Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: Isolation, cultivation, and identification of GMECs. (A) Morphological observation of adherent cells. Mammary epithelial cells on day 7 of isolation (left panel, scale bar = 400 μm); mammary epithelial cells after 5 passages of purification (middle panel, scale bar = 800 μm); mammary epithelial cells after 5 passages of purification (right panel, scale bar = 200 μm). (B) Cell identification by Oil Red O Staining. Scale bars correspond to 1,000 μm (left panel), 700 μm (middle panel), 400 μm (right panel). (C) Immunofluorescence detection of cytokeratin 18 and β-casein (scale bar = 500 μm). GMECs, goat mammary epithelial cells.

Article Snippet: Cells were washed with phosphate buffer solution, fixed with 4% paraformaldehyde, permeabilized with Triton, and blocked with 5% bovine serum albumin at room temperature for 1 h. Subsequently, the cells were incubated with Cytokeratin 18 (66187; Proteintech; 1:800) and β-casein antibody (bs-0466R; Bioss; 1:500) for 1 h in the dark.

Techniques: Isolation, Purification, Staining, Immunofluorescence

Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: ELF5 regulate casein synthesis in GMECs. (A, B) The expression of αS1-casein, αS2-casein, β-casein, κ-casein, phosphorylated JAK2 and STAT5 after transfection with pcDNA3.1-ELF5 or pcDNA3.1-NC for 48 h. (C, D) The expression of αS1-casein, αS2-casein, β-casein, κ-casein, p-JAK2, and p-STAT5 after transfection with siRNA-ELF5 or siRNA-NC (100 nM) for 48 h. The relative protein abundance of αS1-casein, αS2-casein, β-casein, and κ-casein was normalized to β-tubulin. The relative protein abundance of p-JAK2 and p-STAT5 was normalized to total JAK2 and STAT5, respectively. Values are presented as mean±SEM. * p<0.05, ** p<0.01. siRNA, small interfering ribonucleic acid; NC, negative control; GMECs, goat mammary epithelial cells; SEM, standard error of the mean.

Article Snippet: Cells were washed with phosphate buffer solution, fixed with 4% paraformaldehyde, permeabilized with Triton, and blocked with 5% bovine serum albumin at room temperature for 1 h. Subsequently, the cells were incubated with Cytokeratin 18 (66187; Proteintech; 1:800) and β-casein antibody (bs-0466R; Bioss; 1:500) for 1 h in the dark.

Techniques: Expressing, Transfection, Quantitative Proteomics, Negative Control

Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: The protein abundance of caseins after JAK2 and STAT5 inhibition in GMECs. (A) The expression of αS1-casein, αS2-casein, β-casein, κ-casein, and phosphorylated JAK2 and STAT5 after JAK2 inhibitor Tyrphosting AG490 (30 μM) treatment for 48 h. (B) The expression of αS1-casein, αS2-casein, β-casein, κ-casein, and p-STAT5 after STAT5 inhibitor STAT5-IN-1 (50 μM) treatment for 48 h. The relative protein abundance of αS1-casein, αS2-casein, β-casein, and κ-casein was normalized to β-tubulin. The relative protein abundance of p-JAK2 and p-STAT5 was normalized to total JAK2 and STAT5, respectively. Values are presented as mean±SEM. ** p<0.01. GMECs, goat mammary epithelial cells; SEM, standard error of the mean.

Article Snippet: Cells were washed with phosphate buffer solution, fixed with 4% paraformaldehyde, permeabilized with Triton, and blocked with 5% bovine serum albumin at room temperature for 1 h. Subsequently, the cells were incubated with Cytokeratin 18 (66187; Proteintech; 1:800) and β-casein antibody (bs-0466R; Bioss; 1:500) for 1 h in the dark.

Techniques: Quantitative Proteomics, Inhibition, Expressing

Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: ELF5 mediates casein synthesis by STAT5 activity in GMECs. (A) Cells were treated with STAT5-IN-1 (50 μM) or DMSO, followed by pcDNA3.1-ELF5 or pcDNA3.1-NC transfection for 48 h. (B) Cells were treated with STAT5-IN-1 (50 μM) or DMSO, followed by siRNA-ELF5 or siRNA-NC (100 nM) transfection for 48 h. The protein abundances of αS1-casein, αS2-casein, β-casein, κ-casein, and p-STAT5 were detected. The relative protein abundance of αS1-casein, αS2-casein, β-casein, and κ-casein was normalized to β-tubulin. The relative protein abundance of p-STAT5 was normalized to total STAT5. Values are presented as mean±SEM. a–d Different lowercase letters represent significant differences (* p<0.05). siRNA, small interfering ribonucleic acid; NC, negative control; GMECs, goat mammary epithelial cells; SEM, standard error of the mean.

Article Snippet: Cells were washed with phosphate buffer solution, fixed with 4% paraformaldehyde, permeabilized with Triton, and blocked with 5% bovine serum albumin at room temperature for 1 h. Subsequently, the cells were incubated with Cytokeratin 18 (66187; Proteintech; 1:800) and β-casein antibody (bs-0466R; Bioss; 1:500) for 1 h in the dark.

Techniques: Activity Assay, Transfection, Quantitative Proteomics, Negative Control

Journal: Animal Bioscience

Article Title: ELF5 modulates casein synthesis in goat mammary epithelial cells via JAK2/STAT5 signaling pathway

doi: 10.5713/ab.25.0181

Figure Lengend Snippet: ELF5 mediates casein synthesis by JAK2 and STAT5 activity in GMECs. (A) Cells were treated with Tyrphosting AG490 (30 μM) or DMSO, followed by pcDNA3.1-ELF5 or pcDNA3.1-NC transfection for 48 h. (B) Cells were treated with Tyrphosting AG490 (30 μM) or DMSO, followed by siRNA-ELF5 or siRNA-NC (100 nM) transfection for 48 h. The protein abundances of αS1-casein, αS2-casein, β-casein, κ-casein, p-JAK2, and p-STAT5 were detected. The relative protein abundance of αS1-casein, αS2-casein, β-casein, and κ-casein was normalized to β-tubulin. The relative protein abundance of p-JAK2 and p-STAT5 was normalized to total JAK2 and STAT5, respectively. Values are presented as mean±SEM. a–d Different lowercase letters represent significant differences (* p<0.05). NC, negative control; siRNA, small interfering ribonucleic acid; GMECs, goat mammary epithelial cells; SEM, standard error of the mean.

Article Snippet: Cells were washed with phosphate buffer solution, fixed with 4% paraformaldehyde, permeabilized with Triton, and blocked with 5% bovine serum albumin at room temperature for 1 h. Subsequently, the cells were incubated with Cytokeratin 18 (66187; Proteintech; 1:800) and β-casein antibody (bs-0466R; Bioss; 1:500) for 1 h in the dark.

Techniques: Activity Assay, Transfection, Quantitative Proteomics, Negative Control

Journal: Journal of Extracellular Biology

Article Title: Workflow for the Isolation and Characterisation of Human Milk Extracellular Vesicles (HMEVs) and Their Inflammatory Biomarker Profile

doi: 10.1002/jex2.70110

Figure Lengend Snippet: Evaluation of different beta‐casein removal approaches. Beta‐casein bands from Western blot analysis (top) and total protein content determined by ATR‐FTIR (bottom). Control = skimmed HM. Error bars indicate standard deviation ( N = 3). ATR‐FTIR, attenuated total reflectance–Fourier transform infrared; CaCl 2 , calcium chloride; HAc, acetic acid; HCl, hydrochloric acid; KH 2 PO 4 , potassium dihydrogen phosphate.

Article Snippet: Primary antibodies against CD81, Alix, β‐casein and secondary antibody against IgG were purchased from Santa Cruz Biotech (USA).

Techniques: Western Blot, Control, Standard Deviation, Fourier Transform Infrared Spectroscopy

Journal: Journal of Extracellular Biology

Article Title: Workflow for the Isolation and Characterisation of Human Milk Extracellular Vesicles (HMEVs) and Their Inflammatory Biomarker Profile

doi: 10.1002/jex2.70110

Figure Lengend Snippet: (Top) EV concentrations for the four tested processing and storage procedures. Note : Each condition was evaluated using two independent replicates; *one‐way ANOVA with Tukey's post hoc test was used for identifying significant differences (i.e., p values <0.05); EV concentrations were normalised to HM volume. (Bottom) Western blot analysis for the method (from HM‐DP to HMS2‐dUC) and casein positive control. DP, direct processing; dUC, differential ultracentrifugation; EV, extracellular vesicle; HM, human milk; HMS, HM storage.

Article Snippet: Primary antibodies against CD81, Alix, β‐casein and secondary antibody against IgG were purchased from Santa Cruz Biotech (USA).

Techniques: Western Blot, Positive Control