Journal:

Article Title: Regulation of Transcription by Hypoxia Requires a Multiprotein Complex That Includes Hypoxia-Inducible Factor 1, an Adjacent Transcription Factor, and p300/CREB Binding Protein

doi:

Figure Lengend Snippet: Effect of CREB overexpression and CREBM1 on the LDH-A promoter. (A) Hypoxic induction of the transfected GH reporter, relative to α-globin control (α), was measured by RNase protection assay. Hypoxic induction was increased by cotransfection of a CREB expression plasmid and decreased by expression of a dominant negative protein, CREBM1. (B) To test whether hypoxia influences the phosphorylation of CREB, Western blot analyses were performed with nuclear extracts from HeLa cells incubated in normoxia (N), hypoxia (H), or 20 μM forskolin (F). Blots were probed with an anti-phospho-CREB antibody (UBI) or an anti-CREB antibody (25C10G; Santa Cruz).

Article Snippet: Blots were probed with an anti-phospho-CREB antibody (UBI) or an anti-CREB antibody (25C10G; Santa Cruz).

Techniques: Over Expression, Transfection, Rnase Protection Assay, Cotransfection, Expressing, Plasmid Preparation, Dominant Negative Mutation, Western Blot, Incubation

Journal: Oncogene

Article Title: Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes.

doi: 10.1038/sj.onc.1209255

Figure Lengend Snippet: Figure 3 CREB-1, from lymphocyte nuclear extracts, binds to an oligonucleotide from the cyclin D2 promoter. Supershift analysis of the 320 to 270 bp region of the cyclin D2 promoter using CREB-1 and CREB-2 antibodies. Nuclear extracts from (a) IL-2 stimulated Kit225 or (b) EPV-immortalized B cells were untreated (lanes 1 and 6) or incubated with specific antibodies raised to CREB-1 (lanes 2, 3, 7 and 8) or CREB-2 (lanes 4, 5, 9 and 10). The different lanes marked CREB1 and CREB2 represent incubation with two different antibodies. They were then incubated with an oligonucleotide corresponding to the 320 to 270 bp region of the cyclin D2 promoter (lanes 1–5) or a CREB-1 consensus oligonu- cleotide (lanes 6–10). This figure is representative of four experiments. (c) A DNA affinity precipitation was performed using nuclear extracts from EPV-immortalized B cells with oligonucleotides corresponding to consensus STAT, NF-kB or CREB oligonucleotides, as indicated or with an oligonucleotide corresponding to the 320 to 270 bp region of the cyclin D2 promoter. NE designates nuclear extract on which no precipitation has been performed. The precipitated proteins were separated by SDS–PAGE followed by Western blotting. CREB-1 was detected using a specific antibody.

Article Snippet: For supershift assays, the nuclear extracts were preincubated for 30min with 1ml of antibodies for CREB1 or CREB2 from Santa Cruz Biotechnology: CREB-1 (C-21, sc-186); CREB-1 (24H4B, sc-7583); CREB-2 (C20, sc-186) and CREB-2 (C19, sc-7583).

Techniques: Incubation, SDS Page, Western Blot

Journal: Oncogene

Article Title: Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes.

doi: 10.1038/sj.onc.1209255

Figure Lengend Snippet: Figure 4 CREB-1 is phosphorylated and its phosphorylation and binding are important for cyclin D2 promoter activity. (a) Kit225 cells were cotransfected with the human D2 promoter and phRL-SV40 together with various amounts of an expression vector for CREB-1 or S133A CREB-1. The amount of DNA was kept constant by the addition of empty vector (pcDNA3). Cells were stimulated with IL-2 for 18 h. Data were normalized using the Renilla luciferase and is expressed as a fold activation induced by IL-2. Error bars represent the mean and standard error of the mean from three independent experiments. (b) Protein extracts were generated from Kit225 cells that had been pretreated with LY294002 for 30 min, stimulated with IL-2 for 1 h. Protein extracts were analysed by SDS– PAGE, and probed with specific antibodies as indicated. (c) To investigate the phosphorylation status of DNA bound CREB-1, a DNA affinity precipitation was performed on Kit225 cell nuclear extracts, with an oligonucleotide corresponding to the 320 to 270 bp region of the cyclin D2 promoter. The precipitated proteins were resolved by SDS–PAGE followed by Western blotting and detection with specific antibodies. (d) Lymphoblastoid cells were treated with various doses of LY294002 as indicated for 1 h. Protein extracts were generated and analysed as in (b). Representative experiments are shown from at least four experiments.

Article Snippet: For supershift assays, the nuclear extracts were preincubated for 30min with 1ml of antibodies for CREB1 or CREB2 from Santa Cruz Biotechnology: CREB-1 (C-21, sc-186); CREB-1 (24H4B, sc-7583); CREB-2 (C20, sc-186) and CREB-2 (C19, sc-7583).

Techniques: Phospho-proteomics, Binding Assay, Activity Assay, Expressing, Plasmid Preparation, Luciferase, Activation Assay, Generated, SDS Page, Western Blot

Journal: Oncogene

Article Title: Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes.

doi: 10.1038/sj.onc.1209255

Figure Lengend Snippet: Figure 5 Loss of the CREB-1-binding site in the cyclin D2 promoter activity causes a dramatic loss of activity. Two doses of each of the four cyclin D2 promoters, two of which contained a mutation within the CREB-1-binding site (illustrated in schematic (a)), were transfected into Kit225 cells. After 4 h, cells were lysed and luciferase activity was determined (b). IB4 lymphoblastoid cells were transiently transfected with the full-length cyclin D2 promoter and the promoter containing a mutation in the CREB-1 site after 16 h (c).

Article Snippet: For supershift assays, the nuclear extracts were preincubated for 30min with 1ml of antibodies for CREB1 or CREB2 from Santa Cruz Biotechnology: CREB-1 (C-21, sc-186); CREB-1 (24H4B, sc-7583); CREB-2 (C20, sc-186) and CREB-2 (C19, sc-7583).

Techniques: Binding Assay, Activity Assay, Mutagenesis, Transfection, Luciferase

Journal: Oncogene

Article Title: Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes.

doi: 10.1038/sj.onc.1209255

Figure Lengend Snippet: Figure 6 CREB phosphorylation and proliferation of EPV- immortalized cells is inhibited by a PKA inhibitor. (a) EPV- immortalized cells were incubated for 1 h with a variety of protein kinase inhibitors. The inhibitors were Ro-31–8220 (5 mM), Genis- tein (500 mM), KN-93 (10 mM), KT5720 (500 mM), staurosporine (100 nM) and U0126 (500 mM). Protein extracts were generated and CREB phosphorylation was measured with specific antibodies following SDS–PAGE and Western blotting. (b) A range of doses of staurosporine and KT5720 were used to treat cells and CREB phosphorylation was measured as before. (c) EBV-immortalized cells were treated with four doses of KT5720 for 72 h. Cells were counted by flow cytometry. The results are expressed as the percentage of values from untreated cells and are the mean and standard error of the mean of three independent experiments.

Article Snippet: For supershift assays, the nuclear extracts were preincubated for 30min with 1ml of antibodies for CREB1 or CREB2 from Santa Cruz Biotechnology: CREB-1 (C-21, sc-186); CREB-1 (24H4B, sc-7583); CREB-2 (C20, sc-186) and CREB-2 (C19, sc-7583).

Techniques: Phospho-proteomics, Incubation, Generated, SDS Page, Western Blot, Cytometry

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: bioRxiv

Article Title: Differential antitumor activity of compounds targeting the ubiquitin-proteasome machinery in gastrointestinal stromal tumor (GIST) cells

doi: 10.1101/791426

Figure Lengend Snippet: (A, B) Immunoblot analysis of GIST cells treated with delanzomib at the indicated concentrations for 72 h (A) or with 0.1 μM delanzomib for the indicated times (B) and probed for phospho-H2AX (S139) and total H2AX as well as phospho-KIT (Y719) and total KIT. Grouped immunoblot images are either cropped from different parts of the same gel or from a separate gel run with another aliquot of the same protein lysate. (C, D) RT-PCR (C) and quantitative RT-PCR (qRT-PCR) amplification (D) of KIT mRNA after treating GIST cells with DMSO or 0.1 μM delanzomib in comparison to bortezomib (BO) for 48 h. GIST882 cells were also treated with the RNA polymerase II inhibitor α-amanitin (α -ama) or H 2 O solvent control. (E) Immunofluorescence microscopic analysis of GIST882 and GIST48 cells treated with DMSO or 0.1 μM delanzomib for 72 h and stained for the transcriptional co-activator CREB-binding protein (CBP; green). Nuclei were stained with DAPI. Bar, 20 μm.

Article Snippet: Primary antibodies used for immunoblotting, immunofluorescence and immunohistochemistry were actin (Sigma), cleaved caspase 3 (Cell Signaling), CREB binding protein (CBP; Santa Cruz), cyclin A (Novocastra), pH2AX S139 (Millipore), H2AX (Bethyl), phospho-histone H3 S10, phospho-KIT Y719 (both Cell Signaling), Ki-67 (Thermo Scientific Neomarkers), KIT (Dako/Agilent), p27 Kip1 (Fisher/BD Biosciences Pharmingen), PARP (Invitrogen), cleaved PARP (Abcam) and mono-ubiquitin (BD Pharmingen).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Comparison, Solvent, Control, Immunofluorescence, Staining, Binding Assay

Journal: Nature

Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer.

doi: 10.1038/s41586-018-0018-1

Figure Lengend Snippet: Fig. 3 | SRC-3 phosphorylation by PFKFB4 enhances gene expression of metabolic enzymes. a, Relative levels of metabolites altered by shRNAs against PFKFB4 or SRC-3 compared to control non-targeting shRNA in MDA-MB231 cells. n = 3 biologically independent samples. **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. b, Relative proliferation of MDA-MB-231 and MCF-7 cells 4 days after treatment with siRNA targeting GFP (control) or SRC-3 under the conditions indicated. Ade, adenine; Gua, guanine. n = 5 biologically independent replicates. ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. c, mRNA expression of metabolic enzymes TKT, XDH and AMPD1 in MDA-MB-231 cells after treatment with siRNAs targeting GFP (control), PFKFB4 or SRC-3. n = 3 biologically independent samples. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. d, Immunoprecipitation (IP) of ATF4 from MDA-MB-231 cells

Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

Techniques: Phospho-proteomics, Gene Expression, Control, shRNA, Expressing, Immunoprecipitation

Journal: Gut Microbes

Article Title: Activation of ectopic olfactory receptor 544 induces GLP-1 secretion and regulates gut inflammation

doi: 10.1080/19490976.2021.1987782

Figure Lengend Snippet: Olfr544 activation induces GLP-1 secretion via CREB phosphorylation in mice small intestine. (a) Olfr544 and Olfr545 expression by qPCR in mouse small intestine and colon tissues. (b) Olfr544, Gcg , and ChgA expression by RT-PCR in GLUTag cells. (c) Acute AzA injection induced GLP-1 secretion in mice in both ex vivo and in vivo conditions in wild-type C57BL/6 J mice (n = 4–5) but not in Olfr544-knockout mice (n = 5–8). (d) Immunoblot analysis of CREB and phosphorylated CREB ( p -CREB) (n = 3). Ctrl, control; AzA, azelaic acid (50 μM). The data are presented as the mean ± SEM. One-way ANOVA followed by Tukey’s HSD test and Student t-test were performed for multiple- and two group comparisons. Different asterisks indicate a significant difference at * P < .05, ** P < .01, *** P < .01 compared with controls

Article Snippet: Primary antibodies against β-actin (SC-47778, 1:1000), α-tubulin (SC-5286, 1:1000), CREB (SC-377154, 1:500), p -CREB (Ser133; SC-101663, 1:500) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Ex Vivo, In Vivo, Knock-Out, Western Blot, Control

Journal: Gut Microbes

Article Title: Activation of ectopic olfactory receptor 544 induces GLP-1 secretion and regulates gut inflammation

doi: 10.1080/19490976.2021.1987782

Figure Lengend Snippet: Activation of Olfr544 by AzA activates the cAMP-PKA signaling axis and induces GLP-1 secretion in GLUTag cells. (a) Olfr544 activation by AzA induces GLP-1 secretion (n = 6). (b) AzA increases cAMP concentrations (n = 5), but not intracellular calcium (n = 9) or inositol phosphate (IP) levels (n = 5). HTRF ratio, homogeneous time-resolved fluorescence ratio (665 nm/620 nm). (c) GLP-1 secretion by GLUTag cells incubated with a PKA inhibitor (H89) or an EPAC inhibitor (ESI-09) (n = 3). (d) Relative PKA activity (n = 4), and (e) immunoblot analysis of CREB and phosphorylated CREB ( p -CREB) (n = 3). Ctrl, control; F/I, 10 μM forskolin and 10 μM IBMX; FSK, forskolin (10 μM); AzA, azelaic acid (50 μM); A-23187 (10 μM), a calcium-ionophore positive control. The data are presented as the mean ± SEM. One-way ANOVA with Bonferroni’s test and Student t-test were performed for multiple- and two group comparisons. * P < .05, ** P < .01, *** P < .001 compared with controls

Article Snippet: Primary antibodies against β-actin (SC-47778, 1:1000), α-tubulin (SC-5286, 1:1000), CREB (SC-377154, 1:500), p -CREB (Ser133; SC-101663, 1:500) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Fluorescence, Incubation, Activity Assay, Western Blot, Control, Positive Control

Journal: Gut Microbes

Article Title: Activation of ectopic olfactory receptor 544 induces GLP-1 secretion and regulates gut inflammation

doi: 10.1080/19490976.2021.1987782

Figure Lengend Snippet: Silencing Olfr544 expression abrogates the effect of AzA on GLUTag cells. (a) Olfr544 knockdown in GLUTag cells transfected with shRNA against Olfr544. The mRNA level of Olfr544 was determined by RT-PCR analysis (n = 3). (b) Intracellular cAMP levels (n = 4) and PKA activity (n = 4) in GLUTag cells with Olfr544 gene knockdown. (c) CREB, p -CREB expression, and p -CREB/CREB ratio (n = 3), and (d) GLP-1 secretion levels in GLUTag cells with Olfr544 gene knockdown (n = 3). Scr, scrambled shRNA; Ctrl, control; F/I, 10 μM forskolin and 10 μM IBMX; FSK, forskolin (10 μM); AzA, azelaic acid (50 μM). The data are presented as the mean ± SEM. One-way ANOVA followed by Tukey’s HSD test and Student t-test were performed for multiple- and two group comparisons. * P < .05, ** P < .01, *** P < .001 compared with controls

Article Snippet: Primary antibodies against β-actin (SC-47778, 1:1000), α-tubulin (SC-5286, 1:1000), CREB (SC-377154, 1:500), p -CREB (Ser133; SC-101663, 1:500) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Knockdown, Transfection, shRNA, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Control