rabbit anti-camp response element-binding protein (creb)  (Cell Signaling Technology Inc)

Journal: Neurochemical Research

Article Title: Oxytocin Protects Against Corticosterone-Induced DA Dysfunction: An Involvement of the PKA/CREB Pathway

doi: 10.1007/s11064-024-04294-7

Figure Lengend Snippet: Proposed mechanism underlying the neuroprotective effects of OXT against CORT-induced dopamine dysfunction in SH-SY5Y cells. OXT binds to its receptor, resulting in augmented stimulation of cAMP, which serves as an activator of PKA. Subsequently, PKA phosphorylates TH at Ser40 and CREB at Ser133. Phosphorylated CREB can bind to downstream coactivators to orchestrate the transcriptional activity of its target, including the TH gene. This regulatory process contributes to the protective effects of OXT against CORT-induced toxicity, thereby conferring neuroprotection. (Created with BioRender.com/Mahidol University)

Article Snippet: Polyclonal rabbit anti-phospho-tyrosine hydroxylase (Ser40) antibody (pTH, Cat. No. 2791 S), monoclonal rabbit anti-phospho-cAMP-response-element-binding-protein (Ser133) antibody (pCREB; Cat. No. 9198 S), and polyclonal rabbit anti-catalytic-protein kinase A-α subunit antibody (PKAc-α; (Cat. No. 4782 S) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay

Journal: Antioxidants

Article Title: Ecklonia cava Ameliorates Cognitive Impairment on Amyloid β-Induced Neurotoxicity by Modulating Oxidative Stress and Synaptic Function in Institute of Cancer Research (ICR) Mice

doi: 10.3390/antiox13080951

Figure Lengend Snippet: List of primary and secondary antibodies information used in this study.

Article Snippet: p-cAMP-response element binding protein (p-CREB) , #9198 , Cell Signaling Tech.

Techniques: Binding Assay

Journal: Antioxidants

Article Title: Ecklonia cava Ameliorates Cognitive Impairment on Amyloid β-Induced Neurotoxicity by Modulating Oxidative Stress and Synaptic Function in Institute of Cancer Research (ICR) Mice

doi: 10.3390/antiox13080951

Figure Lengend Snippet: Effect of 70% ethanol extract of Ecklonia cava (EE) on BDNF/AKT/CREB signaling pathway in Aβ-induced mice. Western blot band images ( a ). Relative expressions of BDNF ( b ), TrkB ( c ), PI3K ( d ), p-AKT ( e ), and p-CREB ( f ) on the corresponding quantitation to β-actin. Data are represented as mean ± SD ( n = 3). Different lowercase letters on the bar graph indicate significant differences between groups ( p < 0.05).

Article Snippet: p-cAMP-response element binding protein (p-CREB) , #9198 , Cell Signaling Tech.

Techniques: Western Blot, Quantitation Assay

Journal: Stress Biology

Article Title: β-Hydroxybutyric acid improves cognitive function in a model of heat stress by promoting adult hippocampal neurogenesis

doi: 10.1007/s44154-022-00079-6

Figure Lengend Snippet: BHBA activates the Akt-CREB-BDNF and MeCP2 pathways in the heat stress-treated mice. A Representative western blot bands showing the relative protein expression of p-Akt, Akt, p-CREB, CREB, MeCP2, and BDNF. B - E Quantitative analysis showing the ratio of p-Akt/Akt ( B ), p-CREB/CREB ( C ), and the level of MeCP2 ( D ) and BDNF ( E ). Note that the phosphorylation of Akt and CREB and the expression of MeCP2 and BDNF were decreased in the heat stress-treated group, compared to the control group. However, BHBA treatment increased these protein levels in the heat stress-treated mice. Data were normalized to β-actin: expression of the target protein = intensity of target protein band/intensity of β-actin. The results are presented as the ratio of the experimental group to the control group with the value taken as 1. All data analyses were performed using one-way ANOVA with Tukey’s post-test. Data are represented as mean ± SEM. B-E n = 8. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Akt, protein kinase B; B, BHBA; BDNF, brain-derived neurotrophic factor; CREB, cAMP response element binding protein; M, minocycline; MeCP2, methyl-CpG binding protein 2

Article Snippet: The following primary antibodies were used: rabbit anti-spinophilin (1:1000; ab18561, Abcam, Cambridge, MA, USA), mouse anti-synaptophysin (1:1000; ab32127, Abcam, Cambridge, MA, USA), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; #2507, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-eukaryotic initiation factor 2α (eIF2α; 1:1000; ab115822, Abcam, Cambridge, MA, USA), rabbit anti-phospho-eIF2α (Ser51; p-eIF2α; 1:1000; ab32157, Abcam, Cambridge, MA, USA), rabbit anti-C/EBP homologous protein (CHOP; 1:1000; BM4962, BOATER, Wuhan, China), rabbit anti-glucose-regulated protein 78 (GRP78; 1:1000; A0241, ABclonal, Wuhan, China), rabbit anti-activating transcription factor 4 (ATF4; 1:1000; BM5179, BOSTER, Wuhan, China), rabbit anti-caspase-3 (1:1000; #9662, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-cleaved caspase-3 (1:1000; #9661, Cell Signaling Technology, Danvers, MA, USA), rabbit anti- protein kinase B (Akt; 1:1000; A17909, ABclonal, Wuhan, China), rabbit anti-phospho-Akt (Ser473; p-Akt; 1:1000; T40067, Abmart, Shanghai, China), rabbit anti-cAMP response element binding protein (CREB; 1:1000; #9197, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-CREB (Ser133; p-CREB; 1:1000; #9198, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-methyl CpG binding protein 2 (MeCP2; 1:1000; #3456, Cell Signaling Technology, Danvers, MA, USA), mouse anti-BDNF (1:1000; ab205067, Abcam, Cambridge, MA, USA), mouse anti-β-actin (1:1000; #3700, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Expressing, Derivative Assay, Binding Assay

Journal: Molecular Vision

Article Title: Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells

doi:

Figure Lengend Snippet: High NaCl-induced gene expression of various members of the Jun, Fos, ATF, JDP, and CREB protein families. A : Presence of gene transcripts in RPE cells. To confirm the correct lengths of the PCR products, agarose gel electrophoresis was performed using products obtained from two RPE cell lines (1, 2) derived from different post-mortem donors. Negative controls (0) were performed by adding double-distilled water instead of cDNA as template. The β-actin ( ACTB ) mRNA level was used to normalize the c-Fos and c-Jun mRNA levels. B : Gene expression levels after the cells were stimulated for 2, 6, and 24 h (as indicated by the panels of the bars) with high (+ 100 mM) NaCl. mRNA levels were determined with real-time reverse transcription (RT)–PCR and are expressed as folds of the unstimulated control. Each bar represents data obtained in four independent experiments using cell lines from different donors. Significant difference versus unstimulated control: * p <0.05.

Article Snippet: The following antibodies were used: a rabbit anti-human β-actin (1:1,000; Cell Signaling, Frankfurt, Germany), a rabbit anti-c-Fos (1:1,000; Cell Signaling), a rabbit anti-c-Jun (1:1,000; Cell Signaling), a rabbit anti-phosphorylated c-Fos (1:1,000; ThermoFisher Scientific, Waltham, MA), a rabbit anti-phosphorylated c-Jun (1:1,000; Cell Signaling), a rabbit anti-cAMP response element-binding protein (CREB; 1:1,000; Cell Signaling), a rabbit anti-histone H3 (1:1,000; Cell Signaling), and anti-rabbit immunoglobulin G (IgG) conjugated with alkaline phosphatase (1:2,000; Cell Signaling).

Techniques: Expressing, Agarose Gel Electrophoresis, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Vision

Article Title: Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells

doi:

Figure Lengend Snippet: High extracellular NaCl induces elevation in the levels of c-Fos and phosphorylated c-Jun proteins in RPE cells. The cells were stimulated for 2, 4, and 6 h with high (+ 100 mM) NaCl or the hypoxia mimetic CoCl 2 (150 µM). Protein levels were determined with western blot analysis of cell lysates ( A – C ) and nuclear extracts ( D ). A : Example of a western blot analysis in one cell line. Left: The levels of the following proteins were determined: β-actin, c-Fos, phosphorylated c-Fos (P-c-Fos), c-Jun, and phosphorylated c-Jun (P-c-Jun). Right: Negative control obtained with the omission of the first antibodies. B : Cytosolic levels of c-Fos ( left ) and phosphorylated c-Fos proteins ( right ), as determined with densitometric analysis of western blot data. C : Cytosolic levels of c-Jun ( left ) and phosphorylated c-Jun proteins ( right ). The data are normalized to the level of the β-actin protein and are expressed as a percentage of the unstimulated control (100%). Each bar represents data obtained in three to six independent experiments using cell lines from different donors. Significant difference versus unstimulated control: * p<0.05. D : Examples of western blot analysis of the nuclear extracts. The cells were stimulated for 2 and 6 h with high (+ 100 mM) NaCl. Note that the nuclear levels of histone H3 (His H3) and CREB proteins did not change in response to high NaCl.

Article Snippet: The following antibodies were used: a rabbit anti-human β-actin (1:1,000; Cell Signaling, Frankfurt, Germany), a rabbit anti-c-Fos (1:1,000; Cell Signaling), a rabbit anti-c-Jun (1:1,000; Cell Signaling), a rabbit anti-phosphorylated c-Fos (1:1,000; ThermoFisher Scientific, Waltham, MA), a rabbit anti-phosphorylated c-Jun (1:1,000; Cell Signaling), a rabbit anti-cAMP response element-binding protein (CREB; 1:1,000; Cell Signaling), a rabbit anti-histone H3 (1:1,000; Cell Signaling), and anti-rabbit immunoglobulin G (IgG) conjugated with alkaline phosphatase (1:2,000; Cell Signaling).

Techniques: Western Blot, Negative Control

Journal: Molecular Vision

Article Title: Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells

doi:

Figure Lengend Snippet: High extracellular NaCl induces DNA binding of AP-1 in RPE cells, as indicated by the increased level of the complexes of AP-1 protein and labeled oligonucleotides in electrophoretic mobility shift assay (EMSA; above and middle ). Such an increase was not observed under isoosmotic conditions. High NaCl did not induce an increase in the level of the cAMP response element-binding protein (CREB)/oligonucleotide complexes ( below ). The cells were stimulated with the addition of 100 mM NaCl to the culture medium for 2 and 6 h. Control blots were obtained with nuclear extracts of cells cultured for 6 h. The addition of an excess of unlabeled oligonucleotides (Competitor) was used to show the specificity of the AP-1- and CREB/oligonucleotide complexes. Similar results were obtained in four independent experiments using cells from different donors.

Article Snippet: The following antibodies were used: a rabbit anti-human β-actin (1:1,000; Cell Signaling, Frankfurt, Germany), a rabbit anti-c-Fos (1:1,000; Cell Signaling), a rabbit anti-c-Jun (1:1,000; Cell Signaling), a rabbit anti-phosphorylated c-Fos (1:1,000; ThermoFisher Scientific, Waltham, MA), a rabbit anti-phosphorylated c-Jun (1:1,000; Cell Signaling), a rabbit anti-cAMP response element-binding protein (CREB; 1:1,000; Cell Signaling), a rabbit anti-histone H3 (1:1,000; Cell Signaling), and anti-rabbit immunoglobulin G (IgG) conjugated with alkaline phosphatase (1:2,000; Cell Signaling).

Techniques: Binding Assay, Labeling, Electrophoretic Mobility Shift Assay, Cell Culture

Journal: Molecular Vision

Article Title: Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells

doi:

Figure Lengend Snippet: Transcription factor activities involved in mediating NaCl-induced expression of the c-Fos gene in RPE cells. mRNA levels were determined with real-time reverse transcription (RT)–PCR analysis in cells cultured for 2 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and are expressed as folds of the unstimulated control. A : The level of c-Fos mRNA was determined in cells cultured in the presence of the following inhibitory compounds: the AP-1 inhibitor SR11302 (5 µM), the cAMP response element-binding protein (CREB) inhibitor 666–15 (250 nM), a HIF inhibitor (HIF-Inh; 5 µM), the STAT3 inhibitor Stattic (1 µM), and the nuclear factor (NF)-κB inhibitor CAPE (5 µM). B, C : mRNA levels were determined in cells transfected with nuclear factor of activated T cell 5 (NFAT5) siRNA (siNFAT5; 5 nM) and non-targeted siRNA (siNon; 5 nM), respectively. B : Transfection of RPE cells with NFAT5 siRNA for 48 h resulted in a reduction of the NFAT5 mRNA level in cells cultured for 2 h in iso- and hyperosmotic media. Non-targeted siRNA had no effects. As negative control, transfection reagent (TR) without siRNA was tested. C : Knocking down NFAT5 with siRNA reduced the level of c-Fos mRNA under hyperosmotic conditions. Each bar represents data obtained in three to nine independent experiments using cell lines from different donors. Significant difference versus unstimulated control: *p<0.05. Significant difference versus NaCl control: ●p<0.05. ○ p<0.05.

Article Snippet: The following antibodies were used: a rabbit anti-human β-actin (1:1,000; Cell Signaling, Frankfurt, Germany), a rabbit anti-c-Fos (1:1,000; Cell Signaling), a rabbit anti-c-Jun (1:1,000; Cell Signaling), a rabbit anti-phosphorylated c-Fos (1:1,000; ThermoFisher Scientific, Waltham, MA), a rabbit anti-phosphorylated c-Jun (1:1,000; Cell Signaling), a rabbit anti-cAMP response element-binding protein (CREB; 1:1,000; Cell Signaling), a rabbit anti-histone H3 (1:1,000; Cell Signaling), and anti-rabbit immunoglobulin G (IgG) conjugated with alkaline phosphatase (1:2,000; Cell Signaling).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Binding Assay, Transfection, Negative Control