anti-phosphorylated creb (pcreb  (Merck & Co)

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Electroacupuncture Enhances Neuroplasticity by Regulating the Orexin A-Mediated cAMP/PKA/CREB Signaling Pathway in Senescence-Accelerated Mouse Prone 8 (SAMP8) Mice

doi: 10.1155/2022/8694462

Figure Lengend Snippet: EA at 10 Hz regulates the expression of 9 cAMP/PKA/CREB signaling pathway-related proteins in the hippocampus. (a–j) Western blot images and quantification of the protein levels of OxA, cAMP, pPKA/PKA, pCREB/CREB, GluN1, GluN2A, GluA2, SYP, and PSD95 in the hippocampus. The data are presented as the mean ± standard error of the mean ( n = 3; compared with the model group, ∗∗∗ P < 0.001 and ∗∗ P < 0.01; compared with the OxA group or 10 Hz EA group, ### P < 0.001, ## P < 0.01, and # P < 0.05; ns: not significant).

Article Snippet: ImageJ software was used to analyze the levels of OxA, cAMP, PKA, CREB, phosphorylated CREB (pCREB), GluN1, GluN2A, GluA2, synaptophysin (SYP), and postsynaptic density protein-95 (PSD95) (all antibodies are purchased from Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Electroacupuncture Enhances Neuroplasticity by Regulating the Orexin A-Mediated cAMP/PKA/CREB Signaling Pathway in Senescence-Accelerated Mouse Prone 8 (SAMP8) Mice

doi: 10.1155/2022/8694462

Figure Lengend Snippet: EA at 10 Hz improves hippocampal neuroplasticity in SAMP8 mice to alleviate learning and memory deficits. Inhibition of OxA expression promotes the expression of cAMP/PKA/CREB signaling pathway-related proteins, thus improving the synaptic plasticity of glutamate neurons and reversing learning and memory deficits.

Article Snippet: ImageJ software was used to analyze the levels of OxA, cAMP, PKA, CREB, phosphorylated CREB (pCREB), GluN1, GluN2A, GluA2, synaptophysin (SYP), and postsynaptic density protein-95 (PSD95) (all antibodies are purchased from Cell Signaling Technology, Danvers, MA).

Techniques: Inhibition, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: A Mixture of Ginkgo biloba L . Leaf and Hericium erinaceus (Bull.) Pers . Fruit Extract Attenuates Scopolamine-Induced Memory Impairments in Mice

doi: 10.1155/2022/9973678

Figure Lengend Snippet: Neuronal protective effect of the mixture (GH) of Ginkgo biloba L . leaf (GL) and Hericium erinaceus (Bull.) Pers . (HE) fruit extracts on scopolamine- (Sco-) induced SH-SY5Y neuroblastoma cells. (a–d) Western blotting assay of BDNF (a), pGSK3 β /GSK3 β (b), pERK/ERK (c), and pCREB/CREB (d) was carried out. GAPDH was used as a loading control. Quantification was calculated using densitometric analysis using Bio-Rad Quantity software. Data represent the mean ± SEM ( n = 3). # P < 0.05 and ## P < 0.01 vs. control group. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. Sco-treated group.

Article Snippet: Anti-Bax, anti-Bcl-2, anti-cleaved-caspase-3, anti-BDNF, anti-CREB, anti-phosphorylated CREB (pCREB), anti-ERK, anti-pERK, anti-protein kinase B (AKT), anti-pAKT, anti-glycogen synthase kinase 3 beta (GSK3 β ), anti-pGSK3 β , and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Western Blot, Control, Software

Journal: Oxidative Medicine and Cellular Longevity

Article Title: A Mixture of Ginkgo biloba L . Leaf and Hericium erinaceus (Bull.) Pers . Fruit Extract Attenuates Scopolamine-Induced Memory Impairments in Mice

doi: 10.1155/2022/9973678

Figure Lengend Snippet: Effect of the mixture (GH) of Ginkgo biloba L . leaf (GL) and Hericium erinaceus (Bull.) Pers . (HE) fruit extracts on the expressed levels of BDNF, pAKT, pERK, and pCREB in the brain. Whole brain from randomly selected mice in each group was analyzed for western blotting assay. (a–d) Western blotting assay of BDNF (a), pAKT/AKT (b), pERK/ERK (c), and pCREB/CREB (d) was carried out. GAPDH was used as a loading control. Quantification was performed using densitometric analysis with Bio-Rad Quantity software. Data represent the mean ± SEM ( n = 7). # P < 0.05 and ## P < 0.01 vs. control group. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. Sco-treated group.

Article Snippet: Anti-Bax, anti-Bcl-2, anti-cleaved-caspase-3, anti-BDNF, anti-CREB, anti-phosphorylated CREB (pCREB), anti-ERK, anti-pERK, anti-protein kinase B (AKT), anti-pAKT, anti-glycogen synthase kinase 3 beta (GSK3 β ), anti-pGSK3 β , and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Western Blot, Control, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: The Hallucinogenic Serotonin 2A Receptor Agonist, 2,5-Dimethoxy-4-Iodoamphetamine, Promotes cAMP Response Element Binding Protein-Dependent Gene Expression of Specific Plasticity-Associated Genes in the Rodent Neocortex

doi: 10.3389/fnmol.2021.790213

Figure Lengend Snippet: Acute treatment with the 5-HT 2A receptor agonist, DOI regulates neuronal plasticity-associated gene expression via the MAP kinase and CaMKII signaling pathways and enhances phosphorylated CREB (pCREB) expression in vitro. (A) Shown is a schematic of the treatment paradigm for cortical neurons derived from E17.5 rat embryos, allowed to differentiate till day in vitro (DIV) 10, following which neurons were treated with vehicle (DMSO) or the 5-HT 2A receptor agonist, DOI (10 μM), in the presence or absence of CaMKII and MAP kinase signaling pathway inhibitors (CaMKII inhibitor: KN-62; MAPKK inhibitor: U0126). (B) Shown is the relative mRNA expression for plasticity-associated genes following DOI treatment in the presence or absence of the 5-HT 2A receptor antagonist, MDL100,907 or PLC inhibitor, U73122, represented as fold change of vehicle ± SEM. (Representative results from n = 4 wells per treatment group/ N = 2, * p < 0.05 as compared to vehicle, @ p < 0.05 as compared to DOI, one-way ANOVA, Tukey’s post hoc test). (C) Shown is the relative mRNA expression for plasticity-associated genes following DOI treatment in the presence or absence of MAP kinase and CaMKII signaling pathway inhibitors, represented as fold change of vehicle ± SEM. (Representative results from n = 3–5 wells per treatment group/ N = 3, * p < 0.05 as compared to vehicle, @ p < 0.05 as compared to DOI, one-way ANOVA, Tukey’s post hoc test). (D) Shown is a schematic summarizing the putative signaling pathways that may contribute to DOI-induced gene expression. The CaMKII inhibitor, KN-62 and the MAPKK inhibitor, U0126 inhibit the CaMKII and MAP kinase signaling pathways respectively. The DOI-mediated upregulation of Arc , Bdnf1 , Cebpb , and Egr2 mRNA levels was blocked by both the MAPKK and CaMKII inhibitors, whereas the increase in cFos mRNA was blocked by the CaMKII, not the MAPKK, inhibitor and the upregulation of Egr1 mRNA was blocked by the MAPKK, not the CaMKII, inhibitor. (E) Shown are representative immunofluorescence images of rat cortical neurons in vitro with double staining for the neuronal marker MAP2 (red) and 5-HT 2A receptor (green). Scale bar: 30 μm. Magnification: 20X. (F) Shown are representative immunofluorescence images of rat cortical neurons with double staining for pCREB (green) and the neuronal marker MAP2 (red) – upper panel: Vehicle; lower panel: DOI. Scale bar: 30 μm. Magnification: 20X. (G–J) Shown are representative immunoblots for pCREB and CREB protein levels in rat cortical neurons treated with DOI (G) or with DOI in the presence or absence of the MAPKK inhibitor U0126 (H) or the CaMKII inhibitor KN-62 (I) . (J) Quantitative densitometric analysis of pCREB/CREB levels in rat cortical neurons treated with DOI in the presence or absence of U0126 or KN-62. Results are expressed as fold change of vehicle ± SEM. (Representative results from n = 3–5 wells per treatment group/ N = 3, * p < 0.05 as compared to vehicle, @ p < 0.05 as compared to DOI, one-way ANOVA, Tukey’s post hoc test). (K) Shown is a schematic depicting the putative pathway via which pCREB levels are enhanced following DOI administration, indicative of a role for the MAP kinase and CaMKII signaling pathways.

Article Snippet: The tissue was placed in a pre-chilled Dounce homogenizer, sonicated and immunoprecipitated using a phosphorylated CREB (pCREB) antibody (1 μg; Cell Signaling Technology, MA, United States).

Techniques: Gene Expression, Protein-Protein interactions, Expressing, In Vitro, Derivative Assay, Immunofluorescence, Double Staining, Marker, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: The Hallucinogenic Serotonin 2A Receptor Agonist, 2,5-Dimethoxy-4-Iodoamphetamine, Promotes cAMP Response Element Binding Protein-Dependent Gene Expression of Specific Plasticity-Associated Genes in the Rodent Neocortex

doi: 10.3389/fnmol.2021.790213

Figure Lengend Snippet: Acute treatment with DOI enhances both the expression of putative CRE-containing plasticity-associated genes and the enrichment of pCREB within the promoter regions of specific plasticity-associated genes in the neocortex of adult rats. (A) Shown is a schematic of the treatment paradigm wherein adult Sprague-Dawley rats were injected with vehicle or DOI (8 mg/kg) and were sacrificed 2 h following treatment. (B) The bar graph indicates the fold change in mRNA expression of specific plasticity-associated genes in the neocortex of vehicle and DOI-treated rats represented as fold change of vehicle ± SEM ( n = 4–6 per treatment group, * p < 0.05 as compared to vehicle, unpaired Students t -test). (C) Shown are the chromatin immunoprecipitation (ChIP) PCR amplicons with primer locations spanning putative CRE sequences in the upstream gene regulatory sequences for Arc , Bdnf1 , Cebpb , cFos , Egr1 , and Egr2 . (D) Shown is a bar graph for pCREB enrichment at the Arc , Bdnf1 , Cebpb , cFos , Egr1 , and Egr2 promoters based on ChIP analysis performed on tissue derived from the neocortex of vehicle and DOI treated adult rats. Results are expressed as the fold change of vehicle ± SEM. ( n = 7–10 animals per treatment group, * p < 0.05 as compared to vehicle, unpaired Students t -test).

Article Snippet: The tissue was placed in a pre-chilled Dounce homogenizer, sonicated and immunoprecipitated using a phosphorylated CREB (pCREB) antibody (1 μg; Cell Signaling Technology, MA, United States).

Techniques: Expressing, Injection, Chromatin Immunoprecipitation, Derivative Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: The Hallucinogenic Serotonin 2A Receptor Agonist, 2,5-Dimethoxy-4-Iodoamphetamine, Promotes cAMP Response Element Binding Protein-Dependent Gene Expression of Specific Plasticity-Associated Genes in the Rodent Neocortex

doi: 10.3389/fnmol.2021.790213

Figure Lengend Snippet: DOI-mediated regulation of plasticity-associated gene expression in cortical brain regions is perturbed in CREBαδ knockout (CREBαδ KO) mice. (A) Shown is a schematic of the acute treatment paradigm for wild type (WT) and CREBαδ KO mice, with vehicle (saline) or DOI (8 mg/kg) followed by sacrifice 2 h after treatment. (B) The bar graph depicts quantitative qPCR analysis for 5-HT 2A and 5-HT 2C receptors in the neocortex of WT and CREBαδ KO mice represented as fold change of WT ± SEM. ( n = 3–4 animals per treatment group). (C) The bar graph depicts the quantitation of head-twitch responses evoked in response to acute treatment with the 5-HT 2A receptor agonist, DOI or vehicle in both WT and CREBαδ KO mice ( n = 4 animals per treatment group). (D) Shown are representative autoradiographs for Arc mRNA expression in the neocortex from WT, WT + DOI, CREBαδ KO, and CREBαδ KO + DOI mice, with the outline inset indicating the somatosensory region. Shown are bar graphs for the quantitative densitometric analysis of levels of Arc mRNA expression in the somatosensory (E) and prefrontal (F) cortex following DOI or vehicle treatment to WT and CREBαδ KO mice. Results are represented as percentage of WT and are mean ± SEM ( n = 3–4 animals per group, * p < 0.05 as compared to WT + Veh mice, $ p < 0.05 as compared to CREBαδ KO + Veh mice, @ p < 0.05 as compared to WT + DOI mice, two-way ANOVA, Tukey’s post hoc test). (G–J) Bar graphs depict quantitation of qPCR analysis for mRNA expression of Bdnf1 (G) , Cebpb (H) , cFos (I) , and Egr2 (J) , following acute DOI or vehicle treatment to WT and CREBαδ KO mice. ( n = 3–4 animals per group, * p < 0.05 as compared to WT mice, $ p < 0.05 as compared to CREBαδ KO mice, @ p < 0.05 as compared to WT + DOI mice, two-way ANOVA, Tukey’s post hoc test).

Article Snippet: The tissue was placed in a pre-chilled Dounce homogenizer, sonicated and immunoprecipitated using a phosphorylated CREB (pCREB) antibody (1 μg; Cell Signaling Technology, MA, United States).

Techniques: Gene Expression, Knock-Out, Saline, Quantitation Assay, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: The Hallucinogenic Serotonin 2A Receptor Agonist, 2,5-Dimethoxy-4-Iodoamphetamine, Promotes cAMP Response Element Binding Protein-Dependent Gene Expression of Specific Plasticity-Associated Genes in the Rodent Neocortex

doi: 10.3389/fnmol.2021.790213

Figure Lengend Snippet: Schematic depicting the putative mechanism for the CREB-dependent regulation of neuronal plasticity-associated gene expression by the hallucinogenic 5-HT 2A receptor agonist DOI. DOI, a hallucinogenic agonist of the 5-HT 2A receptor is known to evoke a specific transcriptome signature within the neocortex, including the upregulation of the expression of several plasticity-associated genes. The schematic indicates a putative mechanism through which DOI-mediated stimulation of the Gq-coupled 5-HT 2A receptor results in the recruitment of the phospholipase C (PLC), MAP kinase and CaMKII signaling pathways, which would further result in the phosphorylation of the transcription factor CREB, thus facilitating the CREB-dependent transcription of plasticity-associated genes, Arc, Bdnf1 , Cebpb , cFos . This raises the intriguing possibility that CREB-dependent regulation of gene expression could contribute to the effects of the hallucinogenic 5-HT 2A receptor agonist DOI, on neuronal plasticity, synaptogenesis and cell survival.

Article Snippet: The tissue was placed in a pre-chilled Dounce homogenizer, sonicated and immunoprecipitated using a phosphorylated CREB (pCREB) antibody (1 μg; Cell Signaling Technology, MA, United States).

Techniques: Gene Expression, Expressing, Protein-Protein interactions, Phospho-proteomics