rabbit anti-phospho-ser133-creb Cell Signaling Technology Inc Search Results


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  • 97
    Cell Signaling Technology Inc phospho creb
    EPA reversed the inhibitory effects of IL-1β on the expression of BDNF via the <t>Akt/CREB</t> pathways in cultured rat hippocampal neurons. a BDNF mRNA and protein, b expression were measured by real-time PCR and western blotting, respectively. c Relative levels of BDNF protein were determined by densitometry of the immunoblots. Data from PCR and western blotting were normalized by taking the value of the control group as 1. ** P
    Phospho Creb, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1227 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc rabbit anti phospho creb
    The effect of N-methyl-D-aspartate receptor (NMDAR) blockade on the long-term plasticity, gene expression and protein synthesis. (A) In the presence of 2-amino-5-phosphonovaleric acid (APV), the plasticity map (that was obtained as in Figure 1 ) shows that TBS does no longer induce synaptic plasticity. The histogram confirms the almost complete absence of changes in the slice. (B) In the presence of APV, the pseudocolor maps of <t>P-CREB</t> and Creb levels (that were obtained as in Figure 2 ) do not show remarkable differences between a control slice and a slice that has received TBS. Histograms show that no significant differences occur between control slices and slices that have received TBS (mean and SEM, n = 4; 120 min). (C) In the presence of APV, the pseudocolor maps of <t>c-FOS</t> and c-Fos levels (that were obtained as in Figure 2 ) do not show remarkable differences between a control slice and a slice that has received TBS. Histograms show that no significant differences occur between control slices and slices that have received TBS (mean and SEM, n = 4; 120 min).
    Rabbit Anti Phospho Creb, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc rabbit anti phospho creb ser133
    Development-dependent retinal <t>CREB</t> activity is mediated by cGKII in vivo . Phospho-CREB and cGKII colocalize in retinal neurons. Cultured purified neurons ( a .1) or retinal sections ( a .2) were stained against CREB (green (E6) or cyan (E8)) and cGKII (red (E6) or magenta (E8)) and observed in a confocal microscope. Calibration bars=10 μ m in a .1 and 50 μ m in a .2. In b and c , NO decreases or increases nuclear CREB activity via cGKII. E6C3 and E8C3 (respectively) mixed retinal cultures were immunostained against pCREB <t>Ser133</t> and processed for nuclear colocalization with DAPI. In d , cGKII is knocked down in vivo . Lentiviruses coding for cGKII shRNAs or the empty vector pLKO were injected in the air chamber of chicken eggs with E5 embryos and incubated up to E9–E11. Retinas were immunostained for cGKII. Knockdown51.5% in E5–E9 and 78.3% in E8–E11. Calibration bar=50 μ m. In e – k , NO differentially regulates phospho-CREB in retinal layers via cGKII. Lentiviruses coding for cGKII shRNAs were injected (as in d ), and then SNAP was administrated at E6 (upper panels) or at E8 (bottom panels). Eggs were incubated for further 3 days (E9 or E11, respectively) and pCREB immunohistochemistry was performed. Calibration bars=50 μ m. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. In k , NO regulates in vivo retinal tissue apoptotic labeling via cGKII. cGKII shRNAs were injected (as above), and then SNAP was administrated at E6 (upper) or at E8 (bottom). Cleaved caspase-3 staining=red staining in E6–E9 and green staining in E8-E11. Calibration bars=50 μ m. Data represent the mean±S.E.M. (error bars). * P
    Rabbit Anti Phospho Creb Ser133, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc rabbit anti phospho creb ser133 antibody
    Development-dependent retinal <t>CREB</t> activity is mediated by cGKII in vivo . Phospho-CREB and cGKII colocalize in retinal neurons. Cultured purified neurons ( a .1) or retinal sections ( a .2) were stained against CREB (green (E6) or cyan (E8)) and cGKII (red (E6) or magenta (E8)) and observed in a confocal microscope. Calibration bars=10 μ m in a .1 and 50 μ m in a .2. In b and c , NO decreases or increases nuclear CREB activity via cGKII. E6C3 and E8C3 (respectively) mixed retinal cultures were immunostained against pCREB <t>Ser133</t> and processed for nuclear colocalization with DAPI. In d , cGKII is knocked down in vivo . Lentiviruses coding for cGKII shRNAs or the empty vector pLKO were injected in the air chamber of chicken eggs with E5 embryos and incubated up to E9–E11. Retinas were immunostained for cGKII. Knockdown51.5% in E5–E9 and 78.3% in E8–E11. Calibration bar=50 μ m. In e – k , NO differentially regulates phospho-CREB in retinal layers via cGKII. Lentiviruses coding for cGKII shRNAs were injected (as in d ), and then SNAP was administrated at E6 (upper panels) or at E8 (bottom panels). Eggs were incubated for further 3 days (E9 or E11, respectively) and pCREB immunohistochemistry was performed. Calibration bars=50 μ m. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. In k , NO regulates in vivo retinal tissue apoptotic labeling via cGKII. cGKII shRNAs were injected (as above), and then SNAP was administrated at E6 (upper) or at E8 (bottom). Cleaved caspase-3 staining=red staining in E6–E9 and green staining in E8-E11. Calibration bars=50 μ m. Data represent the mean±S.E.M. (error bars). * P
    Rabbit Anti Phospho Creb Ser133 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit anti phospho creb ser 133 antibody
    Development-dependent retinal <t>CREB</t> activity is mediated by cGKII in vivo . Phospho-CREB and cGKII colocalize in retinal neurons. Cultured purified neurons ( a .1) or retinal sections ( a .2) were stained against CREB (green (E6) or cyan (E8)) and cGKII (red (E6) or magenta (E8)) and observed in a confocal microscope. Calibration bars=10 μ m in a .1 and 50 μ m in a .2. In b and c , NO decreases or increases nuclear CREB activity via cGKII. E6C3 and E8C3 (respectively) mixed retinal cultures were immunostained against pCREB <t>Ser133</t> and processed for nuclear colocalization with DAPI. In d , cGKII is knocked down in vivo . Lentiviruses coding for cGKII shRNAs or the empty vector pLKO were injected in the air chamber of chicken eggs with E5 embryos and incubated up to E9–E11. Retinas were immunostained for cGKII. Knockdown51.5% in E5–E9 and 78.3% in E8–E11. Calibration bar=50 μ m. In e – k , NO differentially regulates phospho-CREB in retinal layers via cGKII. Lentiviruses coding for cGKII shRNAs were injected (as in d ), and then SNAP was administrated at E6 (upper panels) or at E8 (bottom panels). Eggs were incubated for further 3 days (E9 or E11, respectively) and pCREB immunohistochemistry was performed. Calibration bars=50 μ m. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. In k , NO regulates in vivo retinal tissue apoptotic labeling via cGKII. cGKII shRNAs were injected (as above), and then SNAP was administrated at E6 (upper) or at E8 (bottom). Cleaved caspase-3 staining=red staining in E6–E9 and green staining in E8-E11. Calibration bars=50 μ m. Data represent the mean±S.E.M. (error bars). * P
    Rabbit Anti Phospho Creb Ser 133 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Cell Signaling Technology Inc rabbit anti phosphorylated creb
    Immunohistochemistry for <t>pCREB</t> in the dentate gyrus of control ( a ), vehicle- ( b ), control-HSP70- ( c ), and Tat-HSP70-treated ( d ) mice. In all groups, pCREB-positive nuclei (arrows) are mainly observed in the subgranular zone of the dentate gyrus. Note that pCREB-positive nuclei are strongly observed in the dentate gyrus of Tat-HSP70-treated mice. GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Scale bar = 50 μm. e The number of pCREB-positive nuclei per section for each group are shown and f values from western blot analysis is expressed as a ratio of pCREB and <t>CREB</t> immunoblot band in control group ( n = 5 per group; a P
    Rabbit Anti Phosphorylated Creb, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    EPA reversed the inhibitory effects of IL-1β on the expression of BDNF via the Akt/CREB pathways in cultured rat hippocampal neurons. a BDNF mRNA and protein, b expression were measured by real-time PCR and western blotting, respectively. c Relative levels of BDNF protein were determined by densitometry of the immunoblots. Data from PCR and western blotting were normalized by taking the value of the control group as 1. ** P

    Journal: BMC Neuroscience

    Article Title: Involvement of Akt/CREB signaling pathways in the protective effect of EPA against interleukin-1β-induced cytotoxicity and BDNF down-regulation in cultured rat hippocampal neurons

    doi: 10.1186/s12868-018-0455-7

    Figure Lengend Snippet: EPA reversed the inhibitory effects of IL-1β on the expression of BDNF via the Akt/CREB pathways in cultured rat hippocampal neurons. a BDNF mRNA and protein, b expression were measured by real-time PCR and western blotting, respectively. c Relative levels of BDNF protein were determined by densitometry of the immunoblots. Data from PCR and western blotting were normalized by taking the value of the control group as 1. ** P

    Article Snippet: Akt (catalog No. 9272), phospho-Akt (catalog No. 5012S, Ser473), CREB (catalog No. 4820) and phospho-CREB (catalog No. 9198, Ser133) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction

    The effect of EPA on Akt and CREB phosphorylation was blocked by inhibition of the Akt signal, in the presence of IL-1β in cultured rat hippocampal neurons. a Cells pretreated with KRX-0401 and then treated with EPA and IL-1β, and the proteins expression was measured by western blotting. b Relative levels of proteins were determined by densitometry of the immunoblots. Data were normalized by taking the value of the control group as 1. ** P

    Journal: BMC Neuroscience

    Article Title: Involvement of Akt/CREB signaling pathways in the protective effect of EPA against interleukin-1β-induced cytotoxicity and BDNF down-regulation in cultured rat hippocampal neurons

    doi: 10.1186/s12868-018-0455-7

    Figure Lengend Snippet: The effect of EPA on Akt and CREB phosphorylation was blocked by inhibition of the Akt signal, in the presence of IL-1β in cultured rat hippocampal neurons. a Cells pretreated with KRX-0401 and then treated with EPA and IL-1β, and the proteins expression was measured by western blotting. b Relative levels of proteins were determined by densitometry of the immunoblots. Data were normalized by taking the value of the control group as 1. ** P

    Article Snippet: Akt (catalog No. 9272), phospho-Akt (catalog No. 5012S, Ser473), CREB (catalog No. 4820) and phospho-CREB (catalog No. 9198, Ser133) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Inhibition, Cell Culture, Expressing, Western Blot

    Expression and activation state of signalling mediators involved in regulation of smooth muscle tone. Western blot analysis was performed for cortical and subcortical brain arterioles from 13 sheep, as described in the methods section. Differences of expression levels of signalling proteins involved in control of smooth muscle tone: (A) nNOS, P-eNOS and eNOS-protein, (B) P-CREB and CREB protein, (C) P-ERK and ERK protein was detected in relation to β-actin. As these data were not normally distributed they are presented as box plots, where boxes represent 25th and 75th percentiles, respectively. Medians are indicated by horizontal lines. Whiskers indicate 10th and 90th percentiles, respectively. 1+2, samples from two different sheep; Cx, cortex; Scx, subcortex; AU, arbitrary units; Ref. reference sample.

    Journal: PLoS ONE

    Article Title: Underlying mechanism of subcortical brain protection during hypoxia and reoxygenation in a sheep model - Influence of α1-adrenergic signalling

    doi: 10.1371/journal.pone.0196363

    Figure Lengend Snippet: Expression and activation state of signalling mediators involved in regulation of smooth muscle tone. Western blot analysis was performed for cortical and subcortical brain arterioles from 13 sheep, as described in the methods section. Differences of expression levels of signalling proteins involved in control of smooth muscle tone: (A) nNOS, P-eNOS and eNOS-protein, (B) P-CREB and CREB protein, (C) P-ERK and ERK protein was detected in relation to β-actin. As these data were not normally distributed they are presented as box plots, where boxes represent 25th and 75th percentiles, respectively. Medians are indicated by horizontal lines. Whiskers indicate 10th and 90th percentiles, respectively. 1+2, samples from two different sheep; Cx, cortex; Scx, subcortex; AU, arbitrary units; Ref. reference sample.

    Article Snippet: These include: anti-NOS1 (nNOS; sc-8309 (H-299)) and anti-NOS3 (eNOS; sc-8311 (H-159)), both from Santa Cruz Biotechnology (Dallas, TX, USA); anti-phospho-eNOS (P-Ser1177; 9570), anti-CREB (rabbit monoclonal 48H2; 9197), anti-phospho-CREB (P-Ser133; rabbit monoclonal 87G3; 9198), anti-ERK1/2 (rabbit monoclonal 137F5; 4695) and anti-phospho-ERK1/2 (P-Thr202/Tyr204; rabbit monoclonal D13.14.4; 4370), all from Cell Signaling Technologies (Frankfurt am Main, Germany).

    Techniques: Expressing, Activation Assay, Western Blot

    Effect of rolipram on phosphorylated CREB (pCREB) and total (tCREB) expression in the testis after pelvic irradiation (IR) (2 Gy). (A) Western blot analysis of pCREB and tCREB expression after 12 hours and 35 days following pelvic irrdiation (2 Gy). Values are reported as the mean±standard deviation of five mice in each group (*p

    Journal: The World Journal of Men's Health

    Article Title: Protective Effect of Administered Rolipram against Radiation-Induced Testicular Injury in Mice

    doi: 10.5534/wjmh.2015.33.1.20

    Figure Lengend Snippet: Effect of rolipram on phosphorylated CREB (pCREB) and total (tCREB) expression in the testis after pelvic irradiation (IR) (2 Gy). (A) Western blot analysis of pCREB and tCREB expression after 12 hours and 35 days following pelvic irrdiation (2 Gy). Values are reported as the mean±standard deviation of five mice in each group (*p

    Article Snippet: The sections were hydrated and blocked in a normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA, USA) for 60 minutes, and the testis sections were incubated with rabbit monoclonal anti-phosphorylated CREB (pCREB) (Ser133; 87G3; 1:200; Cell Signaling Technology, Beverly, MA, USA) and rabbit monoclonal anti-Ki-67 (DRM004; 1:200; Acris Antibodies GmbH, Herford, Germany) antibodies at 4℃ overnight.

    Techniques: Expressing, Irradiation, Western Blot, Standard Deviation, Mouse Assay

    Effects of CSPGs on activities of p90RSK, CREB and LKB1 in N2A and CGN cultures. PTPσ/LAR transfected N2A cells or CGNs derived from PTPσ or LAR KO mice were treated with purified CSPGs (1.5 μg/ml) at several time points and the levels of phosphorylated p90RSK (p-p90RSK Thr573, active form), CREB (p-CREB Ser133, active form) and LKB1 (p-LKB1 Ser431, active form) in the supernatants of cell lysates were measured by Western blots. CSPGs did not alter levels of p-p90RSK in either PTPσ or LAR transfected N2A cells ( a ). CSPGs significantly decreased levels of p-CREB in PTPσ, not LAR, transfected N2A cells ( b ) and in PTPσ+/+, not PTPσ−/−, CGNs ( c ). In contrast, CSPGs stimulation decreased levels of p-LKB1 in LAR, not PTPσ, transfected N2A cells ( d ) and in LAR+/+, not LAR−/−, CGNs ( e ). The full-length blots are included in the Supplementary Information file.

    Journal: Scientific Reports

    Article Title: Two PTP receptors mediate CSPG inhibition by convergent and divergent signaling pathways in neurons

    doi: 10.1038/srep37152

    Figure Lengend Snippet: Effects of CSPGs on activities of p90RSK, CREB and LKB1 in N2A and CGN cultures. PTPσ/LAR transfected N2A cells or CGNs derived from PTPσ or LAR KO mice were treated with purified CSPGs (1.5 μg/ml) at several time points and the levels of phosphorylated p90RSK (p-p90RSK Thr573, active form), CREB (p-CREB Ser133, active form) and LKB1 (p-LKB1 Ser431, active form) in the supernatants of cell lysates were measured by Western blots. CSPGs did not alter levels of p-p90RSK in either PTPσ or LAR transfected N2A cells ( a ). CSPGs significantly decreased levels of p-CREB in PTPσ, not LAR, transfected N2A cells ( b ) and in PTPσ+/+, not PTPσ−/−, CGNs ( c ). In contrast, CSPGs stimulation decreased levels of p-LKB1 in LAR, not PTPσ, transfected N2A cells ( d ) and in LAR+/+, not LAR−/−, CGNs ( e ). The full-length blots are included in the Supplementary Information file.

    Article Snippet: Sources of compounds Antibodies against the following proteins were used: mouse mAb phospho-Akt (Ser473, 587F11), rabbit mAb phospho-S6 ribosomal protein (Ser235/236), rabbit mAb phospho-Erk1/2 (p44/42), rabbit pAb phospho-CRMP2 (Thr514), rabbit pAb phospho-cofilin (Ser3), rabbit mAb phospho-4E- BP1 (eukaryotic initiation factor 4E-binding protein 1, Thr37/46, 236B4), rabbit mAb phospho-CREB (Ser133, 87G3), rabbit mAb phospho-PKA C (Thr197, D45D3), rabbit pAb phospho-90 kDa ribosomal S6 kinase (p90RSK, Thr573), rabbit pAb phospho-PKCζ/λ (Thr410/403), rabbit mAb phospho-PKC (pan) (zeta Thr410, 190D10), rabbit mAb phospho-LKB1 (Ser428, C67A3) (all from Cell Signaling Technology), rabbit pAb phospho-MAP1B (Thr1265, Millipore), rabbit pAb phospho-APC (Ser2054, Abcam), mouse anti-actin clone C4 (MP Biomedicals) and mouse mAb against RhoA (sc-418; from Santa Cruz Biotechnology).

    Techniques: Transfection, Derivative Assay, Mouse Assay, Purification, Western Blot

    Model for the regulation and functions of CREB activity under hypoxic conditions in nucleus and mitochondria Hypoxia increases the activity of CREB (phosphorylation at Ser133 but not Ser121) by the MEK-ERK pathway, leading to an increased expression of pro-angiogenic factors (VEGF). This mechanism can counteract hypoxia. Hyperphosphorylation of CREB under hypoxia causes protein degradation by ubiquitination, while CREB can be stabilizied by SUMOylation. Import of modified CREB into mitochondria is enhanced under decreased oxygen supply, which in return can promote mitochondrial biogenesis and mitochondrial functions by binding to the mitochondrial promoter (D-LOOP) as well as regulating the expression of Ppargc1a.

    Journal: Oncotarget

    Article Title: Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization

    doi: 10.18632/oncotarget.10474

    Figure Lengend Snippet: Model for the regulation and functions of CREB activity under hypoxic conditions in nucleus and mitochondria Hypoxia increases the activity of CREB (phosphorylation at Ser133 but not Ser121) by the MEK-ERK pathway, leading to an increased expression of pro-angiogenic factors (VEGF). This mechanism can counteract hypoxia. Hyperphosphorylation of CREB under hypoxia causes protein degradation by ubiquitination, while CREB can be stabilizied by SUMOylation. Import of modified CREB into mitochondria is enhanced under decreased oxygen supply, which in return can promote mitochondrial biogenesis and mitochondrial functions by binding to the mitochondrial promoter (D-LOOP) as well as regulating the expression of Ppargc1a.

    Article Snippet: For evaluation of localization of CREB in human breast cancer tissue, formalin-fixed paraffin embedded tissue samples that contained necrotic areas indicative of localized hypoxia were stained with the phospho-CREB (Ser133) (87G3) Rabbit mAb (Cell Signaling) in the indicated concentration using the Ventana Discovery Autostainer (Ventana).

    Techniques: Activity Assay, Expressing, Modification, Binding Assay

    Hypoxia-mediated post-transcriptional modification and altered distribution of CREB A. Comparative analysis of different pCREB and CREB modifications under normoxia and early phase hypoxia (up to 12 h). Western blot analyses were performed as described using CREB and pCREB Ser133 -specific mAb. The size of the modified CREB protein is given. The blots represent one of three biological replicates. Extracellular pH (pH [ex]) of the cell free media was directly measured with a pH electrode after harvesting the cells. B. CREB phosphorylation at serine residue 133 and 121 as well as CREB protein expression was analysed under late phase hypoxia (up to 5 d). The photos represent one of two independent biological replicates. The pH of the culture media was measured with a pH electrode directy after harvesting the cells. C. , D. Ubiquitination was analysed by CREB immune precipitation (C) and ubiquitin pull down (D) as described in Materials and Methods by loading the complete supernatant on 10% (C) or 12% (D) gels. The proteins were identified by using anti-CREB- or anti-ubiquitin-specific mAb. Results represent data of three (C) or two (D) biological replicates. E. Influence of the proteasome inhibitor MG-132 and the ubiquitin inhibitor PYR-41 on CREB modifications. Parental HER-2/neu + cells were either left untreated or treated with different concentrations of MG-132 (10, 25, 50 μM) or PYR-41 (10, 20, 50 μM) for 4 h under hypoxia. Following Western blot analysis using anti-CREB-specific antibodies as described above, the appearance of highly modified CREB molecules was determined. The blot represents one of two biological experiments. F. Cells were cultivated under hypoxia for the indicated time and SUMO-1 modified proteins in the cell lysate were precipitated by immunoprecipitation. The proteins were loaded onto a 10% SDS Gel and CREB was detected with specific antibodies.

    Journal: Oncotarget

    Article Title: Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization

    doi: 10.18632/oncotarget.10474

    Figure Lengend Snippet: Hypoxia-mediated post-transcriptional modification and altered distribution of CREB A. Comparative analysis of different pCREB and CREB modifications under normoxia and early phase hypoxia (up to 12 h). Western blot analyses were performed as described using CREB and pCREB Ser133 -specific mAb. The size of the modified CREB protein is given. The blots represent one of three biological replicates. Extracellular pH (pH [ex]) of the cell free media was directly measured with a pH electrode after harvesting the cells. B. CREB phosphorylation at serine residue 133 and 121 as well as CREB protein expression was analysed under late phase hypoxia (up to 5 d). The photos represent one of two independent biological replicates. The pH of the culture media was measured with a pH electrode directy after harvesting the cells. C. , D. Ubiquitination was analysed by CREB immune precipitation (C) and ubiquitin pull down (D) as described in Materials and Methods by loading the complete supernatant on 10% (C) or 12% (D) gels. The proteins were identified by using anti-CREB- or anti-ubiquitin-specific mAb. Results represent data of three (C) or two (D) biological replicates. E. Influence of the proteasome inhibitor MG-132 and the ubiquitin inhibitor PYR-41 on CREB modifications. Parental HER-2/neu + cells were either left untreated or treated with different concentrations of MG-132 (10, 25, 50 μM) or PYR-41 (10, 20, 50 μM) for 4 h under hypoxia. Following Western blot analysis using anti-CREB-specific antibodies as described above, the appearance of highly modified CREB molecules was determined. The blot represents one of two biological experiments. F. Cells were cultivated under hypoxia for the indicated time and SUMO-1 modified proteins in the cell lysate were precipitated by immunoprecipitation. The proteins were loaded onto a 10% SDS Gel and CREB was detected with specific antibodies.

    Article Snippet: For evaluation of localization of CREB in human breast cancer tissue, formalin-fixed paraffin embedded tissue samples that contained necrotic areas indicative of localized hypoxia were stained with the phospho-CREB (Ser133) (87G3) Rabbit mAb (Cell Signaling) in the indicated concentration using the Ventana Discovery Autostainer (Ventana).

    Techniques: Modification, Western Blot, Expressing, Immunoprecipitation, SDS-Gel

    The effect of N-methyl-D-aspartate receptor (NMDAR) blockade on the long-term plasticity, gene expression and protein synthesis. (A) In the presence of 2-amino-5-phosphonovaleric acid (APV), the plasticity map (that was obtained as in Figure 1 ) shows that TBS does no longer induce synaptic plasticity. The histogram confirms the almost complete absence of changes in the slice. (B) In the presence of APV, the pseudocolor maps of P-CREB and Creb levels (that were obtained as in Figure 2 ) do not show remarkable differences between a control slice and a slice that has received TBS. Histograms show that no significant differences occur between control slices and slices that have received TBS (mean and SEM, n = 4; 120 min). (C) In the presence of APV, the pseudocolor maps of c-FOS and c-Fos levels (that were obtained as in Figure 2 ) do not show remarkable differences between a control slice and a slice that has received TBS. Histograms show that no significant differences occur between control slices and slices that have received TBS (mean and SEM, n = 4; 120 min).

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Activation of the CREB/c-Fos Pathway during Long-Term Synaptic Plasticity in the Cerebellum Granular Layer

    doi: 10.3389/fncel.2017.00184

    Figure Lengend Snippet: The effect of N-methyl-D-aspartate receptor (NMDAR) blockade on the long-term plasticity, gene expression and protein synthesis. (A) In the presence of 2-amino-5-phosphonovaleric acid (APV), the plasticity map (that was obtained as in Figure 1 ) shows that TBS does no longer induce synaptic plasticity. The histogram confirms the almost complete absence of changes in the slice. (B) In the presence of APV, the pseudocolor maps of P-CREB and Creb levels (that were obtained as in Figure 2 ) do not show remarkable differences between a control slice and a slice that has received TBS. Histograms show that no significant differences occur between control slices and slices that have received TBS (mean and SEM, n = 4; 120 min). (C) In the presence of APV, the pseudocolor maps of c-FOS and c-Fos levels (that were obtained as in Figure 2 ) do not show remarkable differences between a control slice and a slice that has received TBS. Histograms show that no significant differences occur between control slices and slices that have received TBS (mean and SEM, n = 4; 120 min).

    Article Snippet: Immunohistochemistry Double immunofluorescent labeling was performed as follows: (1) sections were dried 30 min at room temperature and washed with Tris-buffered saline; (2) sections were blocked 1 h in Tris-buffered saline containing 10% normal horse serum (NHS) and 0.3% Triton X-100 (TX-100) at room temperature; (3) sections were incubated overnight at 4°C in Tris-buffered saline/1% NHS/0.3% TX-100 containing a mixture of a goat anti-c-FOS antibody (sc-52, diluted 1:400; Santa Cruz) and a rabbit anti-phospho-CREB (P-CREB, Ser133) antibody (87G3, diluted 1:100, Cell Signaling); (4) sections were rinsed in Tris-buffered saline and incubated 1 h at room temperature in Tris-buffered saline /1% NHS/0.3% TX-100 containing a mixture of Alexa Fluor 488 conjugated donkey anti-goat IgG antibody (1:300) and Alexa Fluor 594 conjugated donkey anti-rabbit IgG antibody (1:300, Life Technologies); and (5) sections were rinsed in Tris-buffered saline and covered with Prolong with DAPI.

    Techniques: Expressing

    Development-dependent retinal CREB activity is mediated by cGKII in vivo . Phospho-CREB and cGKII colocalize in retinal neurons. Cultured purified neurons ( a .1) or retinal sections ( a .2) were stained against CREB (green (E6) or cyan (E8)) and cGKII (red (E6) or magenta (E8)) and observed in a confocal microscope. Calibration bars=10 μ m in a .1 and 50 μ m in a .2. In b and c , NO decreases or increases nuclear CREB activity via cGKII. E6C3 and E8C3 (respectively) mixed retinal cultures were immunostained against pCREB Ser133 and processed for nuclear colocalization with DAPI. In d , cGKII is knocked down in vivo . Lentiviruses coding for cGKII shRNAs or the empty vector pLKO were injected in the air chamber of chicken eggs with E5 embryos and incubated up to E9–E11. Retinas were immunostained for cGKII. Knockdown51.5% in E5–E9 and 78.3% in E8–E11. Calibration bar=50 μ m. In e – k , NO differentially regulates phospho-CREB in retinal layers via cGKII. Lentiviruses coding for cGKII shRNAs were injected (as in d ), and then SNAP was administrated at E6 (upper panels) or at E8 (bottom panels). Eggs were incubated for further 3 days (E9 or E11, respectively) and pCREB immunohistochemistry was performed. Calibration bars=50 μ m. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. In k , NO regulates in vivo retinal tissue apoptotic labeling via cGKII. cGKII shRNAs were injected (as above), and then SNAP was administrated at E6 (upper) or at E8 (bottom). Cleaved caspase-3 staining=red staining in E6–E9 and green staining in E8-E11. Calibration bars=50 μ m. Data represent the mean±S.E.M. (error bars). * P

    Journal: Cell Death and Differentiation

    Article Title: The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation

    doi: 10.1038/cdd.2014.11

    Figure Lengend Snippet: Development-dependent retinal CREB activity is mediated by cGKII in vivo . Phospho-CREB and cGKII colocalize in retinal neurons. Cultured purified neurons ( a .1) or retinal sections ( a .2) were stained against CREB (green (E6) or cyan (E8)) and cGKII (red (E6) or magenta (E8)) and observed in a confocal microscope. Calibration bars=10 μ m in a .1 and 50 μ m in a .2. In b and c , NO decreases or increases nuclear CREB activity via cGKII. E6C3 and E8C3 (respectively) mixed retinal cultures were immunostained against pCREB Ser133 and processed for nuclear colocalization with DAPI. In d , cGKII is knocked down in vivo . Lentiviruses coding for cGKII shRNAs or the empty vector pLKO were injected in the air chamber of chicken eggs with E5 embryos and incubated up to E9–E11. Retinas were immunostained for cGKII. Knockdown51.5% in E5–E9 and 78.3% in E8–E11. Calibration bar=50 μ m. In e – k , NO differentially regulates phospho-CREB in retinal layers via cGKII. Lentiviruses coding for cGKII shRNAs were injected (as in d ), and then SNAP was administrated at E6 (upper panels) or at E8 (bottom panels). Eggs were incubated for further 3 days (E9 or E11, respectively) and pCREB immunohistochemistry was performed. Calibration bars=50 μ m. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. In k , NO regulates in vivo retinal tissue apoptotic labeling via cGKII. cGKII shRNAs were injected (as above), and then SNAP was administrated at E6 (upper) or at E8 (bottom). Cleaved caspase-3 staining=red staining in E6–E9 and green staining in E8-E11. Calibration bars=50 μ m. Data represent the mean±S.E.M. (error bars). * P

    Article Snippet: Primary antibodies: mouse anti-CREB (#9197), mouse anti-phospho-CREB Ser133 (#9196), rabbit anti-phospho-CREB Ser133 (#9191) were from Cell Signaling (Danvers, MA, USA) and their specificity for chick CREB has been described previously , ; rabbit anti-cGKII (ab110124) was from Abcam (Cambridge, MA, USA), rabbit anti-phospho-AKT Ser473 (#9271) was from Cell Signaling and the specificity of both antibodies in the chick was previously determined ; rabbit anti-phospho-nNOS Ser1417 (07–544) was from Millipore (Merck/Millipore) and its specificity was previously determined.

    Techniques: Activity Assay, In Vivo, Cell Culture, Purification, Staining, Microscopy, Plasmid Preparation, Injection, Incubation, Immunohistochemistry, Labeling

    Immunohistochemistry for pCREB in the dentate gyrus of control ( a ), vehicle- ( b ), control-HSP70- ( c ), and Tat-HSP70-treated ( d ) mice. In all groups, pCREB-positive nuclei (arrows) are mainly observed in the subgranular zone of the dentate gyrus. Note that pCREB-positive nuclei are strongly observed in the dentate gyrus of Tat-HSP70-treated mice. GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Scale bar = 50 μm. e The number of pCREB-positive nuclei per section for each group are shown and f values from western blot analysis is expressed as a ratio of pCREB and CREB immunoblot band in control group ( n = 5 per group; a P

    Journal: Laboratory Animal Research

    Article Title: Heat shock protein 70 increases cell proliferation, neuroblast differentiation, and the phosphorylation of CREB in the hippocampus

    doi: 10.1186/s42826-019-0020-2

    Figure Lengend Snippet: Immunohistochemistry for pCREB in the dentate gyrus of control ( a ), vehicle- ( b ), control-HSP70- ( c ), and Tat-HSP70-treated ( d ) mice. In all groups, pCREB-positive nuclei (arrows) are mainly observed in the subgranular zone of the dentate gyrus. Note that pCREB-positive nuclei are strongly observed in the dentate gyrus of Tat-HSP70-treated mice. GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Scale bar = 50 μm. e The number of pCREB-positive nuclei per section for each group are shown and f values from western blot analysis is expressed as a ratio of pCREB and CREB immunoblot band in control group ( n = 5 per group; a P

    Article Snippet: To reduce background staining, the membrane was incubated with 5% non-fat dry milk in PBS containing 0.1% Tween-20 for 45 min at 25 °C, which was followed by incubation with rabbit anti-polyhistidine primary antibody (1:2000, His-probe, SantaCruz Biotechnology), rabbit anti-doublecortin (DCX) antibody (1:10,000; Abcam, Cambridge, UK), rabbit anti-phosphorylated CREB at Ser133 (pCREB; 1:1000; Cell Signaling Technology, Inc., Beverly, MA, USA), or rabbit anti-CREB (1:1000; Cell Signaling Technology, Inc.), peroxidase-conjugated goat anti-rabbit IgG (1:5000, SantaCruz Biotechnology), and an ECL chemiluminescent kit (Pierce; Thermo Fisher Scientific, Inc.).

    Techniques: Immunohistochemistry, Mouse Assay, Western Blot