Journal: Journal of dairy science

Article Title: Negative regulation of α S1 -casein (CSN1S1) improves β-casein content and reduces allergy potential in goat milk.

doi: 10.3168/jds.2020-18595

Figure Lengend Snippet: Figure 1. αS1-Casein and β-casein content in goat milk and mammary gland tissue. (A) Content of αS1-casein and β-casein in goat milk (n = 40) was determined by ELISA. (B) Total protein content in goat milk was determined with a milk component analyzer. The percentage of casein in total protein is expressed as the ratio of casein content to total protein content in goat milk. (C) αS1-Casein gene (CSN1S1) and β-casein gene (CSN2) mRNA expression profiles in goat mammary tissue at different stages of lactation. Mammary tissue samples were collected from Xinong Saanen goats (n = 5) during early lactation, peak lactation, mid lactation, end of lactation, and dry period. Data were normalized to the end of lactation. Values are presented as means ± SEM. Different letters represent significant differences (P < 0.05) in gene expression.

Article Snippet: Secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (CW0103, CW Biotech; Beijing, China; 1:5,000) was used for primary antibodies αS1-casein (SAB1401093, Sigma-Aldrich; 1:1,000), αS2-casein (bs-10034R, Bioss, Beijing, China; 1:500), β-casein (orb2053, Biorbyt, Cambridgeshire, UK; 1:500), κ-casein (GTX60145, GeneTex, Irvine, CA; 1:500), JAK2 (3230, Cell Signaling Technology, Danvers, MA; 1:1,000), p-JAK2 Tyr1007/1008 (ab32101, Abcam, Cambridge, UK; 1:1,000), mTOR(2972, Cell Signaling Technology; 1:1,000) and p-mTOR Ser2448 (2971; Cell Signaling Technology; 1:1,000).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Gene Expression

Journal: Journal of dairy science

Article Title: Negative regulation of α S1 -casein (CSN1S1) improves β-casein content and reduces allergy potential in goat milk.

doi: 10.3168/jds.2020-18595

Figure Lengend Snippet: Figure 2. Overexpression of CSN1S1 suppressed β-casein expression in primary goat mammary epithelial cells (GMEC). (A) Fluorescent images of adenovirus with green fluorescent protein (Ad-GFP) or adenovirus Ad-CSN1S1 [multiplicity of infection (MOI) of 200] infection of GMEC for 48 h. The image on the left panel is in bright field, and the image on the right panel is green fluorescence in the same field. Scale bar is 500 μm. (B) CSN1S1 mRNA expression level in GMEC infected with Ad-GFP or Ad-CSN1S1 (MOI of 200) for 48 h. (C) mRNA expres- sion levels of caseins and main whey proteins in GMEC infected with Ad-GFP or Ad-CSN1S1 (MOI of 200) for 48 h. (D) Protein abundance of caseins in GMEC infected with Ad-GFP or Ad-CSN1S1 (MOI of 200) for 48 h. Relative protein abundance was normalized to β-actin. Values are presented as means ± SEM from 3 individual cultures (n = 3 goats). *P < 0.05 and **P < 0.01 compared with control (Ad-GFP).

Article Snippet: Secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (CW0103, CW Biotech; Beijing, China; 1:5,000) was used for primary antibodies αS1-casein (SAB1401093, Sigma-Aldrich; 1:1,000), αS2-casein (bs-10034R, Bioss, Beijing, China; 1:500), β-casein (orb2053, Biorbyt, Cambridgeshire, UK; 1:500), κ-casein (GTX60145, GeneTex, Irvine, CA; 1:500), JAK2 (3230, Cell Signaling Technology, Danvers, MA; 1:1,000), p-JAK2 Tyr1007/1008 (ab32101, Abcam, Cambridge, UK; 1:1,000), mTOR(2972, Cell Signaling Technology; 1:1,000) and p-mTOR Ser2448 (2971; Cell Signaling Technology; 1:1,000).

Techniques: Over Expression, Expressing, Infection, Fluorescence, Quantitative Proteomics, Control

Journal: Journal of dairy science

Article Title: Negative regulation of α S1 -casein (CSN1S1) improves β-casein content and reduces allergy potential in goat milk.

doi: 10.3168/jds.2020-18595

Figure Lengend Snippet: Figure 3. Interference of CSN1S1 increases β-casein expression primary goat mammary epithelial cells (GMEC). (A) Interference efficiency detection of small interfering (si)CSN1S1–47, siCSN1S1–195 and siCSN1S1–507 (100 nM) targeting CNS1S1. (B) mRNA expression levels of ca- seins and main whey proteins in GMEC transfected with negative control siRNA (siNC) or siCSN1S1–507 (100 nM) for 48 h. (C) Protein abun- dance of caseins in GMEC transfected with siNC or siCSN1S1–507 (100 nM) for 48 h. Relative protein abundance was normalized to β-actin. Values are presented as means ± SEM from 3 individual cultures (n = 3 goats). *P < 0.05 and **P < 0.01 compared with control (siNC).

Article Snippet: Secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (CW0103, CW Biotech; Beijing, China; 1:5,000) was used for primary antibodies αS1-casein (SAB1401093, Sigma-Aldrich; 1:1,000), αS2-casein (bs-10034R, Bioss, Beijing, China; 1:500), β-casein (orb2053, Biorbyt, Cambridgeshire, UK; 1:500), κ-casein (GTX60145, GeneTex, Irvine, CA; 1:500), JAK2 (3230, Cell Signaling Technology, Danvers, MA; 1:1,000), p-JAK2 Tyr1007/1008 (ab32101, Abcam, Cambridge, UK; 1:1,000), mTOR(2972, Cell Signaling Technology; 1:1,000) and p-mTOR Ser2448 (2971; Cell Signaling Technology; 1:1,000).

Techniques: Expressing, Transfection, Negative Control, Quantitative Proteomics, Control

Journal: Journal of dairy science

Article Title: Negative regulation of α S1 -casein (CSN1S1) improves β-casein content and reduces allergy potential in goat milk.

doi: 10.3168/jds.2020-18595

Figure Lengend Snippet: Figure 5. CSN1S1 represses β-casein expression via Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5a (STAT5a) signaling pathway in primary goat mammary epithelial cells (GMEC). (A) GMEC were transfected with pcDNA3.1-NC or pcDNA3.1-STAT5a followed by infection with adenovirus with green fluorescent protein (Ad-GFP) or adenovirus Ad-CSN1S1 (multiplicity of infection of 200), re- spectively. After 48 h of treatment, protein abundances of phosphorylated (p)-STAT5a, STAT5a, αS1-CN, and β-CN were measured. (B) GMEC were treated with dimethyl sulfoxide (DMSO) or JAK2 inhibitor (AG490; 30 μM) followed by negative control small interfering RNA (siNC) or siCSN1S1–507 (100 nM) transfection, respectively. At 48 h treatment, protein abundances of p-STAT5a, STAT5a, αS1-CN, and β-CN were mea- sured. Relative protein abundance was normalized to β-actin or total kinase. Values are presented as means ± SEM from 3 individual cultures (n = 3 goats). The different letters represent significant differences (P < 0.05) in protein abundance.

Article Snippet: Secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (CW0103, CW Biotech; Beijing, China; 1:5,000) was used for primary antibodies αS1-casein (SAB1401093, Sigma-Aldrich; 1:1,000), αS2-casein (bs-10034R, Bioss, Beijing, China; 1:500), β-casein (orb2053, Biorbyt, Cambridgeshire, UK; 1:500), κ-casein (GTX60145, GeneTex, Irvine, CA; 1:500), JAK2 (3230, Cell Signaling Technology, Danvers, MA; 1:1,000), p-JAK2 Tyr1007/1008 (ab32101, Abcam, Cambridge, UK; 1:1,000), mTOR(2972, Cell Signaling Technology; 1:1,000) and p-mTOR Ser2448 (2971; Cell Signaling Technology; 1:1,000).

Techniques: Expressing, Transfection, Infection, Negative Control, Small Interfering RNA, Quantitative Proteomics

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: Primary antibodies contains ELF5 (sc-166653, SantaCruz; 1:1,000), αS2-casein (bs-10034R, Bioss; 1:2,000), κ-casein (bs-10031R; Bioss; 1:1,000), αS1-casein (bs-10033R; Bioss; 1:1,000), β-casein (sc-166530; SantaCruz; 1:1,000), p-JAK2 (ab32101; Abcam; 1:1,000), p-STAT5 (9351S; 1:1,000), JAK2 (3230; CST; 1:1,000), STAT5 (610191; BD; 1:1,000), proliferating cell nuclear antigen (PCNA, 10205-2-APl Proteintech, 1:5,000), cyclin dependent kinase 2 (CDK2, 10122-1-AP; Proteintech, 1:10,000), B-cell lymphoma-2-associated X (BAX, 50599-2-lg; Proteintech, 1:10,000), Caspase3 (19677-1-AP; Proteintech, 1:2,000), β-Tubulin (CW0098M; CWBIO, 1:5,000).

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: Primary antibodies contains ELF5 (sc-166653, SantaCruz; 1:1,000), αS2-casein (bs-10034R, Bioss; 1:2,000), κ-casein (bs-10031R; Bioss; 1:1,000), αS1-casein (bs-10033R; Bioss; 1:1,000), β-casein (sc-166530; SantaCruz; 1:1,000), p-JAK2 (ab32101; Abcam; 1:1,000), p-STAT5 (9351S; 1:1,000), JAK2 (3230; CST; 1:1,000), STAT5 (610191; BD; 1:1,000), proliferating cell nuclear antigen (PCNA, 10205-2-APl Proteintech, 1:5,000), cyclin dependent kinase 2 (CDK2, 10122-1-AP; Proteintech, 1:10,000), B-cell lymphoma-2-associated X (BAX, 50599-2-lg; Proteintech, 1:10,000), Caspase3 (19677-1-AP; Proteintech, 1:2,000), β-Tubulin (CW0098M; CWBIO, 1:5,000).

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: Primary antibodies contains ELF5 (sc-166653, SantaCruz; 1:1,000), αS2-casein (bs-10034R, Bioss; 1:2,000), κ-casein (bs-10031R; Bioss; 1:1,000), αS1-casein (bs-10033R; Bioss; 1:1,000), β-casein (sc-166530; SantaCruz; 1:1,000), p-JAK2 (ab32101; Abcam; 1:1,000), p-STAT5 (9351S; 1:1,000), JAK2 (3230; CST; 1:1,000), STAT5 (610191; BD; 1:1,000), proliferating cell nuclear antigen (PCNA, 10205-2-APl Proteintech, 1:5,000), cyclin dependent kinase 2 (CDK2, 10122-1-AP; Proteintech, 1:10,000), B-cell lymphoma-2-associated X (BAX, 50599-2-lg; Proteintech, 1:10,000), Caspase3 (19677-1-AP; Proteintech, 1:2,000), β-Tubulin (CW0098M; CWBIO, 1:5,000).

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: Primary antibodies contains ELF5 (sc-166653, SantaCruz; 1:1,000), αS2-casein (bs-10034R, Bioss; 1:2,000), κ-casein (bs-10031R; Bioss; 1:1,000), αS1-casein (bs-10033R; Bioss; 1:1,000), β-casein (sc-166530; SantaCruz; 1:1,000), p-JAK2 (ab32101; Abcam; 1:1,000), p-STAT5 (9351S; 1:1,000), JAK2 (3230; CST; 1:1,000), STAT5 (610191; BD; 1:1,000), proliferating cell nuclear antigen (PCNA, 10205-2-APl Proteintech, 1:5,000), cyclin dependent kinase 2 (CDK2, 10122-1-AP; Proteintech, 1:10,000), B-cell lymphoma-2-associated X (BAX, 50599-2-lg; Proteintech, 1:10,000), Caspase3 (19677-1-AP; Proteintech, 1:2,000), β-Tubulin (CW0098M; CWBIO, 1:5,000).

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: The interaction between ELF5 and STAT5 in GMECs. (A) Cells were transfected with ELF5-MYC and STAT5-FLAG for 48 h. Then, total protein of cells was extracted and incubated with MYC (or FLAG) tag antibody and protein G magnetic beads at 4°C overnight. The protein abundance of ELF5 and STAT5 was detected. (B) The mRNA expression of STAT5a and ELF5 gene was measured after pCMV-STAT5a and pCMV-NC transfection for 48 h. Values are presented as mean±SEM. ** p<0.01. GMECs, goat mammary epithelial cells; NC, negative control; SEM, standard error of the mean.

Article Snippet: Primary antibodies contains ELF5 (sc-166653, SantaCruz; 1:1,000), αS2-casein (bs-10034R, Bioss; 1:2,000), κ-casein (bs-10031R; Bioss; 1:1,000), αS1-casein (bs-10033R; Bioss; 1:1,000), β-casein (sc-166530; SantaCruz; 1:1,000), p-JAK2 (ab32101; Abcam; 1:1,000), p-STAT5 (9351S; 1:1,000), JAK2 (3230; CST; 1:1,000), STAT5 (610191; BD; 1:1,000), proliferating cell nuclear antigen (PCNA, 10205-2-APl Proteintech, 1:5,000), cyclin dependent kinase 2 (CDK2, 10122-1-AP; Proteintech, 1:10,000), B-cell lymphoma-2-associated X (BAX, 50599-2-lg; Proteintech, 1:10,000), Caspase3 (19677-1-AP; Proteintech, 1:2,000), β-Tubulin (CW0098M; CWBIO, 1:5,000).

Techniques: Transfection, Incubation, FLAG-tag, Magnetic Beads, Quantitative Proteomics, Expressing, 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: Molecular mechanism of ELF5 promotes casein synthesis by enhancing the activity of JAK2/STAT5 signaling pathway in goat mammary epithelial cells. PRLR stands for prolactin receptor.

Article Snippet: Primary antibodies contains ELF5 (sc-166653, SantaCruz; 1:1,000), αS2-casein (bs-10034R, Bioss; 1:2,000), κ-casein (bs-10031R; Bioss; 1:1,000), αS1-casein (bs-10033R; Bioss; 1:1,000), β-casein (sc-166530; SantaCruz; 1:1,000), p-JAK2 (ab32101; Abcam; 1:1,000), p-STAT5 (9351S; 1:1,000), JAK2 (3230; CST; 1:1,000), STAT5 (610191; BD; 1:1,000), proliferating cell nuclear antigen (PCNA, 10205-2-APl Proteintech, 1:5,000), cyclin dependent kinase 2 (CDK2, 10122-1-AP; Proteintech, 1:10,000), B-cell lymphoma-2-associated X (BAX, 50599-2-lg; Proteintech, 1:10,000), Caspase3 (19677-1-AP; Proteintech, 1:2,000), β-Tubulin (CW0098M; CWBIO, 1:5,000).

Techniques: Activity Assay