Journal: Nature Communications

Article Title: TUSC3 regulates ERMA-mediated Mg 2+ uptake for synaptic function and neurodevelopment

doi: 10.1038/s41467-025-65668-1

Figure Lengend Snippet: a Representative western blot of ER stress proteins in the hippocampus of 4-month-old female WT and TUSC3 KO mice. b – i Quantification of GRP78 ( b ), p-PERK (T982) ( c ), p-eIF2α (S51) ( d ), CHOP ( e ), XBP1s ( f ), p-AKT (S473) ( g ), p-CREB (S133) ( h ), and ATF6 ( i ) normalized to β-actin ( n = 3 mice per group). j Representative western blot analysis of ER stress proteins in the striatum of 4-month-old female WT and TUSC3 KO mice ( n = 3 per group). k – r Quantification of GRP78 ( k ), p-PERK (T982) ( l ), p-eIF2α (S51) ( m ), CHOP ( n ), XBP1s ( o ), p-AKT (S473) ( p ), p-CREB (S133) ( q ), and ATF6 ( r ) normalized to β-actin ( n = 3 mice per group). s Non-ID and ID1/ID2 fibroblasts were chronically treated with MgT (400 μM) for 48 h and analyzed by western blotting (left). Quantification of p-eIF2α (S51) levels normalized to eIF2α (right) ( n = 3 independent biological replicates). t Non-ID and ID-derived fibroblasts were pretreated with or without MgT (400 μM) for 24 h, followed by puromycin (5 μg/ml) treatment for 2 h. Representative western blot using an anti-puromycin antibody (left), and puromycin incorporation was quantified and normalized to β-actin (right) ( n = 3 independent biological replicates). u SH-SY5Y shControl and shTUSC3#3-1 cells were pretreated with MgT (400 μM) for 12 h, then treated with either DMSO, thapsigargin (2 μM), tunicamycin (2 μg/ml), A23187 (2 μM), or etoposide (25 μM) for 24 h. Cells were stained with propidium iodide and Calcein-AM, and double-positive cells were quantified and normalized to Calcein-AM-positive cells. Data represent three independent biological experiments ( n = 3). The exact number of cells analyzed per condition is provided in the Source Data file. v Representative western blot of primary cortical neurons (DIV7) treated with vehicle or 4-PBA (10 mM) for 24 h (left). Quantification of indicated protein levels normalized to TUBA (right) ( n = 3 independent biological replicates). Two-tailed unpaired t -test ( b – i , and k – r ); one-way ANOVA followed by Tukey’s post hoc multiple comparison test ( s , t , v ); two-way ANOVA followed by Tukey’s multiple comparison test ( u ). Data are presented as mean ± S.E.M. or S.D. ( t , u ). Source data are provided as a file.

Article Snippet: The following primary antibodies were used in this study: ACTB (Santa Cruz Biotechnology; sc-47778; lot # J0421; WB 1:5000), TUBA (Santa Cruz Biotechnology; sc-23948; lot # G2921; WB 1:5000), TUSC3 (clone D-9; Santa Cruz Biotechnology; sc-390566; lot # J1218; IF 1:500), GRP78 (Santa Cruz Biotechnology; sc-376768; lot # C0316; WB 1:3000), p-CREB-1-S133 (clone 10E9; Santa Cruz Biotechnology; sc-81486; WB 1:3000), FLAG (Sigma-Aldrich; F1804; lot # 0000375542; WB 1:3000), RFP (MBL; PM005; lot # 048; WB 1:3000), TUSC3 (Proteintech; 16039-1-AP; lot # 00007251; WB 1:500), AKT (Cell Signaling Technology; 9272; lot # 6; WB 1:3000), P-AKT (S473) (Cell Signaling Technology; 4060; lot # 27; WB 1:3000; IF 1:250), CREB (Cell Signaling Technology; 9197; WB 1:1000), CHOP (Cell Signaling Technology; 2895; WB 1:1000), ATF6 (Cell Signaling Technology; 65880; lot # 5; WB 1:3000), MAP2 (Cell Signaling Technology; 4542; IF 1:500), NCAM1 (Cell Signaling Technology; 3576; WB 1:3000), PERK (ABclonal; A18196; lot # 3560572205; WB 1:3000), p-PERK (T982) (ABclonal; AP0886; lot # 5500040024; WB 1:3000), XBP1s (ABclonal; A17007; lot # 1153240401; WB 1:3000), eIF2α (ABclonal; A21221; lot # 360002151; WB 1:3000), p-eIF2α (ABclonal; AP0745; lot # 360003834; WB 1:3000), Puromycin (ABclonal; A23931; lot # 3523033102; WB 1:3000), PSD-93 (ABclonal; A19669; WB 1:3000), PSD-95 (ABclonal; A7889; WB 1:3000), GluA1 (ABclonal; A1826; lot # 3561869003; WB 1:3000; IF 1:250), NMDAR1 (ABclonal; A7677; lot # 5500016744; WB 1:1000), Synaptophysin (ABclonal; A6344; lot # 5500008363; WB 1:1000; IF 1:250), and N-Cadherin (BD Biosciences; 610920; WB 1:3000).

Techniques: Western Blot, Derivative Assay, Staining, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: TUSC3 regulates ERMA-mediated Mg 2+ uptake for synaptic function and neurodevelopment

doi: 10.1038/s41467-025-65668-1

Figure Lengend Snippet: a Representative western blot of PSD-93, PSD-95, GluA1, and phosphorylated CREB (S133) in whole-brain lysates of 4-month-old WT and TUSC3 KO female mice (left). Quantification of indicated protein levels (right) ( n = 3). b , c Representative confocal images of immunohistochemistry for TUSC3, synaptophysin (SYP), and MAP2 in the CA1 and CA3 hippocampal regions and the striatum of 4-month-old WT and TUSC3 KO female mice ( b ), and quantification of SYP intensity normalized to MAP2 ( c ) ( n = 3). Scale bar: 50 μm. d , e Representative confocal images of immunohistochemistry for TUSC3, GluA1, and MAP2 in the CA1 and CA3 hippocampal regions and the striatum of 4-month-old WT and TUSC3 KO female mice ( d ), and quantification of GluA1 intensity normalized to MAP2 ( e ) ( n = 3). Scale bar: 50 μm. f , g Representative dendritic images of primary hippocampal neurons transfected with EGFP ( f ). Scale bar: 50 μm. Sholl analysis of dendritic branching in WT and TUSC3 KO neurons ( n = 20 cells per group) ( g ). h , i Representative confocal images of dendritic spines of primary hippocampal neurons (DIV 12) transfected with EGFP for 48 h ( h ). Violin plot quantifying dendritic spine density (spines per 10 μm) in WT and KO neurons ( n = 20 cells per group) ( i ). Scale bar: 10 μm. j–l Representative traces of sEPSCs recorded from hippocampal slices of 2-month-old WT and TUSC3 KO mice ( j ), quantification of sEPSC amplitude ( k ), and frequency ( l ) in WT ( n = 12 cells from 5 mice; 4 F, 1 M) and TUSC3 KO ( n = 8 cells from 5 mice; 2 F, 3 M). m Representative traces (left) and quantification of the eEPSC amplitude (right) in WT ( n = 8 cells from 5 mice; 4 F, 1 M), TUSC3 KO ( n = 8 cells from 5 mice; 2 F, 3 M). n Representative traces (left) and EPSC-PPR (right) in WT ( n = 8 cells from 5 mice; 3 F, 2 M), TUSC3 KO ( n = 9 cells from 6 mice; 4 F, 2 M). Two-tailed unpaired t -test. Data are presented as mean ± S.E.M. ( a , c , e , and k – n ) or S.D. ( g , i ). Source data are provided as a file.

Article Snippet: The following primary antibodies were used in this study: ACTB (Santa Cruz Biotechnology; sc-47778; lot # J0421; WB 1:5000), TUBA (Santa Cruz Biotechnology; sc-23948; lot # G2921; WB 1:5000), TUSC3 (clone D-9; Santa Cruz Biotechnology; sc-390566; lot # J1218; IF 1:500), GRP78 (Santa Cruz Biotechnology; sc-376768; lot # C0316; WB 1:3000), p-CREB-1-S133 (clone 10E9; Santa Cruz Biotechnology; sc-81486; WB 1:3000), FLAG (Sigma-Aldrich; F1804; lot # 0000375542; WB 1:3000), RFP (MBL; PM005; lot # 048; WB 1:3000), TUSC3 (Proteintech; 16039-1-AP; lot # 00007251; WB 1:500), AKT (Cell Signaling Technology; 9272; lot # 6; WB 1:3000), P-AKT (S473) (Cell Signaling Technology; 4060; lot # 27; WB 1:3000; IF 1:250), CREB (Cell Signaling Technology; 9197; WB 1:1000), CHOP (Cell Signaling Technology; 2895; WB 1:1000), ATF6 (Cell Signaling Technology; 65880; lot # 5; WB 1:3000), MAP2 (Cell Signaling Technology; 4542; IF 1:500), NCAM1 (Cell Signaling Technology; 3576; WB 1:3000), PERK (ABclonal; A18196; lot # 3560572205; WB 1:3000), p-PERK (T982) (ABclonal; AP0886; lot # 5500040024; WB 1:3000), XBP1s (ABclonal; A17007; lot # 1153240401; WB 1:3000), eIF2α (ABclonal; A21221; lot # 360002151; WB 1:3000), p-eIF2α (ABclonal; AP0745; lot # 360003834; WB 1:3000), Puromycin (ABclonal; A23931; lot # 3523033102; WB 1:3000), PSD-93 (ABclonal; A19669; WB 1:3000), PSD-95 (ABclonal; A7889; WB 1:3000), GluA1 (ABclonal; A1826; lot # 3561869003; WB 1:3000; IF 1:250), NMDAR1 (ABclonal; A7677; lot # 5500016744; WB 1:1000), Synaptophysin (ABclonal; A6344; lot # 5500008363; WB 1:1000; IF 1:250), and N-Cadherin (BD Biosciences; 610920; WB 1:3000).

Techniques: Western Blot, Immunohistochemistry, Transfection, 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: 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 sources and dilutions of antibodies were as follows: rabbit anti-Fibronectin (1:1000, Abcam #23750), rabbit anti-SNAI2 (1:1000, Cell Signaling Technology #9585), rabbit anti-CREB (1:1000, Cell Signaling Technology #9197), rabbit anti-p-CREB S133 (1:1000, Cell Signaling Technology #9198), rabbit anti-CBP (1:1000, Cell Signaling Technology #7389), rabbit anti-p300 (1:1000, Cell Signaling Technology #70088), mouse anti-β-Tubulin (1:5000, proteintech, 66240-1-Ig), rabbit anti-α-Actinin(1:5000, proteintech, 11313-2-AP), horseradish peroxidase–conjugated horse anti-rabbit/mouse IgG (1:5000, 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: 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

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Effect of the RA-induced differentiation on DRD1 level, PKA activity, p-CREB S133 , Arc, and BDNF intracellular level/secretion in SH-SY5Y cells. In cultures treated or not with RA (10µM) were determined: a ) The DRD1 level (immunoblot at the bottom); each bar represents the mean ± SEM of DRD1/β-actin ratio from three independent experiments. b ) The PKA activity level; each bar represents the mean ± SEM of Relative activity kinase from three independent experiments in duplicates. c ) The nuclear expression level of p-CREB S133 (immunoblot at the bottom); each bar represents the mean ± SEM of p-CREB S133 /Lamin B ratio from three independent experiments, *** p < 0.001 statistical difference compared RA treatment to w/o treatment. d ) The expression level of Arc (immunoblot at the bottom); each bar represents the mean ± SEM of Arc/β-actin ratio from three independent experiments, * p < 0.05 statistical difference compared RA treatment to w/o treatment. The BDNF ( e ) intracellular and ( f ) secreted level, each bar represents the mean ± SEM of BDNF from three independent experiments by duplicate, ** p < 0.01 and **** p < 0.0001 statistical differences compared RA treatment to w/o treatment. Data were analyzed by t-student test

Article Snippet: PVDF was blocked with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-Tween) for 1 h. Membranes were incubated overnight at 4 °C with antibodies against Syn (1:1000), DRD1 (1:1000), Arc (1:500), BDNF (1:1000), p-CREB S133 (1:500), β-actin (1:5000), and Lamin B1 (1:500) (Santa Cruz Biotech., Santa Cruz, CA, USA). β-actin and Lamin B1 antibodies were used as a total and nuclear loading control, respectively.

Techniques: Activity Assay, Western Blot, Expressing

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Effects of dopamine D1-like receptor activation on p-CREB S133 level,Arc level,and BDNF secretion. a ) A representative immunoblot of p-CREB S133 and Lamin B is at the bottom of the graphic; each bar represents the mean ± SEM of the p-CREB S133 /Lamin B ratio from three independent experiments, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (10 µM) (0.5 h) to cells w/o stimulation (F = 6.517). b ) A representative immunoblot of the Arc and β-actin level is at the bottom of the graphic. Each bar represents the mean ± SEM of the Arc/Actin ratio from three independent experiments, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (0.25 and 6 h) to cells non-stimulated (F = 4.378). c ) ELISA determined the BDNF secretion; each bar represents the mean ± SEM of three independent experiments by duplicated, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (6 and 24 h) to cells w/o stimulation. Results in a ) and b ) were analyzed using a one-way ANOVA test and Dunnet´s post hoc tests. Results of c ) were analyzed using a two-way ANOVA test and Tukey´s post hoc test

Article Snippet: PVDF was blocked with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-Tween) for 1 h. Membranes were incubated overnight at 4 °C with antibodies against Syn (1:1000), DRD1 (1:1000), Arc (1:500), BDNF (1:1000), p-CREB S133 (1:500), β-actin (1:5000), and Lamin B1 (1:500) (Santa Cruz Biotech., Santa Cruz, CA, USA). β-actin and Lamin B1 antibodies were used as a total and nuclear loading control, respectively.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Blocking the dopamine D1-like receptor inhibits the Arc, BDNF, and p-CREB S133 induction. After the pretreatment with antagonist SCH-23390 (10µM), the cells were stimulated with agonist SKF-38393 (10 µM) at maximum induction time for each parameter. a ) A representative immunoblot of p-CREB S133 and Lamin B levels is at the bottom of the graphic; each bar represents the mean ± SEM of p-CREB S133 /Lamin B ratio from three independent experiments, ** p < 0.01 statistical difference compared cells stimulated with SKF-38393 (0.5 h) to cells w/o stimulation, and && p < 0.01 cells treated with SCH-23390 + SKF-38393 to cells stimulated with SKF-38393. b ) A representative immunoblot of Arc and β-Actin level is at the bottom of the graphic; each bar represents the mean ± SEM of the Arc/β-Actin ratio from three independent experiments, ** p < 0.01 statistical difference compared cells stimulated with SKF-38393 (0.25 and 6 h) to cells w/o stimulation, and & p < 0.05 statistical difference compared cells treated with SCH-23390 + SKF-38393 (6 h) to cells stimulated with SKF-38393 (6 h) (F = v4.564 for 0.25 min and F = 14.98 for 6 h). c ) The BDNF secretion level determined by ELISA, each bar represents the mean ± SEM from three independent experiments by duplicated, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 to cells w/o stimulation (F = 11.52). Data were analyzed using a t -test or one-way ANOVA test followed by a Tukey post hoc test

Article Snippet: PVDF was blocked with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-Tween) for 1 h. Membranes were incubated overnight at 4 °C with antibodies against Syn (1:1000), DRD1 (1:1000), Arc (1:500), BDNF (1:1000), p-CREB S133 (1:500), β-actin (1:5000), and Lamin B1 (1:500) (Santa Cruz Biotech., Santa Cruz, CA, USA). β-actin and Lamin B1 antibodies were used as a total and nuclear loading control, respectively.

Techniques: Blocking Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Effects of dopamine D1-like receptor on p-CREB S133 and Arc location . After blocking (or not) the D1-like receptor, the cells were stimulated with SKF-38393 (10 µM) at indicated times. ( a ) After thirty minutes of stimulation, the p-CREB S133 location was immunodetected with an antibody against phosphorylated CREB in Ser − 133 (green); the F-actin was labeled with TRITC-phalloidin (red), and the nucleus was stained by DAPI (blue). For SKF-38,393 (10 µM), the yellow arrowheads indicate the prominent location of p-CREB S133 concurring in the nucleus visualized by the white circles in the merge column. ( b ) After fifteen minutes or six hours of stimulation, the Arc location was detected by immunofluorescence labeling Arc protein (green); the F-actin was labeled using TRITC-phalloidin (red); the nucleus was stained by DAPI (blue). An area (white dotted square) was magnified. The white arrow/squares point out the clusters of Arc near the cell membrane and lamellipodial F-actin structures without colors overlapping in merge at 0.25 h. In contrast, at 6 h, the white arrowheads point out the yellow dots corresponding to Arc location (green) overlapping with F-actin (red)

Article Snippet: PVDF was blocked with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-Tween) for 1 h. Membranes were incubated overnight at 4 °C with antibodies against Syn (1:1000), DRD1 (1:1000), Arc (1:500), BDNF (1:1000), p-CREB S133 (1:500), β-actin (1:5000), and Lamin B1 (1:500) (Santa Cruz Biotech., Santa Cruz, CA, USA). β-actin and Lamin B1 antibodies were used as a total and nuclear loading control, respectively.

Techniques: Blocking Assay, Labeling, Staining, Immunofluorescence, Membrane

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Schematical representation of dynamic changes by dopamine D1-like receptor activation on p-CREB S133 , Arc, BDNF. PKA is activated once the dopamine D1-like receptor is stimulated, and CREB is phosphorylated ( p-CREB S133 ). Arc protein is increased at 15 min, suggesting preexistent mRNA translation; a second increase at 6 suggests a second cycle of transcription and translation. BDNF secretion fluctuates over time, suggesting that in addition to stimulation of secretion, at 24 h, the dopamine D1-like receptor would induce the BDNF endocytosis (scheme was created with BioRender.com )

Article Snippet: PVDF was blocked with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-Tween) for 1 h. Membranes were incubated overnight at 4 °C with antibodies against Syn (1:1000), DRD1 (1:1000), Arc (1:500), BDNF (1:1000), p-CREB S133 (1:500), β-actin (1:5000), and Lamin B1 (1:500) (Santa Cruz Biotech., Santa Cruz, CA, USA). β-actin and Lamin B1 antibodies were used as a total and nuclear loading control, respectively.

Techniques: Activation Assay

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Effect of the RA-induced differentiation on DRD1 level, PKA activity, p-CREB S133 , Arc, and BDNF intracellular level/secretion in SH-SY5Y cells. In cultures treated or not with RA (10µM) were determined: a ) The DRD1 level (immunoblot at the bottom); each bar represents the mean ± SEM of DRD1/β-actin ratio from three independent experiments. b ) The PKA activity level; each bar represents the mean ± SEM of Relative activity kinase from three independent experiments in duplicates. c ) The nuclear expression level of p-CREB S133 (immunoblot at the bottom); each bar represents the mean ± SEM of p-CREB S133 /Lamin B ratio from three independent experiments, *** p < 0.001 statistical difference compared RA treatment to w/o treatment. d ) The expression level of Arc (immunoblot at the bottom); each bar represents the mean ± SEM of Arc/β-actin ratio from three independent experiments, * p < 0.05 statistical difference compared RA treatment to w/o treatment. The BDNF ( e ) intracellular and ( f ) secreted level, each bar represents the mean ± SEM of BDNF from three independent experiments by duplicate, ** p < 0.01 and **** p < 0.0001 statistical differences compared RA treatment to w/o treatment. Data were analyzed by t-student test

Article Snippet: Cells were stained with mouse anti-Arc or mouse anti-p-CREB S133 antibodies (Santa Cruz Biotech., Santa Cruz, CA, USA) (both at 1:30).

Techniques: Activity Assay, Western Blot, Expressing

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Effects of dopamine D1-like receptor activation on p-CREB S133 level,Arc level,and BDNF secretion. a ) A representative immunoblot of p-CREB S133 and Lamin B is at the bottom of the graphic; each bar represents the mean ± SEM of the p-CREB S133 /Lamin B ratio from three independent experiments, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (10 µM) (0.5 h) to cells w/o stimulation (F = 6.517). b ) A representative immunoblot of the Arc and β-actin level is at the bottom of the graphic. Each bar represents the mean ± SEM of the Arc/Actin ratio from three independent experiments, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (0.25 and 6 h) to cells non-stimulated (F = 4.378). c ) ELISA determined the BDNF secretion; each bar represents the mean ± SEM of three independent experiments by duplicated, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (6 and 24 h) to cells w/o stimulation. Results in a ) and b ) were analyzed using a one-way ANOVA test and Dunnet´s post hoc tests. Results of c ) were analyzed using a two-way ANOVA test and Tukey´s post hoc test

Article Snippet: Cells were stained with mouse anti-Arc or mouse anti-p-CREB S133 antibodies (Santa Cruz Biotech., Santa Cruz, CA, USA) (both at 1:30).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Blocking the dopamine D1-like receptor inhibits the Arc, BDNF, and p-CREB S133 induction. After the pretreatment with antagonist SCH-23390 (10µM), the cells were stimulated with agonist SKF-38393 (10 µM) at maximum induction time for each parameter. a ) A representative immunoblot of p-CREB S133 and Lamin B levels is at the bottom of the graphic; each bar represents the mean ± SEM of p-CREB S133 /Lamin B ratio from three independent experiments, ** p < 0.01 statistical difference compared cells stimulated with SKF-38393 (0.5 h) to cells w/o stimulation, and && p < 0.01 cells treated with SCH-23390 + SKF-38393 to cells stimulated with SKF-38393. b ) A representative immunoblot of Arc and β-Actin level is at the bottom of the graphic; each bar represents the mean ± SEM of the Arc/β-Actin ratio from three independent experiments, ** p < 0.01 statistical difference compared cells stimulated with SKF-38393 (0.25 and 6 h) to cells w/o stimulation, and & p < 0.05 statistical difference compared cells treated with SCH-23390 + SKF-38393 (6 h) to cells stimulated with SKF-38393 (6 h) (F = v4.564 for 0.25 min and F = 14.98 for 6 h). c ) The BDNF secretion level determined by ELISA, each bar represents the mean ± SEM from three independent experiments by duplicated, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 to cells w/o stimulation (F = 11.52). Data were analyzed using a t -test or one-way ANOVA test followed by a Tukey post hoc test

Article Snippet: Cells were stained with mouse anti-Arc or mouse anti-p-CREB S133 antibodies (Santa Cruz Biotech., Santa Cruz, CA, USA) (both at 1:30).

Techniques: Blocking Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Effects of dopamine D1-like receptor on p-CREB S133 and Arc location . After blocking (or not) the D1-like receptor, the cells were stimulated with SKF-38393 (10 µM) at indicated times. ( a ) After thirty minutes of stimulation, the p-CREB S133 location was immunodetected with an antibody against phosphorylated CREB in Ser − 133 (green); the F-actin was labeled with TRITC-phalloidin (red), and the nucleus was stained by DAPI (blue). For SKF-38,393 (10 µM), the yellow arrowheads indicate the prominent location of p-CREB S133 concurring in the nucleus visualized by the white circles in the merge column. ( b ) After fifteen minutes or six hours of stimulation, the Arc location was detected by immunofluorescence labeling Arc protein (green); the F-actin was labeled using TRITC-phalloidin (red); the nucleus was stained by DAPI (blue). An area (white dotted square) was magnified. The white arrow/squares point out the clusters of Arc near the cell membrane and lamellipodial F-actin structures without colors overlapping in merge at 0.25 h. In contrast, at 6 h, the white arrowheads point out the yellow dots corresponding to Arc location (green) overlapping with F-actin (red)

Article Snippet: Cells were stained with mouse anti-Arc or mouse anti-p-CREB S133 antibodies (Santa Cruz Biotech., Santa Cruz, CA, USA) (both at 1:30).

Techniques: Blocking Assay, Labeling, Staining, Immunofluorescence, Membrane

Journal: Neurochemical Research

Article Title: Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

doi: 10.1007/s11064-024-04293-8

Figure Lengend Snippet: Schematical representation of dynamic changes by dopamine D1-like receptor activation on p-CREB S133 , Arc, BDNF. PKA is activated once the dopamine D1-like receptor is stimulated, and CREB is phosphorylated ( p-CREB S133 ). Arc protein is increased at 15 min, suggesting preexistent mRNA translation; a second increase at 6 suggests a second cycle of transcription and translation. BDNF secretion fluctuates over time, suggesting that in addition to stimulation of secretion, at 24 h, the dopamine D1-like receptor would induce the BDNF endocytosis (scheme was created with BioRender.com )

Article Snippet: Cells were stained with mouse anti-Arc or mouse anti-p-CREB S133 antibodies (Santa Cruz Biotech., Santa Cruz, CA, USA) (both at 1:30).

Techniques: Activation Assay