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