rabbit anti βcasein  (Bioss)

Journal: Nature Communications

Article Title: VTA dopamine neurons are hyperexcitable in 3xTg-AD mice due to casein kinase 2-dependent SK channel dysfunction

doi: 10.1038/s41467-024-53891-1

Figure Lengend Snippet: a Workflow for cell segmentation analysis following immunohistochemistry. Confocal z-stack images were acquired, then the brightest slice of the channel of interest was chosen for analysis. DA neurons were segmented on TH staining from the same plane using the Cellpose ‘cyto v3’ model following upscaling (see Methods). Intensities within each segmented cell were calculated, quantified as average intensity across single cells, and plotted as cumulative probabilities. b Staining for CK2 indicated a slight rightward shift in the cumulative probability curve ( c ) of casein kinase 2 (CK2) intensity in tyrosine hydroxylase (TH) positive cells from 3xTg mice (Kolmogorov–Smirnov test, D = 0.1183, p < 0.0001, n = 2353 WT and 2187 3xTg cells, 6 mice). d Staining for phosphorylated calmodulin (p-CaM) indicated a moderate rightward shift in the cumulative probability curve ( e Kolmogorov–Smirnov test, D = 0.1202, p < 0.0001, n = 2612 WT and 2193 3xTg neurons, 6 mice). f SK channel upregulation in 3xTg mice was indicated by a strong rightward shift in the cumulative probability of small conductance calcium-activated potassium channel 3 (SK3) intensity in single cells ( g Kolmogorov–Smirnov test, D = 0.2284, p < 0.0001, n = 2914 WT and 3165 3xTg neurons, 6 mice). Red scale bars = 200 µm. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were diluted as follows: chicken anti-tyrosine hydroxylase (TH, 1:750, Abcam), rabbit anti-Kcnn3 (SK3, 1:200, Alomone, ThermoFischer), rabbit anti-casein kinase 2 beta 1 (CK2, 1:200, Invitrogen), rabbit anti-phospo-calmodulin (p-CaM, 1:100, Invitrogen), and rat anti-DAT (1:750, Millipore), and incubated for 72 h at 4 °C.

Techniques: Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: VTA dopamine neurons are hyperexcitable in 3xTg-AD mice due to casein kinase 2-dependent SK channel dysfunction

doi: 10.1038/s41467-024-53891-1

Figure Lengend Snippet: Normally, VTA DA neurons maintain a balance between CK2 and PP2A resulting in low basal phosphorylation of CaM. In 3xTg mice, hyperactive CK2 results in hyperphosphorylated CaM, effectively decreasing SK channel calcium binding affinity and channel opening. This results in increased firing rate and irregularity in 3xTg mice. Created in BioRender. Beckstead, M. (2023) BioRender.com/j31r759.

Article Snippet: Primary antibodies were diluted as follows: chicken anti-tyrosine hydroxylase (TH, 1:750, Abcam), rabbit anti-Kcnn3 (SK3, 1:200, Alomone, ThermoFischer), rabbit anti-casein kinase 2 beta 1 (CK2, 1:200, Invitrogen), rabbit anti-phospo-calmodulin (p-CaM, 1:100, Invitrogen), and rat anti-DAT (1:750, Millipore), and incubated for 72 h at 4 °C.

Techniques: Binding Assay

Journal: Animal bioscience

Article Title: miR-380-3p promotes β-casein expression by targeting αS1-casein in goat mammary epithelial cells.

doi: 10.5713/ab.23.0007

Figure Lengend Snippet: Figure 3. miR-380-3p increases STAT5a activity and β-casein abundance. (A) Expression of αS2-casein, β-casein and κ-casein gene in cells treated with miR-380-3p mimic (50 nM) for 48 h. (B) Expression of αS2-casein, β-casein and κ-casein gene in cells treated with miR-380-3p inhibitor (100 nM) for 48 h. (C) Expression of p-STAT5a and β-casein in cells treated with miR-380-3p mimic (50 nM) or miR-380-3p inhibitor (100 nM) for 48 h. Relative β-casein expression was normalized by comparison with β-actin. Relative p-STAT5a expression was normalized to STAT5a. STAT5a, signal trans ducer and activator of transcription 5a. Data are presented as mean±standard error of the mean. * p<0.05 and ** p<0.01 compared with control.

Article Snippet: Nonspecific sites were blocked using 5% skim milk (232100; BD Biosciences, Franklin Lakes, NJ, USA) at least 2 h, then incubated at least 12 h at 4°C with primary antibody: rabbit αS1-casein (SAB1401093; Sigma-Aldrich, USA; 1:1,000), rabbit β-casein (orb2053; Biorbyt, Cambridge, UK; 1:500), mouse signal transducer and activator of transcription 5a (STAT5a) (610191; BD Biosciences, USA; 1:1,000), mouse p-STAT5a (611964; BD Biosciences, USA; 1:500), or mouse β-actin (CW0096; CW Biotech, Beijing, China; 1:2,000).

Techniques: Activity Assay, Expressing, Comparison, Control

Journal: Animal bioscience

Article Title: miR-380-3p promotes β-casein expression by targeting αS1-casein in goat mammary epithelial cells.

doi: 10.5713/ab.23.0007

Figure Lengend Snippet: Figure 4. miR-380-3p improves β-casein expression by targeting αS1-casein gene. (A, B) Expression of αS1-casein, p-STAT5a and β-casein in cells co-treated with pcDNA3.1-αS1-casein and miR-380-3p mimic (50 nM) for 48 h. (C, D) Expression of αS1-casein, p-STAT5a and β-casein in cells co-treated with si-αS1-Casein (100 nM) and miR-380-3p inhibitor (100 nM) for 48 h. Relative expression of αS1-casein and β-casein was normalized by comparison with β-actin. Relative p-STAT5a expression was normalized to STAT5a. STAT5a, signal transducer and activator of transcription 5a. Data are presented as mean±standard error of the mean.

Article Snippet: Nonspecific sites were blocked using 5% skim milk (232100; BD Biosciences, Franklin Lakes, NJ, USA) at least 2 h, then incubated at least 12 h at 4°C with primary antibody: rabbit αS1-casein (SAB1401093; Sigma-Aldrich, USA; 1:1,000), rabbit β-casein (orb2053; Biorbyt, Cambridge, UK; 1:500), mouse signal transducer and activator of transcription 5a (STAT5a) (610191; BD Biosciences, USA; 1:1,000), mouse p-STAT5a (611964; BD Biosciences, USA; 1:500), or mouse β-actin (CW0096; CW Biotech, Beijing, China; 1:2,000).

Techniques: Expressing, Comparison

Journal: Animal bioscience

Article Title: miR-380-3p promotes β-casein expression by targeting αS1-casein in goat mammary epithelial cells.

doi: 10.5713/ab.23.0007

Figure Lengend Snippet: Figure 6. The proposed model regarding the role and regulation of miR-380-3p in goat mammary epithelial cells. miR-380-3p inhibits αS1-casein expression by targeting 3’UTR of αS1-casein gene. Then, miR-380-3p increases β-casein expression through αS1-casein/STAT5 axis. Moreover, miR-380-3p promotes β-casein transcription by the binding of STAT5 to β-casein gene promoter region. STAT5a, signal transducer and activator of transcription 5a.

Article Snippet: Nonspecific sites were blocked using 5% skim milk (232100; BD Biosciences, Franklin Lakes, NJ, USA) at least 2 h, then incubated at least 12 h at 4°C with primary antibody: rabbit αS1-casein (SAB1401093; Sigma-Aldrich, USA; 1:1,000), rabbit β-casein (orb2053; Biorbyt, Cambridge, UK; 1:500), mouse signal transducer and activator of transcription 5a (STAT5a) (610191; BD Biosciences, USA; 1:1,000), mouse p-STAT5a (611964; BD Biosciences, USA; 1:500), or mouse β-actin (CW0096; CW Biotech, Beijing, China; 1:2,000).

Techniques: Expressing, Binding Assay

Journal: Animal bioscience

Article Title: miR-380-3p promotes β-casein expression by targeting αS1-casein in goat mammary epithelial cells.

doi: 10.5713/ab.23.0007

Figure Lengend Snippet: Figure 5. miR-380-3p activates β-casein transcription via regulating STAT5. (A) Conserved binding motif of transcription factor STAT5. (B) Binding site of STAT5 in β-casein promoter region and construction of pGL3 luciferase reporter vectors. pGL3-WT-β-casein represents the wildtype β-casein promoter vector; pGL3-MUT-β-Casein represents the mutation (red bases) vector on STAT5 binding sequence. (C) Promoter activity of β-casein was measured by luciferase assay. Cells were treated with STAT5-IN-1 (50 μM) followed by pGL3-WT-β-Casein and miR-380-3p mimic (50 nM) co-treatment for 48 h. (D) Relative luciferase activity in cells co-treated with pGL3-β-Casein and miR-380-3p mimic (50 nM) for 48 h. (E) ChIP assay of the STAT5 binding site to β-casein gene promoter by qRT-PCR. Cells were treated with STAT5-IN-1 (50 μM) for 48 h. (F) ChIP assay in cells treated with miR-380-3p mimic (50 nM) for 48 h. STAT5a, signal transducer and activator of transcription 5a; qRT-PCR, quantitative real-time polymerase chain reaction. Data are presented as mean±standard error of the mean. * p<0.05 and ** p<0.01 compared with control.

Article Snippet: Nonspecific sites were blocked using 5% skim milk (232100; BD Biosciences, Franklin Lakes, NJ, USA) at least 2 h, then incubated at least 12 h at 4°C with primary antibody: rabbit αS1-casein (SAB1401093; Sigma-Aldrich, USA; 1:1,000), rabbit β-casein (orb2053; Biorbyt, Cambridge, UK; 1:500), mouse signal transducer and activator of transcription 5a (STAT5a) (610191; BD Biosciences, USA; 1:1,000), mouse p-STAT5a (611964; BD Biosciences, USA; 1:500), or mouse β-actin (CW0096; CW Biotech, Beijing, China; 1:2,000).

Techniques: Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Sequencing, Activity Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Control