Western blots of P2 crude synaptosome fractions of dissected mPFC , which included both prelimbic and infralimbic subregions were conducted as previously described (6 (link), 7 (link), 20 (link), 21 ) using the following antibodies:_pERK, pS6K, pAkt, or p mTORC1 (Cell Signaling, 1:1000) and pTrk B (Santacruz, 1:250). After detection of phospho proteins, membranes were stripped and reprobed with antibodies for total ERK 1/2, S6K, Akt, mTORC1, TrkB (Cell Signaling, 1:1000) overnight. Densitometry was used to quantify protein bands using Image J Software (NIH), and phospho proteins were normalized to their respective total protein.
P trkb
Manufactured by Santa Cruz Biotechnology
Sourced in United States
P-TrkB is a product that detects the phosphorylated form of the TrkB receptor. TrkB is a receptor tyrosine kinase that plays a role in neuronal survival, differentiation, and synaptic plasticity. The phosphorylated form of TrkB is an indicator of TrkB activation.
Automatically generated - may contain errors
Lab products found in correlation
Brain derived neurotrophic factor (bdnf), by Santa Cruz Biotechnology (3 mentions)
Mtorc1, by Cell Signaling Technology (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
P creb, by Santa Cruz Biotechnology (2 mentions)
Sc 546, by Santa Cruz Biotechnology (2 mentions)
Anti trkb, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Isoflurane, by Merck Group (1 mentions)
Genistein, by Merck Group (1 mentions)
Gsk 3β, by Santa Cruz Biotechnology (1 mentions)
P creb, by Cell Signaling Technology (1 mentions)
P gsk3β, by Santa Cruz Biotechnology (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Phosphatase and tensin homolog pten, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
7 protocols using p trkb
1
Molecular Signaling Profiling in mPFC Synapses
2
Genistein and Isoflurane Neuroprotective Mechanisms
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Genistein and isoflurane (0.75%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). For expression analysis, antibodies against CREB, p-CREB, cleaved caspase-3, Bcl-2, Bad, Bcl-xL, Bax, β-actin, phosphatase and tensin homolog (PTEN), and mammalian target of rapamycin complex 1 (mTORc1) were purchased from Cell Signaling Technology (Beverly, MA, USA). Akt, p-Akt, GSK-3β, p- GSK-3β, BDNF, TrkB, and p-TrkB were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PDE4 and Ca2+/calmodulin-dependent kinase IV (CaMKIV) were from Abcam. All other chemicals and reagents used in the present study were purchased from Sigma-Aldrich unless otherwise noted.
3
Trimethyltin-Induced Neurodegeneration Model
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HT-22 cells were provided from Dr. Hyun-Yon Lee (Seowon University, Cheongju, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P/S), and phosphate-bufferedsaline (PBS) were purchased from Gibco (Gaithersburg, MD, USA). Trimethyltin (TMT) was obtained from Sigma (St. Louis, MO, USA). Thiazolyl blue tetrazolium bromide (MTT) was purchased from Alfa Aesar Chemical Inc. (Ward Hill, MA, USA). An Aamplite colorimetric acetylcholinesteraseassay kit was purchased from AAT Bioquest (Sunnyvale, CA, USA).The peroxide-sensitive fluorophore 2,7′-dichlorodihydrofluoresceindiacetate (DCF-DA) was purchased from CELL BIOLABS (San Diego, CA, USA). Unless noted otherwise, all other chemicals were purchased from Sigma (St. Louis, MO, USA). Antibodies specific for PARP, p-CREB, p-TrkB, BDNF, SOD2, NOX4, HO-1, and α-tubulin were obtained from Santa Cruz (Dallas, TX, USA).
4
Western Blot Analysis of Neuroreceptors
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Proteins were separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad, USA). We blocked the membranes and incubated them with anti-NR3C1 (1:500, sc-12763), anti-BDNF (1:500, sc-546), anti-TrkB (1:500, sc-7268), p-TrkB (1:500, sc-8058), which were purchased from Santa Cruz Biotechnology, USA. GAPDH (1:1000, A9044) from Sigma, USA was used as control. After washing, we incubated the blots with HRP-conjugated secondary antibodies. Chemiluminescent was used to quantify the signals.
5
BDNF Signaling Pathway Activation
6
Hippocampal Protein Expression Analysis
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Samples of the hippocampus were homogenized on ice (12,000 rpm, 4°C, 20 min) and quantified using a Bradford assay (BioRad, USA). Nuclear protein was obtained using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (CER, Pierce Biotechnology, USA). The BCA method was used to measure total protein. In total, 50 μg of protein lysate was separated by electrophoresis and transferred to a polyvinylidene difluoride membrane under semidry conditions at 250 mA.
After blocking in TBST buffer, the membranes were incubated with the following primary antibodies: PSD-95 (1 : 500, Proteintech, USA), BDNF (1 : 500, Proteintech, USA), pTrkB (Thr706, 1 : 400, Santa Cruz, USA), TrkB (1 : 1000, Santa Cruz, USA), pAkt (Ser473, 1 : 1000, Proteintech, USA), Akt (1 : 500, Proteintech, USA), pCREB (Ser133, 1 : 200, Santa Cruz, USA), and CREB (1 : 1000, Proteintech, USA). Then, the membranes were incubated with a horseradish peroxidase- (HRP-) conjugated secondary antibody (Abcam, USA). Protein signal intensities were detected with an ECL kit (Thermo) and visualized by exposure to Kodak film. The amount of protein was expressed as a relative value to the levels of β-actin.
After blocking in TBST buffer, the membranes were incubated with the following primary antibodies: PSD-95 (1 : 500, Proteintech, USA), BDNF (1 : 500, Proteintech, USA), pTrkB (Thr706, 1 : 400, Santa Cruz, USA), TrkB (1 : 1000, Santa Cruz, USA), pAkt (Ser473, 1 : 1000, Proteintech, USA), Akt (1 : 500, Proteintech, USA), pCREB (Ser133, 1 : 200, Santa Cruz, USA), and CREB (1 : 1000, Proteintech, USA). Then, the membranes were incubated with a horseradish peroxidase- (HRP-) conjugated secondary antibody (Abcam, USA). Protein signal intensities were detected with an ECL kit (Thermo) and visualized by exposure to Kodak film. The amount of protein was expressed as a relative value to the levels of β-actin.
7
Western Blotting Pathway Analysis
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Western blotting was performed as described previously [28 (link)]. The protein-transferred membranes were incubated overnight at 4°C with primary antibodies for TrkB (1:200, sc-8316, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-TrkB (1:500, ab197072, Cambridge, MA, USA), BDNF (1:200, sc-546, Santa Cruz Biotechnology), MMP-2 (1:200, sc-10736, Santa Cruz Biotechnology), MMP-9 (1:200, sc-6840, Santa Cruz Biotechnology), E-cadherin (1:200, sc-7870, Santa Cruz Biotechnology), vimentin (1:200, sc-6260, Santa Cruz Biotechnology), SLUG (1:200, sc-15391, Santa Cruz Biotechnology), SNAIL (1:200, sc-10433, Santa Cruz Biotechnology), twist (1:200, sc-15393, Santa Cruz Biotechnology), VEGF-C (1:200, sc-7133, Santa Cruz Biotechnology), VEGF-D (1:200, sc-7603, Santa Cruz Biotechnology), p-MEK-1/2 (1:200, sc-7995, Santa Cruz Biotechnology), Erk1/2 (1:200, No9102 Cell signaling Technology), p-Akt1/2/3 (1:200, sc-101629, Santa Cruz Biotechnology), or Akt1/2/3 (1:200, sc-8312, Santa Cruz Biotechnology). Peroxidase-linked secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA) were subsequently added and the membranes were further incubated for 1 h at room temperature. The antibodies for α-tubulin (1:1000, Sigma-Aldich, St. Louis, MO, USA) and β-actin (1:200, sc-47778, Santa Cruz Biotechnology) were used as protein loading controls.
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