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 signal transducer and activator of transcription 5 (STAT5) antibody, rabbit polyclonal anti-human phosphorylated STAT5 (Tyr 694 ) antibody, rabbit polyclonal anti-human PI3K antibody and rabbit polyclonal anti-human phosphorylated PI3K (Tyr 317 ) antibody were purchased from Bioss antibodies (Beijing, China).

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 signal transducer and activator of transcription 5 (STAT5) antibody, rabbit polyclonal anti-human phosphorylated STAT5 (Tyr 694 ) antibody, rabbit polyclonal anti-human PI3K antibody and rabbit polyclonal anti-human phosphorylated PI3K (Tyr 317 ) antibody were purchased from Bioss antibodies (Beijing, China).

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: 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 signal transducer and activator of transcription 5 (STAT5) antibody, rabbit polyclonal anti-human phosphorylated STAT5 (Tyr 694 ) antibody, rabbit polyclonal anti-human PI3K antibody and rabbit polyclonal anti-human phosphorylated PI3K (Tyr 317 ) antibody were purchased from Bioss antibodies (Beijing, China).

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: 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 signal transducer and activator of transcription 5 (STAT5) antibody, rabbit polyclonal anti-human phosphorylated STAT5 (Tyr 694 ) antibody, rabbit polyclonal anti-human PI3K antibody and rabbit polyclonal anti-human phosphorylated PI3K (Tyr 317 ) antibody were purchased from Bioss antibodies (Beijing, China).

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 signal transducer and activator of transcription 5 (STAT5) antibody, rabbit polyclonal anti-human phosphorylated STAT5 (Tyr 694 ) antibody, rabbit polyclonal anti-human PI3K antibody and rabbit polyclonal anti-human phosphorylated PI3K (Tyr 317 ) antibody were purchased from Bioss antibodies (Beijing, China).

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: 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 signal transducer and activator of transcription 5 (STAT5) antibody, rabbit polyclonal anti-human phosphorylated STAT5 (Tyr 694 ) antibody, rabbit polyclonal anti-human PI3K antibody and rabbit polyclonal anti-human phosphorylated PI3K (Tyr 317 ) antibody were purchased from Bioss antibodies (Beijing, China).

Techniques: Protein-Protein interactions, Expressing, Binding Assay

Journal: Redox biology

Article Title: Dysregulation of sphingolipid and cholesterol homeostasis imposes oxidative stress in human spermatozoa.

doi: 10.1016/j.redox.2025.103669

Figure Lengend Snippet: Fig. 2. MβCD increases PI3K phosphorylation, while inhibition of S1PR or NOS impairs MβCD-induced tyrosine phosphorylation. Spermatozoa were incubated for 3.5 h at 37 ◦C under various conditions: untreated (−), treated with 10 % v/v fetal cord serum ultrafiltrate (FCSu), treated with 0.5 mM methyl- β-cyclodextrin (MβCD), and treated with MβCD in combination with (a) 40 μM VPC 23019 (S1PR1/3 inhibitor) or (b) L-NAME (Nitric Oxide Synthase inhibitor). (a) Immunoblotting analysis revealed that VPC 23019 at 40 μM decreased PI3K phosphorylation (P-PI3K) in spermatozoa incubated with 0.5 mM of MβCD. (b) Immunoblotting showed that L-NAME treatment decreased tyrosine phosphorylation (P-Tyr) in sperm samples treated with FCSu and MβCD. The signal intensity for each lane was normalized to the silver-stain optical density value for accurate quantification. α-Tubulin loading controls were performed for direct comparison with the silver stain loading controls. The data represent sperm samples from four different healthy donors (n = 4). Statistical analysis was performed using ANOVA with Tukey’s test: *p ≤0.05, **p ≤0.01, and ***p ≤0.001.

Article Snippet: Rabbit polyclonal anti-phospho-PI3K (P-PI3K) antibodies were purchased from Cell Signaling (#4228) (Beverly, MA, USA).

Techniques: Phospho-proteomics, Inhibition, Incubation, Western Blot, Silver Staining, Comparison