primary antibodies against p-creb s133  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: The transcription factor CREB regulates epithelial-mesenchymal transition of lens epithelial cells by phosphorylation-dependent and phosphorylation-independent mechanisms

doi: 10.1016/j.jbc.2024.108064

Figure Lengend Snippet: CREB loss of function leads to attenuated mesenchymal gene expression in mouse lens epithelial cells. A , cells were treated with 20 ng/ml TGFβ for 24 h, and Western blot analysis was used to examine the protein expression levels of Fibronectin and SNAI2 in mock or TGFβ-treated-αTN4-1 cells overexpressing NC-shRNA, CREB 1#shRNA, and CREB 2#shRNA. B and C , quantification results of Fibronectin and SNAI2 protein levels in ( A ). D , immunofluorescence staining for Fibronectin proteins in mock or TGFβ-treated-Mock-sh and CREB-sh-αTN4-1 cells. Scale bar represents 20 μm. E , quantification results of mean fluorescence of Fibronectin protein in ( D ). F , cells were treated with 20 ng/ml TGFβ for 24 h, and Western blot analysis was used to examine the protein expression levels of Fibronectin and SNAI2. G and H , quantification results of Fibronectin and SNAI2 protein levels in F . I , immunofluorescence staining for Fibronectin proteins in mock or TGFβ-treated-Mock-KO and CREB-KO-αTN4-1 cells. Scale bar represents 20 μm. J , quantification results of mean fluorescence of Fibronectin protein in ( I ). In B , C , E , G , H , and J , the data shown are the averages of three independent biological experiments, and error bars represent the SD of mean. Statistical analysis: Two-way ANOVA followed by Tukey’s post hoc test. ∗∗ p < 0.01, ∗ p < 0.05, n.s, statistically not significant.

Article Snippet: The permeabilized samples were blocked in normal goat serum for 1 h followed by overnight incubation at 4 °C with primary antibodies against p-CREB S133 (1:200, Cell Signaling Technology) and α-SMA (1: 1:100, Cell Signaling Technology).

Techniques: Gene Expression, Western Blot, Expressing, shRNA, Immunofluorescence, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The transcription factor CREB regulates epithelial-mesenchymal transition of lens epithelial cells by phosphorylation-dependent and phosphorylation-independent mechanisms

doi: 10.1016/j.jbc.2024.108064

Figure Lengend Snippet: CREB S133 phosphorylation and CBP interaction mediate mesenchymal genes expression in lens epithelial cells. A , αTN4-1 cells were treated with 20 ng/ml TGFβ for 24 h, and Western blot analysis was used to examine the protein expression levels of p-CREB S133, Fibronectin, and SNAI2 in mock or TGFβ-treated αTN4-1 cells. B , quantification results of Fibronectin, SNAI2, and p-CREB S133 protein levels in A . C , the anterior capsules of one lens of the mouse was punctured with a needle to induce ASC, and the other lens remains as untreated normal. After 3 days of healing, the protein expression levels of lens capsular epithelium from normal and injured-induced ASC mice were examined. Western blot analysis was used to examine the protein expression levels of p-CREB S133 and Fibronectin in the lens epithelium of the normal and injured-induced ASC mice. D , quantification results of Fibronectin and p-CREB S133 protein levels in ( C ). E , immunofluorescence were used to analyze p-CREB S133 ( green ), α-SMA ( red ), and lens epithelial cell nuclei in the lens capsule of the normal and injured-induced ASC mice ( blue ) (n = 3 lenses per group). two-tailed Student’s t test was used in ( B ) and ( D ), The data shown are the averages of three independent biological experiments, and error bars represent the SD of mean. ∗∗ p < 0.01, ∗ p < 0.05, n.s, statistically not significant.

Article Snippet: The permeabilized samples were blocked in normal goat serum for 1 h followed by overnight incubation at 4 °C with primary antibodies against p-CREB S133 (1:200, Cell Signaling Technology) and α-SMA (1: 1:100, Cell Signaling Technology).

Techniques: Phospho-proteomics, Expressing, Western Blot, Capsules, Immunofluorescence, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The transcription factor CREB regulates epithelial-mesenchymal transition of lens epithelial cells by phosphorylation-dependent and phosphorylation-independent mechanisms

doi: 10.1016/j.jbc.2024.108064

Figure Lengend Snippet: CBP gene silencing did not attenuate mesenchymal gene expression in mouse lens epithelial cells. A , mock and TGFβ-treated αTN4-1 cells were cotreated with indicated concentration of CREB-CBP inhibitor or DMSO for 24 h. Western blot analysis of the protein levels of Fibronectin and SNAI2. B and C , quantification results of Fibronectin and SNAI2 protein levels in ( A ). qRT-PCR ( D ) and Western blot analysis ( E ) was used to examine the mRNA and protein expression levels, respectively, of CBP in αTN4-1 cells overexpressing NC-shRNA, CBP 1#shRNA, and CBP 2#shRNA. F , Western blot analysis was used to examine the protein expression levels of Fn1 and SNAI2 in mock or TGFβ-treated-αTN4-1 cells overexpressing NC-shRNA, CBP 1#shRNA, and CBP 2#shRNA. G and H , quantification results of Fibronectin and SNAI2 protein levels in ( F ). I , immunofluorescence staining for Fibronectin proteins in the mock or TGFβ-treated-αTN4-1 cells overexpressing NC-shRNA, CBP 1#shRNA, and CBP 2#shRNA. Scale bar represents 20 μm. J , quantification results of mean fluorescence of Fibronectin protein in I . The data shown are the averages of three independent biological experiments, and error bars represent the SD of mean. Statistical analysis: One-way ANOVA followed by Tukey’s correction was used in ( B ), ( C ), and ( D ). Two-way ANOVA followed by Tukey’s post hoc test was used in ( G ), ( H ), and ( J ). ∗∗ p < 0.01, ∗ p < 0.05. n.s, statistically not significant.

Article Snippet: The permeabilized samples were blocked in normal goat serum for 1 h followed by overnight incubation at 4 °C with primary antibodies against p-CREB S133 (1:200, Cell Signaling Technology) and α-SMA (1: 1:100, Cell Signaling Technology).

Techniques: Gene Expression, Concentration Assay, Western Blot, Quantitative RT-PCR, Expressing, shRNA, Immunofluorescence, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The transcription factor CREB regulates epithelial-mesenchymal transition of lens epithelial cells by phosphorylation-dependent and phosphorylation-independent mechanisms

doi: 10.1016/j.jbc.2024.108064

Figure Lengend Snippet: S133A-CREB serves as a novel transcription factor. A , qRT-PCR was used to examine the mRNA expression levels of Fn1 and Snai2 in αTN4-1 cells overexpressing pCI-neo-vector(Vector), pCI-neo-WT-CREB (WT-CREB), and pCI-neo-S133A-CREB (S133A-CREB). B , Western blot analysis was used to examine the protein expression levels of Fibronectin and SNAI2 in αTN4-1 cells overexpressing Vector, WT-CREB and S133A-CREB. C , αTN4-1 cells overexpressing Vector, WT-CREB or S133A-CREB were treated with 20 ng/ml TGFβ for 24 h. Western blot analysis was used to examine the protein expression levels of Fibronectin and SNAI2. D and E , quantification results of Fibronectin and SNAI2 protein levels in ( C ). F , Western blot analysis was used to examine the protein expression levels of Fibronectin in S133A-CREB heterozygote and the WT littermate mice. G , quantification results of Fibronectin protein levels in ( F ). H and I , ChIP-qPCR assays to demonstrate that S133A-CREB mutant proteins bind to the promoter region of Fn1 and Snai2 in vivo . The data shown are the averages of three independent biological experiments, and error bars represent the SD of mean. Statistical analysis: One-way ANOVA in ( A ). Two-way ANOVA followed by Tukey’s post hoc test was used in ( D ) and ( E ). Two-tailed Student’s t test in ( G ), ( H ), and ( I ). ∗∗ p < 0.01, ∗ p < 0.05, n.s, statistically not significant.

Article Snippet: The permeabilized samples were blocked in normal goat serum for 1 h followed by overnight incubation at 4 °C with primary antibodies against p-CREB S133 (1:200, Cell Signaling Technology) and α-SMA (1: 1:100, Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Western Blot, ChIP-qPCR, Mutagenesis, In Vivo, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The transcription factor CREB regulates epithelial-mesenchymal transition of lens epithelial cells by phosphorylation-dependent and phosphorylation-independent mechanisms

doi: 10.1016/j.jbc.2024.108064

Figure Lengend Snippet: CRTC co-activators are essential for the transactivation of S133A-CREB. αTN4-1 cells overexpressing pCI-neo-vector(Vector), WT-CREB, KCREB, R314A-CREB, S133A-CREB, S133A-KCREB, and S133A/R314A-CREB were treated with 20 ng/ml TGFβ for 24 h. A , Western blot analysis was used to examine the protein expression levels of Fibronectin and SNAI2. B and C , quantification results of Fibronectin and SNAI2 protein levels in ( A ). Error bars represent the SD of the mean (n = 3). Statistical analysis: Two-way ANOVA followed by Tukey’s correction. ∗∗ p < 0.01, ∗ p < 0.05. D , immunofluorescence staining for Fibronectin proteins in mock or TGFβ-treated-Mock-KO and CREB-KO-αTN4-1 cells. Scale bar represents 20 μm. E , quantification results of mean fluorescence of Fibronectin protein in ( D ). The data shown were derived from two different clones, and three independent Western blot experiments were conducted for each clone. The error bars represent theSD of mean. Statistical analysis: Two-way ANOVA followed by Tukey’s post hoc test. ∗∗ p < 0.01, ∗ p < 0.05, n.s, statistically not significant.

Article Snippet: The permeabilized samples were blocked in normal goat serum for 1 h followed by overnight incubation at 4 °C with primary antibodies against p-CREB S133 (1:200, Cell Signaling Technology) and α-SMA (1: 1:100, Cell Signaling Technology).

Techniques: Plasmid Preparation, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Derivative Assay, Clone Assay