Journal: PLoS ONE

Article Title: Sodium-Glucose Cotransporter 2 Inhibitor and a Low Carbohydrate Diet Affect Gluconeogenesis and Glycogen Content Differently in the Kidney and the Liver of Non-Diabetic Mice

doi: 10.1371/journal.pone.0157672

Figure Lengend Snippet: Taqman (R) gene expression assays annotation.

Article Snippet: Mm00501607_m1 , Creb1 , cAMP responsive element binding protein 1 , NM_009952.2.

Techniques: Gene Expression, Binding Assay

Journal: PLoS ONE

Article Title: Sodium-Glucose Cotransporter 2 Inhibitor and a Low Carbohydrate Diet Affect Gluconeogenesis and Glycogen Content Differently in the Kidney and the Liver of Non-Diabetic Mice

doi: 10.1371/journal.pone.0157672

Figure Lengend Snippet: Foxo1 and Creb1 mRNA expression in the kidney (a, b) and liver (c, d) were determined by quantitative RT-PCR. The means ± SEM of mRNA related to NC group are presented. n = 6–10. *p < 0.05, **p < 0.01 vs NC. White bars, NC; hatched white bars, LC; hatched grey bars, NC+Ipra; black bars LC+Ipra.

Article Snippet: Mm00501607_m1 , Creb1 , cAMP responsive element binding protein 1 , NM_009952.2.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Local GHR roles in regulation of mitochondrial function through mitochondrial biogenesis during myoblast differentiation

doi: 10.1186/s12964-023-01166-5

Figure Lengend Snippet: Local GHR regulates mitochondrial biogenesis via IGF1-PI3K/AKT/CREB pathway. a The PPI network of human GHR, IGF1, AKT, CREB and PGC1α. b The PPI network of mouse GHR, IGF1, AKT, CREB and PGC1α. c The PPI network of pig GHR, IGF1, AKT, CREB and PGC1α. d The PPI network of chicken GHR, IGF1, AKT, CREB and PGC1α. Protein–protein interaction was performed by the String database and visualized by Cytoscape (version 3.4.0). e and f Western blots with anti-JAK2, anti-p-JAK2, anti-AKT1, anti-p-AKT1, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection with si- GHR and si-NC. g and h Western blots with anti-AKT1, anti-p-AKT1, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection with si- IGF1 and si-NC. i and j Western blots with anti-AKT1, anti-p-AKT1, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after co-transfection with si- GHR + pcDNA3.1- IGF1 , si- GHR + pcDNA3.1 and si-NC + pcDNA3.1. k LY294002 (PI3K inhibitor) was added at 24 h before measuring the expression of PGC1α by RT-qPCR at 48 h after transfection of pcDNA3.1- IGF1 and pcDNA3.1. l GSK690693 (AKT inhibitor) was added at 24 h before measuring the expression of PGC1α by RT-qPCR at 48 h after transfection of pcDNA3.1- IGF1 and pcDNA3.1. m and n LY294002 or GSK690693 was added at 24 h before measuring the protein levels with anti-PGC1α, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection of pcDNA3.1- IGF1 and pcDNA3.1. o The expression of CREB and PGC1α was measured by RT-qPCR at 48 h after transfection with pcDNA3.1- CREB and pcDNA3.1. p and q Western blots with anti-PGC1α, anti-CREB1, anti-p-CREB1 and anti-β-actin at 48 h after transfection with pcDNA3.1- CREB and pcDNA3.1. r Dual-Luciferase report assays transfected with reporter vectors containing different length of 5′ upstream region of PGC1α . s Dual-Luciferase report assays of CREB overexpression co-transfected with reporter vectors containing different length of 5′ upstream region of PGC1α. Data are shown as mean ± SEM, * p < 0.05, ** p < 0.01

Article Snippet: The antibodies and their dilutions utilized for western blots were as follow: anti-GHR (bs-0654R; Bioss, China; 1:500), anti-PGC1α (bs-1832R; Bioss, China; 1:500), anti-NRF1 (12,482–1-AP; Proteintech, USA; 1:500), anti-TOMM20 (AF1717; Beyotime, China; 1:500), anti-JAK2 (bs-0908R; Bioss, China; 1:1000), anti-p-JAK2 (bsm-52171R; Bioss, China; 1:1000), anti-AKT1 (bs-0115 M; Bioss, China; 1:500), anti-p-AKT1 (66,444–1-Ig; Proteintech, China; 1:500), anti-CREB1 (bs-0035R; Bioss, China; 1:500), anti-p-CREB1 (bs-0036R; Bioss, China; 1:500), anti-β-actin (bs-0061R; Bioss, China; 1:1000), goat anti-rabbit IgG-HRP (bs-0295G; Bioss, China; 1:5000), goat anti-mouse IgG-HRP (bs-0296G; Bioss, China; 1:5000).

Techniques: Western Blot, Transfection, Cotransfection, Expressing, Quantitative RT-PCR, Luciferase, Over Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Local GHR roles in regulation of mitochondrial function through mitochondrial biogenesis during myoblast differentiation

doi: 10.1186/s12964-023-01166-5

Figure Lengend Snippet: Schematic diagram for the mechanistic model of the GHR roles in regulation of mitochondrial function during myoblast differentiation. Local GHR enhances mitochondrial function by promoting mitochondrial biogenesis via IGF1 -PI3K/AKT/CREB pathway during myoblast differentiation. This graphical abstract was created with Biorender.com

Article Snippet: The antibodies and their dilutions utilized for western blots were as follow: anti-GHR (bs-0654R; Bioss, China; 1:500), anti-PGC1α (bs-1832R; Bioss, China; 1:500), anti-NRF1 (12,482–1-AP; Proteintech, USA; 1:500), anti-TOMM20 (AF1717; Beyotime, China; 1:500), anti-JAK2 (bs-0908R; Bioss, China; 1:1000), anti-p-JAK2 (bsm-52171R; Bioss, China; 1:1000), anti-AKT1 (bs-0115 M; Bioss, China; 1:500), anti-p-AKT1 (66,444–1-Ig; Proteintech, China; 1:500), anti-CREB1 (bs-0035R; Bioss, China; 1:500), anti-p-CREB1 (bs-0036R; Bioss, China; 1:500), anti-β-actin (bs-0061R; Bioss, China; 1:1000), goat anti-rabbit IgG-HRP (bs-0295G; Bioss, China; 1:5000), goat anti-mouse IgG-HRP (bs-0296G; Bioss, China; 1:5000).

Techniques:

Journal: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 3. Involvement of EGFR/AKT/CREB pathway in REG4-mediated macrophage polarization to M2. (A) Representative western blots of phosphorylated EGFR, AKT and CREB in macrophage cultures treated with REG4 (10 nM) for different time intervals indicated. (B) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively. Data are mean ± SEM from four independent experiments. *p<0.05, **P<0.01 compared to untreated cells. (C) Representative western blotting images showing the phosphorylation of EGFR, AKT and CREB in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. (D) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively, in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. Data are mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with REG4 treatment alone. (E) Representative western blots of CD163, CD68, and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. (F) Quantitative analysis of the immunoblots of CD163, CD68 and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. Data are mean ± SEM from three independent donors. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Phospho-proteomics, Infection, Control, shRNA

Journal: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 4. The conditioned medium of Panc1 cells induces macrophage polarization to M2. (A) Western blots of REG4 in the culture medium or cell lysate of Panc1, AsPC1 and BxPC3 cells. Shown are representatives of three independent experiments with similar results. (B) Representative western blots of CD206 and CD163 in macrophage cultures treated with or without the conditioned medium of Panc1, AsPC1 and BxPC3 cell cultures, respectively. (C) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with or without the conditioned medium of Panc1 (CMPanc1), AsPC1 (CMAsPC1) and BxPC3 (CMBxPC3) cell cultures, respectively. Data are shown by mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with CMPanc1 treatment. (D) Western blots of REG4 in the culture medium and cell lysate of Panc1 cells infected with or without lentiviral REG4 shRNA. Shown are representatives of three independent experiments with similar results. (E) Representative western blots of CD206 and CD163 in macrophage cultures treated with the conditioned medium Panc1 cells infected with lentiviral control shRNA (CMPanc1/shCon) or REG4 shRNA (CMPanc1/shREG4). (F) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with CMPanc1/shCon or CMPanc1/shREG4. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Infection, shRNA, Control

Journal: Journal of Virology

Article Title: The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association

doi: 10.1128/jvi.01565-23

Figure Lengend Snippet: nsp13 interacts with host PRKACA and CREB. (a) Lysates of HEK293T cells expressing Myc-PRKACA and Flag-nsp13 or Flag-vector were subjected to immunoprecipitation with an anti-Flag antibody and analyzed by immunoblotting. (b) Lysates of HEK293T cells expressing Myc-CREB1 and Flag-nsp13 or Flag-vector were subjected to immunoprecipitation with an anti-Flag antibody and analyzed by immunoblotting. (c) Lysates of HEK293T cells transfected with GFP-nsp13 or GFP-vector were subjected to immunoprecipitation with an anti-GFP antibody, and the immunoprecipitates were analyzed by immunoblotting with anti-CREB1 and anti-PRKACA antibodies. (d) Caco-2 cells infected with SARS-CoV-2 [WIV04; multiplicity of infection (MOI) = 10; upper panel] or untreated cells (lower panel) were fixed at 48 hpi and immunolabeled with anti-nsp13 (green) and anti-PRKACA or anti-CREB1 (red) antibodies. ( e) Lung tissue sections of patients infected with 2019BetaCoV (WIV04) were subjected to an in situ proximity ligation assay (PLA) with anti-nsp13 and anti-PRKACA or anti-CREB1 antibodies. At least three independent replicates of each experiment were performed.

Article Snippet: The membranes were subsequently incubated with purified CREB1 or PRKACA protein (Origene) for 1 h at room temperature.

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Transfection, Infection, Immunolabeling, In Situ, Proximity Ligation Assay

Journal: Journal of Virology

Article Title: The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association

doi: 10.1128/jvi.01565-23

Figure Lengend Snippet: CREB1 interacts directly with the nsp13 2A domain. (a) The interaction of SARS-CoV-2 nsp13 with CREB1 was investigated by a GST pulldown assay. Lysates of HEK293T cells transfected with the Myc-CREB1 plasmid were subjected to precipitation with GST-nsp13 and GST agarose beads. Precipitates and lysates were analyzed by immunoblotting, and GST protein was used as a negative control. (b) Schematic representation of nsp13 domains. Nsp13 is composed of a zinc-binding domain (red), stalk domain (orange), 1B domain (light pink), 1A domain (light blue), and 2A domain (blue). ( c) Purified GST-nsp13 and its mutants were dotted onto nitrocellulose membrane and then incubated with purified CREB1 or PRKACA. The direct binding of the GST fusion proteins was detected using an anti-CREB1 or anti-PRKACA antibody and GST protein was used as a negative control (anti-GST indicates equal sample loading). ( d) Purified GST-nsp13 and its mutants [described in (b)] were resolved by SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with purified CREB1 and then subjected to immunoblotting with an anti-CREB1 antibody. GST protein was used as a negative control. The purity of GST-nsp13 and its mutants were analyzed by Coomassie blue staining. (e) (Left) Three-dimensional modeling structure of the interaction between SARS-CoV-2 nsp13 (PDB ID: 7NIO; yellow) and CREB1 (predicted by Alpha Fold; cyan); the interaction domain of nsp13 is highlighted (orange). (Right) The possible binding site was simulated in silico ; the binding residues of nsp13 are highlighted in red, and CREB1 is shown in cyan. The structural model of the nsp13-CREB1 complex is presented as a cartoon superimposed with a transparent molecular Gaussian surface. Images were created using Mol* at the PDB web app . ( f) The interaction of SARS-CoV-2 nsp13 and its mutants with CREB1 was investigated by a GST pulldown assay. Lysates of HEK293T cells transfected with the Myc-CREB1 plasmid were subjected to precipitation with GST-nsp13, GST-nsp13 (mutant A), GST-nsp13 (mutant B), and GST agarose beads. Precipitates were analyzed by immunoblotting, and GST protein was used as a negative control. The purity of GST-nsp13 and its mutants were analyzed by Coomassie blue staining.

Article Snippet: The membranes were subsequently incubated with purified CREB1 or PRKACA protein (Origene) for 1 h at room temperature.

Techniques: GST Pulldown Assay, Transfection, Plasmid Preparation, Western Blot, Negative Control, Binding Assay, Purification, Membrane, Incubation, SDS Page, Staining, In Silico, Mutagenesis

Journal: Journal of Virology

Article Title: The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association

doi: 10.1128/jvi.01565-23

Figure Lengend Snippet: Host CREB1 promotes the ATPase and helicase activity of SARS-CoV-2 nsp13. ( a, b ) Recombinant GST-nsp13 (500 nM) was incubated with CREB1 or PKA at the indicated concentrations. The ATPase activity was measured based on the released free phosphate by a sensitive colorimetric assay. A reaction without nsp13 was used as a negative control. ( c) Purified GST-nsp13 (100 nM) with or without CREB1 (50 nM) was incubated with 750 nM DNA substrate and 2 mM ATP at 25°C for 20 min, and the Cy3 signal was then measured at an excitation wavelength of 550 nm and an emission wavelength of 570 nm (a.u. = arbitrary units). A reaction containing the DNA substrate and ATP was used as a negative control. (d ) The experiment was performed as described in (c), and the nsp13 Δ2A mutant (aa 1-442) was added instead of nsp13 as indicated. (e) Myc-CREB1 and s133-phosphorylated Myc-CREB1 were purified by a eukaryotic expression system (293T cells), and the phosphorylation level of purified proteins was analyzed by western blotting. ( f) Purified GST-nsp13 (100 nM) and eukaryotic CREB1 (50 nM) were incubated with 750 nM DNA substrate and 2 mM ATP at room temperature for 20 min, and the Cy3 signal was measured as described in c. At least three independent replicates of each experiment were performed. Data are presented as the mean ± SEM (* P < 0.05, ** P < 0.01, **** P < 0.0001).

Article Snippet: The membranes were subsequently incubated with purified CREB1 or PRKACA protein (Origene) for 1 h at room temperature.

Techniques: Activity Assay, Recombinant, Incubation, Colorimetric Assay, Negative Control, Purification, Mutagenesis, Expressing, Western Blot

Journal: Journal of Virology

Article Title: The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association

doi: 10.1128/jvi.01565-23

Figure Lengend Snippet: Host CREB1 potentiates SARS-CoV-2 proliferation. ( a, b) H1299-ACE2 cells were transfected with PKA/CREB1 siRNA or scrambled siRNA at the indicated concentration for 12 h. Then, the cells were infected with trVLPs and Ad-N for 48 h. The effect of the siRNAs on PKA/CREB1 expression (line) and the amounts of trVLPs (column) were determined using quantitative reverse transcription PCR (qRT-PCR). Remdesivir (RDV) (10 µM) was used as a control. ( c, d) H1299-ACE2 cells were pretreated with H89 or 666-15 at the indicated concentration for 24 h. Then, the cells were infected with trVLPs and Ad-N for 48 h, and the inhibitor was added again at the time of infection. Viral replications were determined using qRT-PCR (column). RDV (10 µM) was used as a positive control. The cytotoxicity of H89 and 666-15 was determined by a CCK-8 assay (line). DMSO and 10 µM RDV were used as controls. ( e–h) H1299-ACE2 cells infected with live SARS-CoV-2 (WIV04/Omicron strain) at a MOI of 1 were treated with H89 or 666-15 as indicated. DMSO and RDV were used as controls. ( i) A549-ACE2 cells infected with the live SARS-CoV-2 WIV04 strain at a MOI of 1 were treated with 666-15 as indicated. The percent inhibition of SARS-CoV-2 replication by 666-15 in the A549-ACE2 cell line was determined using qRT-PCR.

Article Snippet: The membranes were subsequently incubated with purified CREB1 or PRKACA protein (Origene) for 1 h at room temperature.

Techniques: Transfection, Concentration Assay, Infection, Expressing, Reverse Transcription, Quantitative RT-PCR, Control, Positive Control, CCK-8 Assay, Inhibition

Journal: Journal of Virology

Article Title: The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association

doi: 10.1128/jvi.01565-23

Figure Lengend Snippet: Graphical view of the mechanism by which the PKA-CREB1 axis regulated viral replication through the nsp13:CREB1 interaction. (1) Following entry and uncoating, the RNA genome of SARS-CoV-2 is translated to generate a viral replication complex from ORF1a/b (including nsp12 and nsp13). (2) The helicase nsp13 directly interacts with the host transcription factor CREB1 at the site of viral replication and transcription, resulting in enhanced helicase and ATPase activities of nsp13. (3) PKA Cα-mediated phosphorylation of CREB1 enhanced its promoting effect on nsp13 helicase activity. (4) Viral nsp13 binds CREB1, resulting in the accumulation of CREB1 in the cytoplasm and preventing it from entering the nucleus. (5) Whether the reduction of CREB1 in the nucleus will affect the life activities of the host and its relationship with the pathogenic mechanism of the virus remains to be studied.

Article Snippet: The membranes were subsequently incubated with purified CREB1 or PRKACA protein (Origene) for 1 h at room temperature.

Techniques: Activity Assay, Virus

Journal: Journal of Hepatocellular Carcinoma

Article Title: Exploring Prognosis, Tumor Microenvironment and Tumor Immune Infiltration in Hepatocellular Carcinoma Based on ATF/CREB Transcription Factor Family Gene-Related Model

doi: 10.2147/jhc.s398713

Figure Lengend Snippet: Figure 11 Expression of 6 core genes in HCC and adjacent tissues and prognosis in HCC. (A–F) ATF1, ATF4, BATF, CREB1, CREB3, and CREB3L1 are overexpressed in the tumor tissues as compared to the paraneoplastic tissues in TCGA-HCC cohort. (G–I) Survival curve showing the impact of expressions of ATF1, ATF4, BATF, CREB1, CREB3, and CREB3L1 on the OS in TCGA-HCC dataset.

Article Snippet: Journal of Hepatocellular Carcinoma 2023:10 https://doi.org/10.2147/JHC.S398713 DovePress 329 Powered by TCPDF (www.tcpdf.org) Experimental Materials Rabbit anti-human ATF1, CREB1, and CREB3 antibodies were purchased from CUSABIO (CSB-MA080223, CSBPA005948ESR1HU, and CSB-PA002269DSR1HU) (50 μL; Wuhan, China).

Techniques: Expressing

Journal: Journal of Hepatocellular Carcinoma

Article Title: Exploring Prognosis, Tumor Microenvironment and Tumor Immune Infiltration in Hepatocellular Carcinoma Based on ATF/CREB Transcription Factor Family Gene-Related Model

doi: 10.2147/jhc.s398713

Figure Lengend Snippet: Figure 12 ATF1, CREB1, and CREB3 expression in HCC tissues and adjacent normal tissues. (A, C and E) qRT-PCR revealed that ATF1, CREB1, and CREB3 expression was higher in HCC tissues than in normal tissues. (B, D and E) Immunohistochemical analysis showed that ATF1, CREB1, and CREB3 expression was significantly higher in HCC tissues than in adjacent normal tissues (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Journal of Hepatocellular Carcinoma 2023:10 https://doi.org/10.2147/JHC.S398713 DovePress 329 Powered by TCPDF (www.tcpdf.org) Experimental Materials Rabbit anti-human ATF1, CREB1, and CREB3 antibodies were purchased from CUSABIO (CSB-MA080223, CSBPA005948ESR1HU, and CSB-PA002269DSR1HU) (50 μL; Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining