Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Extracellular Acidification Activates cAMP Responsive Element Binding Protein via Na+/H+ Exchanger Isoform 1–Mediated Ca 2+ Oscillation in Central Nervous System Pericytes

doi: 10.1161/atvbaha.112.254946

Figure Lengend Snippet: Figure 1. Ca2+ oscillation and phosphorylation of cAMP responsive element–binding protein (CREB) induced by extracellular acidosis or removal of extracellular Na+. A, A representative trace of intracellular Ca2+ concentration ([Ca2+]i) in human brain microvascular pericytes exposed to acidified solution. B, Pericytes were stimulated with the acidified solution, and CREB or phospho-CREB at Ser133 (p-CREB) was detected by Western blotting analysis. *P<0.05 vs control. C, A representative trace of [Ca2+]i in pericytes exposed to Na+-free solu tion. D, Pericytes were stimulated with Na+-free solution, and CREB or p-CREB was detected. *P<0.05 vs control.

Article Snippet: Next , the samples were incubated with primary rabbit anti-platelet-derived growth factor receptor β (PDGFR-β) antibody (1:100, CST) overnight at 4°C for the detection of pericytes, incubated with secondary anti-rabbit IgG antibody (1:250, Alexa Fluor 568, Molecular Probes) for 1 hr at room temperature, blocked again, and incubated with Alexa Fluor 488 conjugated anti-phospho-CREB (Ser133) antibody (1:50, CST), anti-NHE1 antibody (1:50, Santa Cruz Biotechnology), or anti-IL-6 antibody (1:25, R&D systems).

Techniques: Phospho-proteomics, Binding Assay, Concentration Assay, Western Blot, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Extracellular Acidification Activates cAMP Responsive Element Binding Protein via Na+/H+ Exchanger Isoform 1–Mediated Ca 2+ Oscillation in Central Nervous System Pericytes

doi: 10.1161/atvbaha.112.254946

Figure Lengend Snippet: Figure 2. Effects of various inhibitors on the phosphorylation of cAMP responsive element–binding protein (CREB). A, The phosphorylation of CREB induced by extracellular acidification was evaluated in the presence of BAPTA, KN-93, wortmannin, U-0126, SB-203580, or H-89. The cells were incubated for 60 minutes with acidic solution after a 30-minute preincubation with inhibitors. *P<0.05 vs control, †P<0.05 vs pH 6.50. B, The phosphorylation of CREB induced by removal of extracellular Na+ was evaluated in the presence of inhibitors. The cells were incubated for 60 minutes with Na+-free solution after 30 minutes of preincubation with inhibitors. *P<0.05 vs control, †P<0.05 vs Na+ 0 mmol/L. C, The phosphorylation of CREB was detected by immunofluorescence in human central nervous system pericytes. The cells were incubated for 60 minutes with acidic or Na+-free solution with or without KN-93. An anti–platelet-derived growth factor receptor-β antibody (red) was used to stain pericytes, and phospho-CREB (p-CREB) (green) was detected by Alexa Fluor 488–conjugated antibody (Ser133). The right panels show the pericytes treated with forskolin as a positive control. Scale bar, 20 μm.

Article Snippet: Next , the samples were incubated with primary rabbit anti-platelet-derived growth factor receptor β (PDGFR-β) antibody (1:100, CST) overnight at 4°C for the detection of pericytes, incubated with secondary anti-rabbit IgG antibody (1:250, Alexa Fluor 568, Molecular Probes) for 1 hr at room temperature, blocked again, and incubated with Alexa Fluor 488 conjugated anti-phospho-CREB (Ser133) antibody (1:50, CST), anti-NHE1 antibody (1:50, Santa Cruz Biotechnology), or anti-IL-6 antibody (1:25, R&D systems).

Techniques: Phospho-proteomics, Binding Assay, Incubation, Control, Immunofluorescence, Derivative Assay, Staining, Positive Control

Journal: Journal of neurochemistry

Article Title: Serotonergic Psychedelics Rapidly Modulate Evoked Glutamate Release in Cultured Cortical Neurons.

doi: 10.1111/jnc.70020

Figure Lengend Snippet: FIGURE 1 | Serotonergic psychedelics increase nuclear localization of phosphorylated CREB after 24 h treatment. Representative images of im- munofluorescent detection of pCREB Ser133 (pseudocoloured, range shown in the rectangle below) and dendritic marker MAP2 (red) overlaid with DAPI nuclear staining (blue) in mature cortical cultures treated with CTRL or drugs for 30 min (a), 2 h (c) or 24 h (e). Scale bar is 15 μm. Quantification of nuclear immunofluorescence (IF) of pCREB after treatment with CTRL or drugs for 30 min (b), 2 h (d) or 24 h (f), number of visual fields (each consisting of 10–15 analysed nuclei) from 2 to 3 independent experiments in brackets. Data did not pass Shapiro–Wilk test for normality, and were assessed by Kruskal–Wallis test (30 min: H(3,92) = 7.020, p = 0.071; 2 h: H(3,108) = 1.003, p = 0.801; 24 h: H(3,128) = 40.26, p < 0.001) and Dunn's multiple comparisons test of CTRL versus each drug (24 h: p < 0.001 for all comparisons).

Article Snippet: Antibodies used for immunocytochemistry experiments: phospho- CREB (Ser133; 1:700, Cell Signaling Technology, RRID:AB_2561044) and MAP2 (1:500, Sigma- Aldrich, RRID:AB_477193).

Techniques: Marker, Staining, Immunofluorescence