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: Methods in molecular biology (Clifton, N.J.)

Article Title: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood

doi: 10.1007/978-1-0716-2553-8_10

Figure Lengend Snippet: Whole blood phosphoflow panel 1

Article Snippet: 165 Ho , pCREB [S133] , 87G3 , Fluidigm 3165009A.

Techniques:

Journal: iScience

Article Title: Prenatal low-dose methylmercury exposure causes premature neuronal differentiation and autism-like behaviors in a rodent model

doi: 10.1016/j.isci.2023.106093

Figure Lengend Snippet: Prenatal MeHg exposure stimulates CREB activity specifically in RGPs (A) Dot plot of downstream CREB target genes ( Jund , Fos , Dusp1 , Egr1 ) and their expression from scRNA-seq data in RGP1 and RGP2 cell populations for control (0ppm) and 0.2ppm MeHg groups. (B) Violin plot from scRNA-seq showing Fos (upper panels) and Jund (lower panels) expressions in RGP1 and RGP2 cell populations in control (0ppm) and 0.2ppm MeHg treatment groups, n > 800 cells for each group, Mann–Whitney U test, ∗∗∗∗p < 0.0001, ns (non-significant). (C) Schematic of cortical percussors isolated from E11-12 CD1 mice, created with BioRender.com . Cells were exposed to 0 nM (control) or 250 nM MeHg with no metformin or metformin (500 μM) for 4 h, at which point they were lysed for western blot or PLA analysis. (D–I) Western blot analysis of E12 cortical precursors cultured for 4 hours in the absence or presence of 250 nM MeHg. (D, G) Blots images were probed for pS133-CREB (D), pT410/403-aPKC (G), total CREB (D), total aPKC (G) and GAPDH (D, G) as a loading control. (E-F) Quantitative analysis of pS133-CREB (E) and total CREB (F) over GAPDH. n = 5 independent experiments, Student’s t test, ∗p < 0.05, ns (non-significant) (H-I) Quantitative analysis of pT410/403-aPKC(H) and aPKC (I) over GAPDH, n = 4 independent experiments, Student’s t test, ∗p < 0.05, ns (non-significant). (J) Images of PLA + interactions (lower panels, red) between pS133-CREB and CBP, counterstained with Hoechst (blue) in cultured cortical precursors of four treatment groups i) control (0ppm MeHg and 0 μM metformin), ii) 250 nM MeHg, iii) 500 μM metformin, and iv) co-treatment of 500 μM metformin +250 nM MeHg, scale bar: 5 μm. Images of negative control PLA (upper panels) in the absence of anti-pS133-CREB and anti-CBP (upper left) or absence of PLA probes (upper right). (K) Quantitative analysis of pS133- CREB and CBP interaction signals (red) within nuclei (blue). n = 100 cells/group, n = 4 independent experiments, two-way ANOVA (group × metformin interaction F(1, 396) = 11.34, p = 0.0008, group F(1, 396) = 1.463, p = 0.2272, metformin F(1, 396) = 25.01, p < 0.0001, n = 400) with post hoc test, ∗p < 0.05, ∗∗p < 0.01. Error bars indicate the standard error of the mean (SEM). See also Figure S6 .

Article Snippet: rabbit anti- p -CREB (S133) , Cell Signaling , Cat#9198S; RRID: AB_2561044.

Techniques: Activity Assay, Expressing, Control, MANN-WHITNEY, Isolation, Western Blot, Cell Culture, Negative Control

Journal: iScience

Article Title: Prenatal low-dose methylmercury exposure causes premature neuronal differentiation and autism-like behaviors in a rodent model

doi: 10.1016/j.isci.2023.106093

Figure Lengend Snippet:

Article Snippet: rabbit anti- p -CREB (S133) , Cell Signaling , Cat#9198S; RRID: AB_2561044.

Techniques: Recombinant, Proximity Ligation Assay, In Situ, Software, Microscopy

Journal: Environment international

Article Title: Exposure to dithiocarbamate fungicide maneb in vitro and in vivo: Neuronal apoptosis and underlying mechanisms.

doi: 10.1016/j.envint.2022.107696

Figure Lengend Snippet: Fig. 5. Changes in apoptosis-related proteins expression in SH-SY5Y cells exposed to maneb. (A, B) Expression levels of mitochondrial apoptosis pathway-related proteins in SH-SY5Y cells following maneb exposure. (C, D) Expression levels of PKA/CREB signaling pathway-related proteins in SH-SY5Y cells following maneb exposure. Data are presented as mean ± SEM, n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: The following antibodies and reagents were used: Bax (Proteintech, 50599–2-Ig), Bcl-2 (Cell Signaling Technology, 3498S), cytochrome c (Abcam, ab133504), caspase-3 (Proteintech, 19677-1-AP), GAPDH (Proteintech, 60004-1- Ig), PKA (Proteintech, 55382-1-AP), phosphodiesterase 10A (PDE10A) (Proteintech, 18078-1-AP), CREB (Proteintech, 12208-1-AP), p-CREB (ABclonal, AP0333), COX IV (Abcam, ab16056), DAPI (Biofroxx, EZ3412B205), MAP-2 (ThermoFisher, SF254293), HRP-conjugated antirabbit IgG (BIO-RAD, 170-6515), HRP-conjugated anti-mouse IgG (BIORAD, 170-6516), Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, A-11001), Cell Counting Kit-8 Viability Assay (CCK-8) (Solarbio, CK04), Apoptosis and Necrosis Assay Kit (Beyotime Biotechnology, C1056), TUNEL BrightRed Apoptosis Detection Kit (Vazyme, A113), Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, C1062), Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute, E004-1-1), N-acetylcysteine (NAC) (SigmaAldrich, A7250), JC-10 (Solarbio, CA1310), Dibutyryl-cAMP (dbcAMP) (Selleck, S7858).

Techniques: Expressing

Journal: Environment international

Article Title: Exposure to dithiocarbamate fungicide maneb in vitro and in vivo: Neuronal apoptosis and underlying mechanisms.

doi: 10.1016/j.envint.2022.107696

Figure Lengend Snippet: Fig. 6. Maneb exposure induces apoptosis and changes the expression of apoptosis-related proteins in mouse brains. (A) TUNEL staining and immuno fluorescence of MAP-2 in mouse brain slides. Scale bars = 50 μm. (B, C) Expression levels of mitochon drial apoptosis pathway-related proteins in mouse brains following maneb exposure. (D, E) Expression levels of PKA/CREB signaling pathway-related pro teins in mouse brains following maneb exposure. Data are presented as mean ± SEM, n = 4, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: The following antibodies and reagents were used: Bax (Proteintech, 50599–2-Ig), Bcl-2 (Cell Signaling Technology, 3498S), cytochrome c (Abcam, ab133504), caspase-3 (Proteintech, 19677-1-AP), GAPDH (Proteintech, 60004-1- Ig), PKA (Proteintech, 55382-1-AP), phosphodiesterase 10A (PDE10A) (Proteintech, 18078-1-AP), CREB (Proteintech, 12208-1-AP), p-CREB (ABclonal, AP0333), COX IV (Abcam, ab16056), DAPI (Biofroxx, EZ3412B205), MAP-2 (ThermoFisher, SF254293), HRP-conjugated antirabbit IgG (BIO-RAD, 170-6515), HRP-conjugated anti-mouse IgG (BIORAD, 170-6516), Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, A-11001), Cell Counting Kit-8 Viability Assay (CCK-8) (Solarbio, CK04), Apoptosis and Necrosis Assay Kit (Beyotime Biotechnology, C1056), TUNEL BrightRed Apoptosis Detection Kit (Vazyme, A113), Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, C1062), Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute, E004-1-1), N-acetylcysteine (NAC) (SigmaAldrich, A7250), JC-10 (Solarbio, CA1310), Dibutyryl-cAMP (dbcAMP) (Selleck, S7858).

Techniques: Expressing, TUNEL Assay, Staining, Fluorescence

Journal: Environment international

Article Title: Exposure to dithiocarbamate fungicide maneb in vitro and in vivo: Neuronal apoptosis and underlying mechanisms.

doi: 10.1016/j.envint.2022.107696

Figure Lengend Snippet: Fig. 7. Schematic diagram of the mechanisms underlying maneb-induced apoptosis. The mitochondrial apoptosis pathway (Bcl-2, Bax, cytochrome c, caspase-3) and the PKA/CREB pathway (PKA, PDE10A, CREB, p-CREB) are involved in maneb-induced apoptosis. In addition, maneb induces mitochon drial dysfunction and ROS accumulation, contributing to the neurotoxicity of maneb.

Article Snippet: The following antibodies and reagents were used: Bax (Proteintech, 50599–2-Ig), Bcl-2 (Cell Signaling Technology, 3498S), cytochrome c (Abcam, ab133504), caspase-3 (Proteintech, 19677-1-AP), GAPDH (Proteintech, 60004-1- Ig), PKA (Proteintech, 55382-1-AP), phosphodiesterase 10A (PDE10A) (Proteintech, 18078-1-AP), CREB (Proteintech, 12208-1-AP), p-CREB (ABclonal, AP0333), COX IV (Abcam, ab16056), DAPI (Biofroxx, EZ3412B205), MAP-2 (ThermoFisher, SF254293), HRP-conjugated antirabbit IgG (BIO-RAD, 170-6515), HRP-conjugated anti-mouse IgG (BIORAD, 170-6516), Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, A-11001), Cell Counting Kit-8 Viability Assay (CCK-8) (Solarbio, CK04), Apoptosis and Necrosis Assay Kit (Beyotime Biotechnology, C1056), TUNEL BrightRed Apoptosis Detection Kit (Vazyme, A113), Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, C1062), Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute, E004-1-1), N-acetylcysteine (NAC) (SigmaAldrich, A7250), JC-10 (Solarbio, CA1310), Dibutyryl-cAMP (dbcAMP) (Selleck, S7858).

Techniques:

Journal: Aging (Albany NY)

Article Title: Dl-3-n-butylphthalide inhibits neuroinflammation by stimulating foxp3 and Ki-67 in an ischemic stroke model

doi: 10.18632/aging.202338

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Anti-pCREB [S133] (87G3)-176Yb , Fluidigm , Cat# 3176005A.

Techniques:

Journal: Biomedicines

Article Title: Inhibition of AKT/GSK3β/CREB Pathway Improves the Responsiveness to AMPA Receptor Antagonists by Regulating GRIA1 Surface Expression in Chronic Epilepsy Rats

doi: 10.3390/biomedicines9040425

Figure Lengend Snippet: Primary antibodies used in the present study.

Article Snippet: p-CREB S133 , Rabbit , Novus biologicals (NB110-55727) , 1:5000 (WB).

Techniques:

Journal: Biomedicines

Article Title: Inhibition of AKT/GSK3β/CREB Pathway Improves the Responsiveness to AMPA Receptor Antagonists by Regulating GRIA1 Surface Expression in Chronic Epilepsy Rats

doi: 10.3390/biomedicines9040425

Figure Lengend Snippet: The effects of perampanel (PER) and GYKI 52466 (GYKI) on total Ca 2+ /cAMP response element-binding protein (CREB) expression and its S133 phosphorylation in chronic epilepsy rats. ( A ) Representative images for Western blot of CREB and its S133 phosphorylation level in the hippocampal tissues. ( B – D ) Quantifications of CREB (B), p-CREB S133 ( C ), and p-CREB/CREB ratio ( D ) in the hippocampal tissues. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM ( * ,# p < 0.05 vs. control (Cont) and vehicle (Veh)-treated animals, respectively; one-way ANOVA with post hoc Bonferroni’s multiple comparison).

Article Snippet: p-CREB S133 , Rabbit , Novus biologicals (NB110-55727) , 1:5000 (WB).

Techniques: Binding Assay, Expressing, Phospho-proteomics, Western Blot, Control, Comparison

Journal: Biomedicines

Article Title: Inhibition of AKT/GSK3β/CREB Pathway Improves the Responsiveness to AMPA Receptor Antagonists by Regulating GRIA1 Surface Expression in Chronic Epilepsy Rats

doi: 10.3390/biomedicines9040425

Figure Lengend Snippet: The effects of 3CAI co-treatment with perampanel (PER) and GYKI 52466 (GYKI) on expression levels of GSK3β, CREB, and PICK1 and the phosphorylation levels of GSK3β and CREB in non-responders. ( A ) Representative images for Western blot of total GSK3β, CREB, and PICK1 levels and the phosphorylation levels of GSK3β and CREB. ( B – H ) Quantifications of GSK3β ( B ), p-GSK3β S9 ( C ), p-GSK3β S9/GSK3β ratio ( D ), CREB ( E ), p-CREB S133 ( F ), p-CREB S133/CREB ratio ( G ), and PICK1 ( H ) in the hippocampal tissues. Open circles indicate each individual value. Horizontal bars indicate mean value. Error bars indicate SEM ( *, # p < 0.05 vs. control (Cont) and vehicle (Veh)-treated animals, respectively; one-way ANOVA with post hoc Bonferroni’s multiple comparison).

Article Snippet: p-CREB S133 , Rabbit , Novus biologicals (NB110-55727) , 1:5000 (WB).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Comparison