Journal: The International Journal of Neuropsychopharmacology

Article Title: Activation of signaling pathways downstream of the brain-derived neurotrophic factor receptor, TrkB, in the rat brain by vagal nerve stimulation and antidepressant drugs

doi: 10.1017/s1461145713000977

Figure Lengend Snippet: Fig. 6. Western blot analysis of hippocampus CREB (≈50 kDa) phosphorylation at S133 after chronic 14 d VNS, sertraline (7.5 mg/ kg/d i.p.) or DMI (10 mg/kg/d i.p.). *p<0.01, Student’s t-test. The number of rats in each group is shown in the bars.

Article Snippet: Membranes were incubated at 4 °C overnight with the following primary antibodies: anti-pY515 (1:1000 in 2.5% BSA in TBST, Abcam, USA), anti-pY705 TrkB (1:1000 in 2.5% BSA in TBST, Abcam, USA), anti-pY816 TrkB (1:4000 in 2.5% BSA in TBST, Abcam, USA), and anti-TrkB (full length, 1:10000 in 2.5% BSA in TBST, Neuromics, USA), anti-PLCγ1 (1:1000), anti-PLC γ1 Y783 (1:250 in 5% BSA in TBST, Cell Signaling Technology, Inc, USA), anti-pERK T202/Y204 (1:1000 in 5% BSA in TBST, Cell Signaling Technology, Inc, USA), anti-ERK (1:2000 in 5% BSA in TBST, Cell Signaling Technology, Inc, USA), anti-AKT (1:1000 in 5% BSA in TBST, Cell Signaling Technology, Inc, USA), anti-pAKT T308 (1:1000 in 5% BSA in TBST, Cell Signaling Technology, Inc, USA), anti-CREB (1:2000 in 5% BSA in TBST, Cell Signaling Technology, Inc, USA), anti-pCREB S133 (1:1000 in 5% BSA in TBST, Cell Signaling Technology, Inc, USA), anti- pp70 S6 kinase T389 (1:1000 in 5% BSA in TBST, Cell Signaling Technology, Inc, USA) and anti-p70 S6 kinase (1:1000 in 5% BSA in TBST, Cell Signaling Technology, Inc, USA).

Techniques: Western Blot, Phospho-proteomics

Journal: Biology

Article Title: Brain-Derived Estrogen Regulates Neurogenesis, Learning and Memory with Aging in Female Rats.

doi: 10.3390/biology12060760

Figure Lengend Snippet: Figure 3. FBN-ARO-KO Impaired Hippocampal Neurons and Cognitive Function. (A,B) Western blot analysis for the indicated proteins in the hippocampus of 1-Mon KO and WT female rats. Data analyses of were expressed as ratios to total CREB or GAPDH (a loading control). Data were expressed as means ± SEM, n = 5–6 for each group. (B–D) Representative images of immunofluorescence staining for MBP2 (red, a marker of myelin sheath) and NeuN (green, a marker of surviving neuron) and quantity of MBP2 fluorescent intensity in the hippocampal subregions (CA1, CA3 and DG). (E,F) Representative images of immunofluorescence staining for MAP2 (green, a dendritic marker) and quantity of MAP2 fluorescent intensity in the hippocampal subregions. DAPI was used to conterstain nuclei (blue). Scale bar, 50 µm; magnification, 40×; Data are shown as means ± SEM. n = 5 in each group. (G,I) 1 Mon intact female rats and (M,O) 6 Mon OVX rats. The latency trial (D,G,M) and probe trail (I,O). Representative tracking plots in the escape latency (H,N) and probe trail (J,P). Open field test both in 1 Mon (K,Q) and 6 Mon rats were performed with grooming times, total grooming time, rearing times, total rearing time, as well as the time of entering the center zone (CZ). (L,R) Representative tracking plots of open field test. Data are shown as means ± SEM. n = 6–8 in each group. n.s., no significance. The full WB figures refer to the Supplementary Materials Figure S1.

Article Snippet: The following primary antibodies were used in this study: Estradiol (BioGenex, San Francisco, CA, USA #AR038), NeuN (Millipore Biotechnology, Massachusetts American #NG1857584), DCX (Santa, TX, USA #sc8066), SOX2 (Abcam, Cambridge, UK #ab97959), GFAP (Abcam, #ab53554), Iba1 (Abcam, #ab175076), MAP2 (Santa, #sc8066), MBP2 (Proteintech, Chicago, IL, #NO.10458-1-AP), GAPDH (Proteintech, #NO.60004-1-1g), p-CREB (Cellsignaling, Boston, MA, USA #9198s), CREB (Abclonal, Wuhan, China #56653), BDNF (Arigo, Shanghai, China #56653), PSD95 (Abcam, #ab1825), Neurofilament (Abcam, #ab7794), Spinophilin (Abcam, #ab13359) and SNAP25 (Abcam, #ab5666).

Techniques: Western Blot, Control, Staining, Marker

Journal: Biology

Article Title: Brain-Derived Estrogen Regulates Neurogenesis, Learning and Memory with Aging in Female Rats.

doi: 10.3390/biology12060760

Figure Lengend Snippet: Figure 4. Letrozole Inhibited Neurogenesis and Neuroprotection in the Hippocampal DG Region of 1-Mon Female Rats. (A,B) Confocal microscope for DCX+ (red), NeuN (green) and data analysis in the hippocampal DG region of control (WT) and letrozole-treated rats. (C) Western blot analyses of p-CREB/CREB, neurofilament, spinophilin, SNAP25, PSD95 an BDNF in the hippocampus. Quantifi- cation of Western blot data shown in (D). n = 5–6 for each group. Let: letrozole. The full WB figures refer to the Supplementary Materials Figure S2.

Article Snippet: The following primary antibodies were used in this study: Estradiol (BioGenex, San Francisco, CA, USA #AR038), NeuN (Millipore Biotechnology, Massachusetts American #NG1857584), DCX (Santa, TX, USA #sc8066), SOX2 (Abcam, Cambridge, UK #ab97959), GFAP (Abcam, #ab53554), Iba1 (Abcam, #ab175076), MAP2 (Santa, #sc8066), MBP2 (Proteintech, Chicago, IL, #NO.10458-1-AP), GAPDH (Proteintech, #NO.60004-1-1g), p-CREB (Cellsignaling, Boston, MA, USA #9198s), CREB (Abclonal, Wuhan, China #56653), BDNF (Arigo, Shanghai, China #56653), PSD95 (Abcam, #ab1825), Neurofilament (Abcam, #ab7794), Spinophilin (Abcam, #ab13359) and SNAP25 (Abcam, #ab5666).

Techniques: Microscopy, Control, Western Blot

Journal: Bone

Article Title: Complex intrinsic abnormalities in osteoblast lineage cells of X-linked hypophosphatemia: Analysis of human iPS cell models generated by CRISPR/Cas9-mediated gene ablation.

doi: 10.1016/j.bone.2024.117044

Figure Lengend Snippet: Fig. 8. Enhanced phosphorylation of CREB and increased expression of PTHRP in osteoblast lineage cells differentiated from PHEX-KO #1 iPSCs. (A-D) PHEX-KO #1 and isogenic control iPSCs were induced to differentiate into the osteoblast lineage, and cells were harvested at the indicated time points to examine the phosphorylation of FRS2α at Tyr196 (A), ERK1/2 at Tyr180/Tyr182 (B), CREB at Ser133 (C), and Smad1 and Smad5 at Ser463 and Ser465 (D) by Western blotting. The results of densitometry are shown in the bottom graphs. (E) Real-time PCR for the expression of PTHRP in osteoblast lineage cells derived from PHEX-KO #1 and isogenic control iPSCs. (F) The expression of Pthrp in osteoblast (OB)-rich and osteocyte (OCy)-rich cells isolated from WT and Hyp mice. Data in graphs are shown as the mean ± SD (n = 3). #, P < 0.05; ##, P < 0.01 vs Day 14. *, p < 0.05.

Article Snippet: After blocking with Blocking One P reagent (Nacalai Tesque Inc.) or Block Ace reagent (Dainippon Pharmaceuticals, Osaka, Japan), the membranes were incubated at 4 ◦C overnight with the following primary antibodies: antiDMP1 rabbit polyclonal antibody (TaKaRa, Shiga, Japan), anti-OPN rabbit polyclonal antibody (ProteinTech, Rosemont, IL, USA), antiPiT1 (SLC20A1) rabbit polyclonal antibody (H-130; Santa Cruz Biotechnology, Santa Cruz, CA), anti-SLC20A2 (PiT-2) rabbit polyclonal antibody (ProteinTech), anti-GAPDH goat polyclonal antibody (V-18; Santa Cruz Biotechnology), anti-phosphorylated ERK1/2 antibody, antiERK1/2 antibody, anti-phosphorylated CREB (Ser133) antibody, antiCREB antibody, anti-phosphorylated FRS2α antibody (Cell Signaling Technology, Beverly, MA, USA), and anti-FRS2α antibody (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction, Derivative Assay, Isolation

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