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:

Article Title: Mitogen-Regulated RSK2-CBP Interaction Controls Their Kinase and Acetylase Activities

doi: 10.1128/MCB.21.20.7089-7096.2001

Figure Lengend Snippet: Stimulation of cells with TPA and UV leads to RSK2-CBP complex dissociation. COS-1 cells were cotransfected with RSK2 and CBP and treated or not with TPA for 15 min, UV light for 30 min, or EGF for 10 min. Induction of RSK2 phosphorylation following treatment was determined using the P-T577 antibody. Anti-phospho-CREB antibody (P-CREB) was also used to show the effectiveness of the various treatments.

Article Snippet: Anti-CBP (A-22) and anti-RSK2 (E1 and C-19) antibodies were from Santa Cruz Biotechnology Inc., anti-HA antibody was from Boehringer-Mannheim, anti-ERK, anti-phosphorylated ERK, anti-CREB, and anti-phosphorylated CREB antibodies were from New England Biolabs, and anti-H3 and anti-phosphorylated H3 antibodies were from Upsate Biotechnology.

Techniques:

Journal:

Article Title: Mitogen-Regulated RSK2-CBP Interaction Controls Their Kinase and Acetylase Activities

doi: 10.1128/MCB.21.20.7089-7096.2001

Figure Lengend Snippet: RSK2-CBP complex formation inhibits RSK2 kinase activity. (A) RSK2 kinase activity is inhibited by the presence of ectopic CBP. COS-1 cells transfected with RSK2 or with both RSK2 and CBP and stimulated or not with EGF for 10 min. Nuclear extracts were prepared and immunoprecipitated (IP) with anti-RSK2 C-19 antibody, and an in vitro kinase assay was performed using the S6 peptide as the substrate. The total levels of RSK2 protein were verified by Western analysis. (B) Basal phosphorylation levels of CREB and histone H3 are downregulated in the presence of ectopic CBP. COS-1 cells were treated as in panel A, and the phosphorylation status of endogenous CREB and histone H3, two targets of RSK2, was detected using phospho-CREB and phospho-H3 antibodies. Western analysis was performed on similar amounts of total proteins, as shown by using anti-CREB antibody. In addition, equivalent amounts of ectopic RSK2 were present in cells overexpressing CBP or not.

Article Snippet: Anti-CBP (A-22) and anti-RSK2 (E1 and C-19) antibodies were from Santa Cruz Biotechnology Inc., anti-HA antibody was from Boehringer-Mannheim, anti-ERK, anti-phosphorylated ERK, anti-CREB, and anti-phosphorylated CREB antibodies were from New England Biolabs, and anti-H3 and anti-phosphorylated H3 antibodies were from Upsate Biotechnology.

Techniques: Activity Assay, Transfection, Immunoprecipitation, In Vitro, Kinase Assay, Western Blot

Journal:

Article Title: Mitogen-Regulated RSK2-CBP Interaction Controls Their Kinase and Acetylase Activities

doi: 10.1128/MCB.21.20.7089-7096.2001

Figure Lengend Snippet: Schematic representation of the phosphorylation-regulated association of CBP and RSK2. In quiescent cells, the two proteins are found associated in a complex with low kinase and HAT activities. In serum-starved cells, the basal phosphorylation levels of two RSK2 substrates, CREB and histone H3, are also low. Upon EGF mitogenic stimulation and consequent activation by phosphorylation of RSK2 at Ser227, CBP and RSK2 dissociate. This results in increased Ser133 CREB and Ser10 H3 phosphorylation (21, 43, 51). The newly phosphorylated CREB associates with CBP, whose HAT activity is also increased. The coordinated activation of both kinase and HAT activities may converge at the histone H3 tail, where concerted modifications of Ser10 and Lys14 have been reported previously (12, 35). This possible scenario does not take into account additional signaling routes which could influence the function of the various components. While it is likely that physical interactions among other kinases and HAT molecules will be found, deciphering the regulatory pathways controlling their functions is essential to the understanding of the molecular events involved in activation of gene expression. Ac-K14, acetylated residue K14; P-S10, phosphorylated residue S10.

Article Snippet: Anti-CBP (A-22) and anti-RSK2 (E1 and C-19) antibodies were from Santa Cruz Biotechnology Inc., anti-HA antibody was from Boehringer-Mannheim, anti-ERK, anti-phosphorylated ERK, anti-CREB, and anti-phosphorylated CREB antibodies were from New England Biolabs, and anti-H3 and anti-phosphorylated H3 antibodies were from Upsate Biotechnology.

Techniques: Activation Assay, Activity Assay, Expressing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB

doi: 10.1152/ajpgi.00177.2014

Figure Lengend Snippet: cAMP response element-binding protein (CREB) expression and phosphorylation (pCREB) in normal and ulcerated esophageal tissues. A, top: Western blot analyses of pCREB and total CREB (phosphorylated + unphosphorylated) protein expression in normal and ulcerated esophageal tissue 3, 6, and 9 days after ulcer induction. Esophageal ulceration induced phosphorylation of CREB. Bottom: quantitative analysis of pCREB. B, top: Western blot analyses of pCREB and total CREB protein expression in esophagus of rats treated intragastrically with either a single 50 μg/kg dose of misoprostol (MS) or its vehicle (VH). Misoprostol treatment further increased pCREB induced by esophageal ulceration. Bottom: quantitative analysis of pCREB. CREB phosphorylation was determined by calculating the ratio between pCREB and total CREB protein levels. Values are means ± SD. For each column (n = 6).

Article Snippet: The membranes were incubated with rabbit polyclonal anti-EP1, anti-EP2, anti-EP3, or anti-EP4 antibodies (Cayman Chemical, Ann Arbor, MI), mouse monoclonal anti-phosphorylated CREB (pCREB) (Cell Signaling Technology, Beverly, MA), or anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies at room temperature for 1 h. Membranes probed with pCREB were stripped and reprobed using rabbit polyclonal anti-CREB antibody (Santa Cruz Biotechnology), which recognizes both pCREB and unphosphorylated CREB.

Techniques: Binding Assay, Expressing, Phospho-proteomics, Western Blot

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB

doi: 10.1152/ajpgi.00177.2014

Figure Lengend Snippet: cAMP mediates misoprostol-induced CREB phosphorylation and stimulation of VEGF expression in HET-1A cells. Cells were treated with either the cAMP analog Sp-cAMP (100 μmol), misoprostol (10 μmol), or PBS for 30 min (A) or 3 h (B). Misoprostol-treated cells were pretreated with either PBS or inhibitor of cAMP-dependent PKA, Rp-cAMP (500 μmol) for 30 min. A, top: Western blot analyses of pCREB and total CREB (phosphorylated + unphosphorylated) protein expression. Sp-cAMP induced CREB phosphorylation in HET-1A cells, and Rp-cAMP inhibited this effect. Bottom: quantitative analysis of CREB phosphorylation. CREB phosphorylation was determined by calculating the ratio between pCREB and total CREB protein levels. B, top: RT-PCR analysis of VEGF mRNA expression. Sp-cAMP induced VEGF mRNA expression in HET-1A cells, and Rp-cAMP inhibited this effect. Bottom: quantitative analysis of VEGF mRNA expression. Each signal was normalized against the corresponding β-actin signal, and the results are expressed as VEGF/β-actin. All values are means ± SD of 3 separate experiments performed in duplicate.

Article Snippet: The membranes were incubated with rabbit polyclonal anti-EP1, anti-EP2, anti-EP3, or anti-EP4 antibodies (Cayman Chemical, Ann Arbor, MI), mouse monoclonal anti-phosphorylated CREB (pCREB) (Cell Signaling Technology, Beverly, MA), or anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies at room temperature for 1 h. Membranes probed with pCREB were stripped and reprobed using rabbit polyclonal anti-CREB antibody (Santa Cruz Biotechnology), which recognizes both pCREB and unphosphorylated CREB.

Techniques: Phospho-proteomics, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction