rabbit anti-human mtor polyclonal antibody  (Spring Bioscience)

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

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

Journal: Frontiers in Immunology

Article Title: β 2 -integrins control HIF1α activation in human neutrophils

doi: 10.3389/fimmu.2024.1406967

Figure Lengend Snippet: Adherent neutrophils engage the mTOR pathway in HIF1α translation. (A) Freshly isolated neutrophils cultured on FN were treated with GM-CSF (20 ng/ml) or left untreated (US) for 30 min. Samples were assessed by immunoblotting for total and phosphorylated mTOR at Ser2448 (n=4). Pre-incubation with 100 nM mTOR inhibitor rapamycin (RAP) for 30 min on ice suppressed mTOR phosphorylation. Representative immunoblots and the corresponding statistics are given. (B, C) Freshly isolated neutrophils cultured on FN were stimulated with 20 ng/ml GM-CSF or left unstimulated (US) with buffer control (BU) or 15 µM ROX for 4 h in the absence or presence of 100 nM RAP (n=4). Protein and total RNA were isolated to detect (B) HIF1α protein and (C) HIF1α mRNA, respectively. A representative immunoblot and the statistics are depicted. The ROX condition was set as a reference for ΔΔCT calculations and statistical analysis. (A–C) Statistical analysis was performed by repeated measure one-way ANOVA with Šidák’s multiple comparison test. ns, not significant. p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****).

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-human IgG HIF1α (1:500, #3716, RRID:AB_2116962, Cell Signaling Technologies, Leiden, The Netherlands), monoclonal mouse anti-human IgG HIF2α clone ep190b (1:500, #NB100-132, RRID:AB_10000898, Novus Biologicals, Wiesbaden, Germany), polyclonal rabbit anti-human IgG HIF2α (1:500, #NB100-122, RRID:AB_535687, Novus Biologicals), polyclonal rabbit anti-human IgG HIF2α (1:500, # PA1-16510, RRID:AB_2098236, Thermo Fisher Scientific), monoclonal rabbit anti-human IgG β-actin clone 13E5 (1:2000, #4970, RRID:AB_2223172), monoclonal rabbit anti-human IgG phospho-STAT3 (Tyr705) clone D3A7 (1:1000, #9145, RRID:AB_2491009), monoclonal mouse anti-human IgG STAT3 clone 124H6 (1:1000, #9139, RRID:AB_331757), polyclonal rabbit anti-human IgG phospho-eIF4E (Ser209) (1:1000, #9741, RRID:AB_331677), monoclonal rabbit anti-human IgG phospho-4EBP1 (Ser65) clone D9G1Q (1:1000, #9451, RRID:AB_330947), monoclonal rabbit anti-human IgG mTOR clone 7C10 (1:1000, #2983, RRID:AB_2105622), and polyclonal rabbit anti-human IgG phospho-mTOR (Ser2448) (1:1000, #2971, RRID:AB_330970, all from Cell Signaling Technologies).

Techniques: Isolation, Cell Culture, Western Blot, Incubation, Phospho-proteomics, Control, Comparison

Journal: Animal Nutrition

Article Title: MRCKα is a novel regulator of prolactin-induced lactogenesis in bovine mammary epithelial cells

doi: 10.1016/j.aninu.2022.06.001

Figure Lengend Snippet: Immunocytochemical characterization of the primary culture of bovine mammary epithelial cells (BMEC). (A) Isolated BMEC were cultured on coverslips for 24 h and analyzed by immunocytochemistry using anti-CK18 monoclonal antibody. Images show CK18 (green), nuclear DNA (DAPI, blue) and merged fluorescence confocal microscope emissions. (B) In parallel, cultured BMEC were lysed after 24 h and subjected to Western blot analysis using anti-CK18 monoclonal or β-casein polyclonal antibody. Equal amounts of total protein (25 μg) were loaded in each lane. BMEC-1 and BMEC-2 represent 2 independent batches of BMEC. DAPI, 4′,6-diamidino-2-phenylindole, a blue-fluorescent DNA stain. CK18 = cytokeratin 18.

Article Snippet: Rabbit polyclonal anti-human phosphorylated Ser2448-mTOR antibody (YT2913; 1:1,000) and rabbit polyclonal anti-human mTOR antibody (YP1134; 1:1,000) were obtained from ImmunoWay Biotechnology Company (Plano, TX).

Techniques: Isolation, Cell Culture, Immunocytochemistry, Fluorescence, Microscopy, Western Blot, Staining

Journal: Animal Nutrition

Article Title: MRCKα is a novel regulator of prolactin-induced lactogenesis in bovine mammary epithelial cells

doi: 10.1016/j.aninu.2022.06.001

Figure Lengend Snippet: Prolactin stimulates the expression of both β-casein and MRCKα. (A) Confluent bovine mammary epithelial cells (BMEC) were stimulated with prolactin (PRL, 0.6 μg/mL) or left untreated (Control) for 24 h, lysed and subjected to Western blot analysis using polyclonal antibodies against β-casein, MRCKα or β-actin. (B) Immunopositive bands were quantified by densitometry, normalized to β-actin and presented as protein expression levels relative to their corresponding control. (C) β-casein protein expression in BMEC tended to correlate positively with that of MRCKα. (D) Abundance of β-casein and MRCKα mRNA measured by RT-qPCR in BMEC upon stimulation with prolactin (PRL, 0.6 μg/mL) or vehicle (Control) for 24 h. (E) β-casein mRNA expression in BMEC correlated positively with that of MRCKα. Values are presented as mean ± SEM of 3 independent experiments. Means without a common letter differ, P < 0.05. MRCKα = myotonic dystrophy-related Cdc42-binding kinase alpha.

Article Snippet: Rabbit polyclonal anti-human phosphorylated Ser2448-mTOR antibody (YT2913; 1:1,000) and rabbit polyclonal anti-human mTOR antibody (YP1134; 1:1,000) were obtained from ImmunoWay Biotechnology Company (Plano, TX).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: Animal Nutrition

Article Title: MRCKα is a novel regulator of prolactin-induced lactogenesis in bovine mammary epithelial cells

doi: 10.1016/j.aninu.2022.06.001

Figure Lengend Snippet: MRCKα positively regulates the synthesis of β-casein in bovine mammary epithelial cells (BMEC). (A) Confluent BMEC were transfected with MRCKα siRNA (SI), scrambled siRNA (control, SC) or left untreated (control, Blank) for 24 h, lysed and subjected to Western blot analysis using polyclonal antibodies directed against MRCKα, β-casein and β-actin. Immunopositive bands were analyzed by densitometry. The resulted protein levels were normalized to β-actin and the average fold-change over Blank for MRCKα (B) and β-casein (C) protein expression are presented of 3 independent experiments. (D) Confluent BMEC were transfected with empty vector pcDNA3.1 (EV), MRCKα-FLAG (OE), or left untreated (control, Blank). After 24 h, cells were lysed and analyzed by immunoblotting using polyclonal antibodies against MRCKα and β-casein. Immunopositive bands were analyzed by densitometry. Protein levels were normalized to β-actin and the average fold-change over Blank for MRCKα (E) and β-casein (F) protein expression is presented of 3 independent experiments. Means without a common letter differ, P < 0.05. MRCKα = myotonic dystrophy-related Cdc42-binding kinase alpha.

Article Snippet: Rabbit polyclonal anti-human phosphorylated Ser2448-mTOR antibody (YT2913; 1:1,000) and rabbit polyclonal anti-human mTOR antibody (YP1134; 1:1,000) were obtained from ImmunoWay Biotechnology Company (Plano, TX).

Techniques: Transfection, Western Blot, Expressing, Plasmid Preparation, Binding Assay