkA, anti-pTrkA, anti-AKT1/2/3, anti-phospho-AKT1s473/2s472/3s474 anti-ERK1/2, anti-phospho ERK1/2Y204 antibodies, and enhanced chemiluminescence kits were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin antibodies and horseradish peroxidase-conjugated antibodies were purchased from Abcam (Cambridge, MA, USA). Formalin was purchased from Ecros (Saint Petersburg, Russia). GSB-106 and GSB-214 were dissolved in water. Then solvents were diluted in culture media in equivalent amounts.
2,3,5-triphenyltetrazolium chloride (TTC) and Nembutal were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA, USA). Poly-
6
Immunodetection of Neurotrophin Signaling
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The following primary antibodies were used: anti-p75NTR, anti-NGF, anti-TrkA, anti-p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-JNK; (Cell Signaling Technology, Milan, Italy).
Biotinylated goat-anti-rabbit, anti-mouse, anti-donkey IgGs were used as secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Biotinylated goat-anti-rabbit, anti-mouse, anti-donkey IgGs were used as secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
7
Immunofluorescence Staining of TrkA, p75, and NGF
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Eight micrometer cryosections were blocked and incubated with primary antibodies: Anti-TrkA (Santa Cruz Biotechnology; sc-118) or anti-p75 (Santa Cruz Biotechnology; sc-5634). Rhodamine-labeled secondary antibodies (Jackson Immuno-Research 111-026-045) were used followed by 4´,6-diamidino-2-phenylindole (DAPI). Paraffin-embedded liver samples were also incubated with anti-NGF (Santa Cruz Biotechnology; sc-549 clone M-20). FITC-labeled secondary (Jackson Immuno-Research 115-095-047) were used followed by DAPI. Immunofluorescence controls without the primary antibody were also performed. Sections were analyzed using an immunofluorescence microscope (Axio Imager.M2, Carl Zeiss, Oberkochen, Germany).
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