Journal: The Tohoku journal of experimental medicine

Article Title: Low-intensity aerobic exercise mitigates exercise-induced bronchoconstriction by improving the function of adrenal medullary chromaffin cells in asthmatic rats.

doi: 10.1620/tjem.234.99

Figure Lengend Snippet: Fig. 5. Low- or moderate-intensity aerobic exercise inhibits the ERK/CREB signaling and downstream gene expression in rat adrenal medulla. The relative levels of phosphorylated ERK and CREB were characterized by Western blot, and the relative levels of c-FOS, EGFR1, c-JUN, JUNB, and FOSB mRNAs to the control β-actin in the adrenal medullary tissues of individual rats were characterized by qRT-PCR. Data are representative images or expressed as the mean ± s.d. of individual groups of rats (n = 4 per group) from three separate experiments. (A) The relative levels of phosphorylated ERK and CREB. (B) The relative levels of c-FOS, EGFR1, c-JUN, JUNB, and FOSB mRNAs. Control: Control rats; Low: The rats with low-intensity aerobic exercise; Mod: The rats with moderate-intensity aerobic exercise; OVA: The rats were sensitized and challenged with OVA; OVA + Low: The rats were sensitized and challenged with OVA and subjected to low-intensity aerobic exercise; OVA + Mod: The rats were sensitized and challenged with OVA and subjected to moder- ate-intensity aerobic exercise. *P < 0.05 vs. the control group; ▲P < 0.05 vs. the OVA group; #P < 0.05 vs. the OVA + Low group.

Article Snippet: The animals were sacrificed and studied on day 56. incubated with anti-peripherin (1:20,000), anti-neurofilament-68 (NF68, 1:5,000), anti-chromogranin A (CgA, 1:10,000), anti-PNMT (1:1,000), anti-ERK1/2 (extracellular signal-regulated kinase, 1:1,000), anti-phosphorylated ERK1/2 (anti-p-ERK1/2, 1:500, Abcam, Cambridge, UK), anti-CREB (cAMP responsive element binding protein, 1:1,000), anti-phosphorylated CREB (anti-p-CREB, 1:1,000), or anti-β-actin (1:1,000, Cell Signaling Technology, Beverly, USA) overnight at 4°C.

Techniques: Gene Expression, Western Blot, Control, Quantitative RT-PCR

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: iScience

Article Title: Cell-type specific circadian transcription factor BMAL1 roles in excitotoxic hippocampal lesions to enhance neurogenesis

doi: 10.1016/j.isci.2024.108829

Figure Lengend Snippet:

Article Snippet: Phospho-CREB (Ser133) (87G3) Rabbit mAb , Cell Signaling , Cat#14001; RRID: AB_2798359.

Techniques: TUNEL Assay, Quantitative RT-PCR, Software

Journal: Molecular Medicine Reports

Article Title: Yifei Xuanfei Jiangzhuo formula, a Chinese herbal decoction, improves memory impairment through inhibiting apoptosis and enhancing PKA/CREB signal transduction in rats with cerebral ischemia/reperfusion

doi: 10.3892/mmr.2015.3962

Figure Lengend Snippet: Protein expression of PKA, CREB and p-CREB in the hippocampus. (A) After treatment and subsequent memory evaluation, hippocampi were harvested and the protein expression was analyzed by western blotting. (B-D) Quantified protein levels of (B) PKA (C) CREB and (D) p-CREB normalized to β-actin. Values are expressed as the mean ± standard error. ** P<0.01 vs. SHAM; ## P<0.01 vs. MOD; & P<0.05, && P<0.01 vs. HUA; ▲ P<0.05, ▲▲ P<0.01 vs. PIR. MOD, model group; LDY, low-dose YXJF group; MDY, medium-dose YXJF group; HDY, high-dose YXJF group; HUA, huperzine A group; PIR, piracetam group; YXJF, Yifei Xuanfei Jiangzhuo formula; PKA, protein kinase A; p-CREB, phosphorylated cyclic adenosine monophosphate-responsive element binding protein.

Article Snippet: Antibodies directed to Bax (rabbit anti-rat; cat. no. 2772S; 1:800), c-Jun (mouse anti-rat; cat. no. 2315S; 1:500), Bcl-2 (rabbit anti-rat; cat. no. 2876S; 1:1,000), PKA C-α (rabbit anti-rat; cat. no. 4782S; 1:600), CREB (rabbit anti-rat; cat. no. 4820; 1:800), and phosphorylated CREB (p-CREB; rabbit anti-rat; cat. no. 4276; 1:500) were all from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Binding Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: C-Reactive Protein Decreases Interleukin-10 Secretion in Activated Human Monocyte-Derived Macrophages via Inhibition of Cyclic AMP Production

doi: 10.1161/01.atv.0000241572.05292.fb

Figure Lengend Snippet: Figure 4. A, i, Effect of CRP on intracellular cAMP levels in HMDMs. The cells were incubated with LPS (500 ng/mL for 24 hours) in the absence or presence of CRP (25 g/mL) for 12 hours. The intracellular cAMP levels were measured by ELISA. ANOVA, P0.01; post hoc Tukey test: *P0.001 vs control; **P0.035 vs LPS alone. The results are meanSD of 3 experiments in duplicate. A, ii, Effect of CRP on AC activity in HMDMs. HMDMs on day 7 were pretreated with CRP for 12 hours followed by LPS challenge for 24 hours in serum-free medium. Membrane fractions were used to measure AC activity by the conversion of 32P-ATP to 32P-cAMP. Data are pre- sented as meanSD of 3 experiments in duplicate. The results are expressed as 32P-cAMP counts/mg protein. *P0.05 vs control, **P0.05 vs LPS alone. B, Effect of cAMP agonists on CRP-mediated IL-10 inhibition. Cells were treated with Db-cAMP (50 mol/L) or Br-cAMP (50 mol/L) 1 hour before CRP treatment (12 hours) followed by LPS challenge for 24 hours. IL-10 was measured in superna- tants. The results are meanSD of 3 experiments in duplicate. ANOVA, §P0.01; post hoc Tukey test: P0.001 vs control; *P0.01 vs LPS; **P0.01 vs CRPLPS. C, Effect of CRP on pCREB/CREB levels. Cells were treated with cAMP agonists 30 minutes before cell harvesting following CRPLPS treatment. Cell lysates were prepared as described in Materials and Methods. C, i, Effect of CRP on ratio of pCREB/CREB. Protein (50 g) was used for measurement of CREB and pCREB by ELISA and ratio of pCREB/CREB was cal- culated. ANOVA, P0.05; post hoc Tukey test: §P0.02 vs control; *P0.03 vs LPS alone; **P0.01 vs CRPLPS. The results are meanSD of 3 experiments in duplicate. C, ii, Western blot analysis depicting effect of CRP on pCREB and CREB done in cell lysates as described in Materials and Methods. Lane 1, control; lane 2, LPS; lane 3, CRPLPS; lane 4, CRPLPS Db-cAMP; lane 5, CRPLPSBr-cAMP. §P0.04 vs control, *P0.05 vs LPS alone, **P0.002 vs CRPLPS (n3 experiments for densitometric ratios).

Article Snippet: CREB as well as pCREB ELISA kits and antibodies were from Biosource and Cell Signaling Technology respectively.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Control, Activity Assay, Membrane, Inhibition, Cell Harvesting, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: C-Reactive Protein Decreases Interleukin-10 Secretion in Activated Human Monocyte-Derived Macrophages via Inhibition of Cyclic AMP Production

doi: 10.1161/01.atv.0000241572.05292.fb

Figure Lengend Snippet: Figure 5. Effect of transfection in THP-1 with indicated CREB plasmids on CRP- mediated IL-10 inhibition. THP-1 cells were grown in 12-well plates and trans- fected with indicated CREB plasmids as detailed in Materials and Methods. Forty- eight hours after transfection, THP-1 cells were pretreated with CRP (25 g/mL) for 12 hours followed by LPS (500 ng/mL) for 24 hours. A, Superna- tants were used for IL-10 measurement *P0.05 vs control, **P0.05 vs LPS alone, €P0.01 vs control in transfected cells. B, Cells were used for the mea- surement of total and pCREB. The results are expressed as pCREB/CREB ratio. *P0.03 vs control, **P0.045 vs LPS alone, €P0.029 vs control in transfected cells.

Article Snippet: CREB as well as pCREB ELISA kits and antibodies were from Biosource and Cell Signaling Technology respectively.

Techniques: Transfection, Inhibition, Control

Journal: Scientific Reports

Article Title: Celecoxib exerts protective effects in the vascular endothelium via COX-2-independent activation of AMPK-CREB-Nrf2 signalling

doi: 10.1038/s41598-018-24548-z

Figure Lengend Snippet: Celecoxib induces HO-1 in HUVEC and HAEC, by activating AMPK and CREB. ( A ) HUVECs were treated with vehicle (veh) or celecoxib (10 μM) for up to 60 mins, prior to lysis and immunoblotting for phospho-CREB Ser133 , total CREB and GAPDH. ( B ) Nuclear lysates, isolated from HUVEC following treatment with celecoxib 10 μM (Cele) or vehicle control for 30 mins, were analysed using a phospho-CREB transcription factor assay kit. Unstimulated Hela cells were used as a positive control (CTRL) and a wild-type oligonucleotide sequence (WT) was used for competitive binding. CREB-binding is expressed relative to vehicle-treated cells. ( C , D ) HUVECs were left untransfected or transfected with control siRNA (CTRL) or CREB siRNA (50 nM) and cultured for 48 h prior to treatment with vehicle or celecoxib (10 µM) for ( C ) 15 mins followed by immunoblotting for phospho-CREB and GAPDH, or for 24 h followed by analysis of ( D ) HO-1 by qRT-PCR and ( E ) HO-1 protein by immunoblotting. ( F ) HAEC were treated with celecoxib (10 μM) or vehicle for 15 mins prior to immunoblotting for: phospho-AMPK Thr172 , phospho-CREB Ser133 and GAPDH. ( G ) HAECs were treated with celecoxib (10 μM) or vehicle for 24 h prior to immunoblotting for HO-1. Data are expressed as the mean ± SEM of at least 3 separate experiments. The histograms show corresponding densitometry data corrected for α-tubulin or GAPDH and normalized to vehicle-treated cells. *P ≤ 0.05, **P ≤ 0.01, **P ≤ 0.001, using the one-way with a Bonferonni correction or two-way ANOVA ( A – E ) and a one-sample t-test. ( F , G ) Immunoblots shown have been cropped to conserve space, please see Supplementary file for original uncropped blots.

Article Snippet: A CREB (Phospho-Ser 133 ) transcription factor assay kit (Cayman Chemicals, Cambridge, UK) was used to determine the transcriptional activation of CREB as per the manufacturers’ instructions.

Techniques: Lysis, Western Blot, Isolation, Control, Transcription Factor Assay, Positive Control, Sequencing, Binding Assay, Transfection, Cell Culture, Quantitative RT-PCR