p mtor ser 2448 cell signaling  (Cell Signaling Technology Inc)

Journal: Clinical Psychopharmacology and Neuroscience

Article Title: Establishment of a Depression Model Using Dexamethasone-treated Three-dimensional Cultured Rat Cortical Cells

doi: 10.9758/cpn.25.1269

Figure Lengend Snippet: Changes in the phosphorylation of mTORC1-mediated proteins and in the expression of synaptic proteins after DEX exposure. Beginning on DIV 10, the neural spheroids were exposed to 100 μM DEX for 5 days. Western blotting and immunofluorescence were performed using each primary antibody (n = 4 biological replicates). (A) Western blotting revealed the levels of phospho-Ser 2448 -mTORC1 (a), phospho- Thr 37/46 -4E-BP1 (b), and phospho-Thr 389 -p70S6K (c). (B) Western blot analysis and representative images of immunoblots of PSD-95 (a) and GluA1 (b) are shown. Synaptic markers: PSD-95 (green) costained with the neuronal marker MAP-2 (red); GluA1 (green) costained with MAP-2 (red). Scale bar: 100 μm. The data are presented as the means ± standard deviations. mTORC1, mechanistic target of rapamycin complex I; DEX, dexamethasone; DIV, days in vitro; 4E-BP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1; PSD-95, postsynaptic density protein-95; GluA1, AMPA receptor subunit glutamate receptor 1; MAP-2, microtubule-associated protein-2; CON, controls. * p < 0.05 or ** p < 0.01 vs. CON.

Article Snippet: p-Ser 2448 -mTORC1 , Cell Signaling Technology (2971) , AB_330970 , WB (1:1,000).

Techniques: Phospho-proteomics, Expressing, Western Blot, Immunofluorescence, Marker, In Vitro, Binding Assay

Journal: Clinical Psychopharmacology and Neuroscience

Article Title: Establishment of a Depression Model Using Dexamethasone-treated Three-dimensional Cultured Rat Cortical Cells

doi: 10.9758/cpn.25.1269

Figure Lengend Snippet: Schematic diagram showing the molecular mechanisms underlying DEX-induced impaired neuroplasticity. Exposure to DEX downregulates signaling pathways that affect neuroplasticity, including BDNF, sirtuin 1, and mTORC1 signaling. Activation of the mTORC1 signaling pathway induces the synthesis of synaptic proteins as well as BDNF. Secreted BDNF interacts with its receptor TrkB, further activating mTORC1 signaling via PI3K/Akt and MEK/ERK1/2. Therefore, synaptic plasticity is enhanced. Sirtuin 1 is also involved in the regulation of neuroplasticity. The original illustration was created using BioRender (biorender.com). Akt, protein kinase B; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; BDNF, brain-derived neurotrophic factor; DEX, dexamethasone; ERK1/2, extracellular signal-regulated kinase 1/2; GluA1, AMPA receptor subunit glutamate receptor 1; MEK, mitogen‑activated protein kinase; mTORC1, mechanistic target of rapamycin complex I; p70S6K, p70S6 kinase; PI3K, phosphatidyl inositol-3 kinase; PSD-95, postsynaptic density protein-95; TrKB, tropomyosin receptor kinase B; 4E-BP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1.

Article Snippet: p-Ser 2448 -mTORC1 , Cell Signaling Technology (2971) , AB_330970 , WB (1:1,000).

Techniques: Protein-Protein interactions, Activation Assay, Derivative Assay, Binding Assay

Journal: Animal Nutrition

Article Title: Myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα) mediates methionine- and leucine-stimulated β-casein synthesis in bovine mammary epithelial cells via targeting mTOR

doi: 10.1016/j.aninu.2025.01.003

Figure Lengend Snippet: Dose-dependent stimulation of β-casein, mTOR phosphorylation, SREBP1 and cyclin D1 by exogenous Met and Leu. Confluent cultures of bovine mammary epithelial cells were treated by adding either Met (0, 0.2, 0.4, 0.6 and 0.8 mM) (Upper panel) or Leu (0, 0.2, 0.4, 0.6 and 0.8 mM) (Lower panel) to the Opti-MEM medium (37 °C). After 24 h, cells were lysed and subjected to Western blot analysis. Protein levels of β-casein, mTOR, SREBP1, cyclin D1, β-actin and p-mTOR were established by densitometric analysis of the immunopositive bands enabling calculation of p-mTOR/mTOR. Protein levels presented were first normalized to β-actin and then to its corresponding no-AA control group. Values are presented as mean ± SEM for three independent experiments. Statistical effects compared with no-AA control group, ∗ P < 0.05, ∗∗ P < 0.01. mTOR = mechanistic target of rapamycin; SREBP1 = sterol regulatory element-binding protein 1.

Article Snippet: Rabbit polyclonal anti-human mTOR antibody and rabbit polyclonal anti-human phosphorylated mTOR (Ser 2448 ) antibody were obtained from ImmunoWay Biotechnology Company (Plano, TX, USA).

Techniques: Phospho-proteomics, Western Blot, Control, Binding Assay

Journal: Animal Nutrition

Article Title: Myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα) mediates methionine- and leucine-stimulated β-casein synthesis in bovine mammary epithelial cells via targeting mTOR

doi: 10.1016/j.aninu.2025.01.003

Figure Lengend Snippet: Silencing of MRCKα inhibits the stimulatory effects of Met and Leu on MRCKα, β-casein, PKB phosphorylation, and mTOR phosphorylation, but not PI3K phosphorylation in bovine mammary epithelial cells. Cells were transfected with scrambled siRNA (small interfering RNA; control, SC) or MRCKα siRNA (SI) and stimulated simultaneously with control (B, basal medium), additional Met (M, Met = 0.6 mM) or Leu (L, Leu = 0.6 mM). After 24 h, cells were lysed, and protein levels were detected by immunoblotting followed by densitometric analysis. All protein levels were first normalized to β-actin and then to SC + B group. Values are presented as mean ± SEM for three independent experiments. Different low case letters above columns indicate significant statistical differences ( P < 0.05). P AA , P -value of amino acid addition (AA); P SI , P -value of silencing of MRCKα (SI); P AA×SI , P -value of AA × SI interaction. MRCKα = myotonic dystrophy-related CDC42-binding kinase alpha; PI3K = phosphatidylinositol 3-kinase; PKB = protein kinase B; mTOR = mechanistic target of rapamycin.

Article Snippet: Rabbit polyclonal anti-human mTOR antibody and rabbit polyclonal anti-human phosphorylated mTOR (Ser 2448 ) antibody were obtained from ImmunoWay Biotechnology Company (Plano, TX, USA).

Techniques: Phospho-proteomics, Transfection, Small Interfering RNA, Control, Western Blot, Binding Assay

Journal: Animal Nutrition

Article Title: Myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα) mediates methionine- and leucine-stimulated β-casein synthesis in bovine mammary epithelial cells via targeting mTOR

doi: 10.1016/j.aninu.2025.01.003

Figure Lengend Snippet: Inhibition of PI3K blocks the stimulatory action of Met and Leu on MRCKα, β-casein, PI3K phosphorylation and mTOR phosphorylation in bovine mammary epithelial cells. Cells were treated with blank (untreated control) or PI3K inhibitor (LY294002, 43 μM) and stimulated simultaneously with control (B, basal medium), additional Met (M, Met = 0.6 mM) or Leu (L, Leu = 0.6 mM). After 24 h, cells were lysed, and protein levels were detected by immunoblotting followed by densitometric analysis. All protein levels were first normalized to β-actin and then to Blank + B group. Values are presented as mean ± SEM for three independent experiments. Different low case letters above columns indicate significant statistical differences ( P < 0.05). P AA , P -value of amino acid addition (AA); P LY , P -value of inhibition of PI3K (LY); P AA×LY , P -value of AA × LY interaction. MRCKα = myotonic dystrophy-related CDC42-binding kinase alpha; PI3K = phosphatidylinositol 3-kinase; PKB = protein kinase B; mTOR = mechanistic target of rapamycin.

Article Snippet: Rabbit polyclonal anti-human mTOR antibody and rabbit polyclonal anti-human phosphorylated mTOR (Ser 2448 ) antibody were obtained from ImmunoWay Biotechnology Company (Plano, TX, USA).

Techniques: Inhibition, Phospho-proteomics, Control, Western Blot, Binding Assay

Journal: Animal Nutrition

Article Title: Myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα) mediates methionine- and leucine-stimulated β-casein synthesis in bovine mammary epithelial cells via targeting mTOR

doi: 10.1016/j.aninu.2025.01.003

Figure Lengend Snippet: Inhibition of protein kinase B (PKB) inhibits the stimulatory effects of MRCKα overexpression on β-casein and mTOR phosphorylation in bovine mammary epithelial cells. Cells were treated with blank (untreated control) or PKB inhibitor (MK2206, 1 μM) and transfected simultaneously with empty vector (control, EV) or MRCKα (OE). After 24 h, cells were lysed, and protein levels were detected by immunoblotting followed by densitometric analysis. All protein levels were first normalized to β-actin and then to Blank + EV group. Values are presented as mean ± SEM for three independent experiments. Different low case letters above columns indicate significant statistical differences ( P < 0.05). P OE , P -value of MRCKα overexpression (OE); P MK , P -value of inhibition of PKB (MK); P OE×MK , P -value of OE × MK interaction. MRCKα = myotonic dystrophy-related CDC42-binding kinase alpha; mTOR = mechanistic target of rapamycin.

Article Snippet: Rabbit polyclonal anti-human mTOR antibody and rabbit polyclonal anti-human phosphorylated mTOR (Ser 2448 ) antibody were obtained from ImmunoWay Biotechnology Company (Plano, TX, USA).

Techniques: Inhibition, Over Expression, Phospho-proteomics, Control, Transfection, Plasmid Preparation, Western Blot, Binding Assay

Journal: Animal Nutrition

Article Title: Myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα) mediates methionine- and leucine-stimulated β-casein synthesis in bovine mammary epithelial cells via targeting mTOR

doi: 10.1016/j.aninu.2025.01.003

Figure Lengend Snippet: Silencing of MRCKα inhibits the stimulatory effects of Met and Leu on mRNA abundance of genes related to milk protein synthesis in bovine mammary epithelial cells. Cells were transfected with scrambled siRNA (control, SC) or MRCKα siRNA (SI) and stimulated simultaneously with control (B, basal medium), additional Met (M, Met = 0.6 mM) or Leu (L, Leu = 0.6 mM). After 24 h, cells were collected and isolated for RT-qPCR analysis. Values are presented as mean ± SEM for three independent experiments. P AA , P -value of amino acid addition (AA); P SI , P -value of silencing of MRCKα (SI); P AA×SI , P -value of AA × SI interaction. CSN2 = β-casein; CSN1S1 = αS1-casein; CSN3 = κ-casein; LALBA = lactalbumin α; MRCKα = myotonic dystrophy-related CDC42-binding kinase alpha; mTOR = mechanistic target of rapamycin; JAK2 = Janus kinase 2; STAT5 = signal transducers and activators of transcription 5.

Article Snippet: Rabbit polyclonal anti-human mTOR antibody and rabbit polyclonal anti-human phosphorylated mTOR (Ser 2448 ) antibody were obtained from ImmunoWay Biotechnology Company (Plano, TX, USA).

Techniques: Transfection, Control, Isolation, Quantitative RT-PCR, Binding Assay

Journal: Animal Nutrition

Article Title: Myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα) mediates methionine- and leucine-stimulated β-casein synthesis in bovine mammary epithelial cells via targeting mTOR

doi: 10.1016/j.aninu.2025.01.003

Figure Lengend Snippet: Amino acids are sensed by PI3K-MRCKα-PKB signaling pathways to activate mTOR and its downstream signaling, which leads to promotion of β-casein synthesis, SREBP1 and Cyclin D1 expression in BMEC. Besides, the mRNA abundance of CSN1S1 , CSN2 and JAK2 are decreased when MRCKα expression is inhibited. PI3K = phosphatidylinositol 3-kinase; MRCKα = myotonic dystrophy-related CDC42-binding kinase alpha; PKB = protein kinase B; mTOR = mechanistic target of rapamycin; CSN1S1 = αS1-casein; CSN2 = β-casein; JAK2 = Janus kinase 2.

Article Snippet: Rabbit polyclonal anti-human mTOR antibody and rabbit polyclonal anti-human phosphorylated mTOR (Ser 2448 ) antibody were obtained from ImmunoWay Biotechnology Company (Plano, TX, USA).

Techniques: Protein-Protein interactions, Expressing, Binding Assay