Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: (A,B) HUVEC were exposed to hypoxia (4 h) or hypoxia followed by reoxygenation (1-24 h) or remained untreated. (A) Cezanne transcript levels were quantified by real-time PCR. Data were pooled from 3 independent experiments. (B) Cytosolic lysates were tested by Western blotting using anti-Cezanne antibodies and by using anti-α-tubulin antibodies to assess total protein levels. Representative blots (upper panels) and results from densitometry analysis of five experiments (lower panel) are shown. (C) HDMEC or HCMEC were exposed to hypoxia (4 h) or remained untreated. Cezanne transcript levels were quantified by real-time PCR. Data were pooled from 3 independent experiments. (D) HUVEC or HCMEC were exposed to 1%, 2%, 5% or normoxia (21%) for 4 h. Cezanne transcript levels were quantified by real-time PCR. Data were pooled from 3 independent experiments. (E,F) HUVEC were exposed to CT8730 (1 μM) or SB202190 (50 μM) for 1 h or were treated with vehicle alone and then exposed to hypoxia (4 h) or remained untreated. (E) Cezanne transcript levels were quantified by real-time PCR. (F) Cytosolic lysates were tested by Western blotting using anti-Cezanne antibodies and by using anti-α-tubulin antibodies to assess total protein levels. n.s., non-specific band. (G) HUVEC were treated with p38α-specific siRNA (si-p38) or with a scrambled, non-targeting sequence and were then exposed to hypoxia (4 h) or hypoxia (4 h) followed by reoxygenation (4 h) or remained untreated (normoxia). Cezanne transcript levels were quantified by real-time PCR.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Sequencing

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: (A) HUVEC or HCMEC were exposed to hypoxia for 15 min-4 h or remained untreated (normoxia). Some cultures were then reoxygenated for 1 h. Cytosolic lysates were tested by Western blotting using anti-phosphorylated ATF2 antibodies or by using anti-α-tubulin antibodies to assess total protein levels. Data are representative of three independent experiments. (B) HUVEC were treated with siRNA sequences that target Cezanne (Cez 1, Dharmacon; Cez 2, SMARTpool), ATF2 (ATF2 1, Dharmacon; ATF2 2, SMARTpool) or SHP2, or with scrambled, non-targeting sequences as a control (Scr). They were then exposed to hypoxia (4 h) or remained untreated (normoxia). Levels of Cezanne or ATF2 were quantified by real-time PCR. Data were pooled from 3 independent experiments.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: HUVEC were treated with Cezanne-specific siRNA (si-Cezanne from Dharmacon) or with a scrambled, non-targeting sequence and were then exposed to hypoxia (4 h) or hypoxia (4 h) followed by reoxygenation (4-20 h) or remained untreated. (A) Levels of E-selectin, VCAM- 1 or ICAM-1 transcripts were quantified by real-time PCR. Data were pooled from three independent experiments. (B) Cytosolic or nuclear lysates were tested by Western blotting using anti-IκBα, anti-RelA or anti-Ser536 phosphorylated RelA antibodies and by using anti-α-tubulin or anti-Lamin B antibodies to assess total protein levels. Data are representative of three independent experiments.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: HUVEC were treated with Cezanne-specific siRNA (si-Cezanne from Dharmacon) or with a scrambled, non-targeting sequence and were then exposed to hypoxia (4 h) or hypoxia (4 h) followed by reoxygenation (2-20 h) or remained untreated. (A) TRAF6 immunoprecipitates or lysates were tested by Western blotting using anti-TRAF6 antibodies. (B) TRAF6 immunoprecipitates were tested by Western blotting using antibodies that recognise Lys63-polyubiquitin. n.s., non-specific. Data are representative of three independent experiments that gave closely similar results.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Sequencing, Western Blot

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: (A,B) Expression levels of Cezanne were assessed in left kidneys of male Fisher rats (n=4) that were exposed to ischemia followed by reperfusion. Basal levels of Cezanne were measured in the uninjured right kidney of each animal. (A) Cezanne transcript levels were quantified in kidneys exposed to ischemia-reperfusion (6 h) and uninjured kidneys by real-time PCR and normalised by quantifying β-actin transcripts. (B) Lysates from kidneys exposed to ischemia-reperfusion (6 h) or uninjured kidneys were tested by Western blotting using anti-Cezanne antibodies or by using anti-α-tubulin antibodies to assess total protein levels. A representative blot and results from densitometry analysis are shown. (C,D) Left kidneys of wild-type or CezanneGT/GT mice (n=6 per group) were exposed to ischemia followed by reperfusion for 6 h whereas contralateral kidneys were uninjured. (C) Cezanne transcript levels were quantified in kidneys exposed to ischemia-reperfusion and uninjured kidneys by real-time PCR and normalised by quantifying β-actin transcripts. (D) Cezanne expression was assessed by immunohistochemistry in kidneys exposed to ischemia-reperfusion. Positive staining was observed in wild-type tissues in vascular and glomerular EC (arrows), in podocytes (arrowhead) and epithelial cells.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Molecular Chaperone αB-Crystallin Is Expressed in the Human Fetal Telencephalon at Midgestation by a Subset of Progenitor Cells

doi: 10.1097/nen.0b013e3181e5f515

Figure Lengend Snippet: FIGURE 5. Colocalization of >B-crystallin (CRYAB), platelet-derived growth factor receptor > (PDGFR>), and glial fibrillary acidic protein (GFAP) (AYD) and CRYAB and heat shock factor 1 (HSF1) (EYG) by laser scanning confocal microscopy. (AYD) Cells in the cingulate subventricular zone at 19 weeks of gestation (wgs) coexpressing CRYAB (A, green), PDGFR> (B, red), and GFAP (C, blue) are marked on a merged image (D) by arrowheads; a cell immunopositive only for CRYAB and GFAP is marked by an arrow. (EYG) Cells expressing CRYAB in the cingulate ventricular zone/subventricular zone at 15 wgs (E, green) show HSF1 expression (F, red), as shown on merged image (G). Arrow points to a cell showing very low level of HSF1 expression. LV, lateral ventricle; SV, subventricular zone; VZ, ventricular zone. Scale bars = (AYG) 20 mm.

Article Snippet: Method controls included omission of the primary antibodies and preincubation of the pAbs to phosphorylated CRYAB with an excess (30 Kg/mL) of adequate synthetic phospho-peptides: either L(pS)PFYLRPPSF (Ser45P+) or FLRAP(Ps)WIDTG (Ser59P+) (both from New England Peptide, Gardner, MA).

Techniques: Derivative Assay, Confocal Microscopy, Expressing

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Molecular Chaperone αB-Crystallin Is Expressed in the Human Fetal Telencephalon at Midgestation by a Subset of Progenitor Cells

doi: 10.1097/nen.0b013e3181e5f515

Figure Lengend Snippet: FIGURE 6. >B-crystallin (CRYAB) distribution and levels in cortical ventricular/subventricular zone of the body of the lateral ventricle of healthy and Down syndrome fetuses at midgestation. Similar distribution (A, C) and levels (B, D) of CRYAB and CRYAB Ser59P+, respectively, in frontoparietal periventricular proliferative zone of healthy and Down syndrome fetuses. In panels B and D, immunoblots from the periventricular frontoparietal proliferative zone are shown in the upper panels and diagrams presenting densitometry analyses of immunoblots in lower panels. Blots were reprobed with monoclonal antibodies to A-actin. C, normal control fetus; DS, Down syndrome subject; wgs, weeks of gestation. Scale bars = (A, C) 40 mm.

Article Snippet: Method controls included omission of the primary antibodies and preincubation of the pAbs to phosphorylated CRYAB with an excess (30 Kg/mL) of adequate synthetic phospho-peptides: either L(pS)PFYLRPPSF (Ser45P+) or FLRAP(Ps)WIDTG (Ser59P+) (both from New England Peptide, Gardner, MA).

Techniques: Western Blot, Bioprocessing, Control

Journal: Molecules

Article Title: Chemometric Investigation and Antimicrobial Activity of Salvia rosmarinus Spenn Essential Oils

doi: 10.3390/molecules27092914

Figure Lengend Snippet: Diameter of inhibition zones of three bacteria strains as a function of the essential oils from the three cooperatives. Each column represented by different letters (a, b, c, d, e, f, and g) represent a significant difference ( p < 0.05) based on Tukey test. Cn EO: essential oil from different cooperatives.

Article Snippet: Two-way ANOVA showed that there was a statistically significant difference among the means of the inhibition diameters and three cooperative EOs for the three strains used. shows that the essential oil from cooperative C1 had the highest activity, with inhibition zones of 16.6 ± 0.5 mm, 31.3 ± 2.1 mm, and 37.1 ± 1.5 mm for E. coli ATCC25922, B. subtilis ATCC 23857, and M. smegmatis MC2 155, respectively.

Techniques: Inhibition, Bacteria

Journal: Molecules

Article Title: Chemometric Investigation and Antimicrobial Activity of Salvia rosmarinus Spenn Essential Oils

doi: 10.3390/molecules27092914

Figure Lengend Snippet: Minimum inhibition concentration of S. rosmarinus essential oils tested.

Article Snippet: Two-way ANOVA showed that there was a statistically significant difference among the means of the inhibition diameters and three cooperative EOs for the three strains used. shows that the essential oil from cooperative C1 had the highest activity, with inhibition zones of 16.6 ± 0.5 mm, 31.3 ± 2.1 mm, and 37.1 ± 1.5 mm for E. coli ATCC25922, B. subtilis ATCC 23857, and M. smegmatis MC2 155, respectively.

Techniques: Inhibition, Concentration Assay