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

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: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 1. Average total fold intensity of phospho-protein signal as compared to median control value. Colored bars (dark blue = 0.5 Gy, light blue = 0.1 Gy and green = 0.05 Gy) indicate values significantly different from controls (yellow). Significance bars are shown for all significant differences between doses and 0 (p ≤0.05). Average fold intensity levels are shown at 2 h post radiation for γH2AX (A), pATF2 (B) and pSMC1 (C). Persistent effects are shown at 24 h for γH2AX (D), pATF2 (E) and pSMC1 (F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques: Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 3. Average total fold pATF2 intensity over median control level versus fluence for various radiation qualities. Average fold intensity levels are shown at 2 h post radiation for Si ions (A),Fe ions (C) and Ti ions (E). Persistent effects are shown at 24 h for Si ions (B), Fe ions (D) and Ti ions (F).

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques: Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 6. Average total fold intensity of pATF2 signal intensity divided by fluence and graphed versus LET. Average fold intensity levels are shown at 2 h post radiation for 0.05 Gy (A), 0.1 Gy (B) and 0.5 Gy (C). Persistent effects are shown at 24 h for 0.05 Gy (D), 0.1 Gy (E) and 0.5 Gy (F).

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques: