Journal: Scientific Reports

Article Title: P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus

doi: 10.1038/srep41841

Figure Lengend Snippet: ( a) Relative frequency of peripheral blood leukocyte populations of 1.5–3 month-old WT and P-Sel −/− mice. ( b , d ) Percentage of IL-10 + conventional dendritic cells (cDC), plasmacytoid DC (pDC), monocytes, granulocytes and B cells; and frequency of IL-10 and IL-17 producing CD4 + and CD8 + T lymphocytes, in 1.5-month-old ( b ) and >18-month-old ( d ) WT and P-Sel −/− mice. ( c) Representative dot plots of IL-10 + cDCs and IL-10 and IL-17 producing CD4 + T cells in 1.5-months old WT and P-Sel −/− mice. ( e) Phenotyping of CD4 + and CD8 + splenic T lymphocytes according to the expression of the naïve/memory/effector markers CD62L and CD44 in 1.5–3 months-old (upper panels) and >18 month-old (lower panels) WT and P-Sel −/− mice. ( f) Representative dot plots showing the distribution of 1.5–3-month-old mice splenic populations according to the expression of L-Selectin and CD44. In all cases, n = 4 mice per group. In all cases, n = 4 mice per group. Bars represent the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.005, by Student’s two tailed t test.

Article Snippet: Consecutive tissue sections of sun-exposed skin biopsies underwent immunohistochemistry with the Dako REAL EnVision Detection System Peroxidase/DAB+kit (Dako), using as primary antibodies anti-human P-Selectin (10 μg/ml) (R&D Systems; Minneapolis, MN, USA) and anti-human CD31 (1/50) (Abcam).

Techniques: Expressing, Two Tailed Test

Journal: Scientific Reports

Article Title: P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus

doi: 10.1038/srep41841

Figure Lengend Snippet: ( a) Representative photomicrographs (20×) of anti-CD31 and anti-P-Selectin stained skin biopsies of healthy donors and SLE patients (upper panels). Black arrows point blood vessels. ( b) 200% magnification of representative blood vessels from the original images are represented (lower panels). ( c) Classification and quantification of CD31+ dermal blood vessels according to the expression level of P-Selectin (healthy controls, n = 4; SLE patients, n = 4); bars show the mean ± SD. **p < 0.01 by Student’s two tailed t test.

Article Snippet: Consecutive tissue sections of sun-exposed skin biopsies underwent immunohistochemistry with the Dako REAL EnVision Detection System Peroxidase/DAB+kit (Dako), using as primary antibodies anti-human P-Selectin (10 μg/ml) (R&D Systems; Minneapolis, MN, USA) and anti-human CD31 (1/50) (Abcam).

Techniques: Staining, Expressing, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The role of kinesin, dynein and microtubules in pancreatic secretion

doi: 10.1007/s00018-009-0052-0

Figure Lengend Snippet: Immunoelectron microscopic localization of kinesin, dynein, and tubulin in resting acinar cells. Ultrathin sections of pancreatic tissue were incubated either with anti-kinesin and anti-tubulin antibodies (a–c, g), or with anti-dynein and anti-tubulin antibodies (d–f) followed by 6 and 12 nm gold-conjugated secondary antibodies (see “Materials and methods”). Lumen of acinar cells (L) and zymogen granules (ZG) are marked. Boxes denote regions of higher magnification shown in b, c, e, f. Neither dynein nor kinesin can be detected in the apical cellular region (c, f) or at the surface of zymogen granules (b, e, g). Tubulin, detected with 6 nm gold particles colocalizes with kinesin and dynein in the perinuclear region (b, e), and can additionally be found near the apical lumen of acinar cells (c, f). Images shown are representative for at least five experiments. Bars 5 μm (a, d), 1 μm (g) or 0.25 μm (b, c, e, f)

Article Snippet: The antibodies (Ab) used were as follows: goat anti-mouse IgG Cy™3-labeled Ab (Jackson Immuno Research, West Grove, PA, USA), goat anti-mouse Biotin-conjugated IgG (Vector Laboratories), goat anti-mouse IgG horseradish peroxidase conjugated (Sigma), goat anti-mouse biotin-SP-conjugated IgG (Jackson Immuno Research), goat anti-mouse IgG labeled with 12 nm colloidal gold (Dianova), goat anti-rabbit IgG labeled with 6 nm colloidal gold (Dianova), goat anti-rabbit Alexa-594 labeled IgG (Molecular Probes, Eugene, OR, USA), mouse monoclonal antibody (mAb) raised against Golgi 58 K protein/formiminotransferase cyclodeaminase (FTCD) (Sigma), anti-dynein mAb cytoplasmic 74 kD intermediate chains (Chemicon International, Temecula, CA, USA), mAb raised against kinesin heavy chain (Chemicon International), anti-β-tubulin mAb (Sigma), mouse IgG (Dianova), rabbit polyclonal antibody (Ab) raised against tubulin (Sigma), sheep FITC-conjugated anti-digoxigenin Ab (Boehringer Mannheim, Germany).

Techniques: Incubation

Journal: Physiological reports

Article Title: Uromodulin deficiency alters tubular injury and interstitial inflammation but not fibrosis in experimental obstructive nephropathy.

doi: 10.14814/phy2.13654

Figure Lengend Snippet: Figure 3. The tubular injury marker KIM-1 is higher in mice lacking in UMOD during obstructive nephropathy. By western blotting, kidney KIM-1 protein levels were increased after UUO (n = 5 for sham groups; n = 7 for UUO groups) at all time-points (days 7, 14, and 21); the levels were significantly higher in the UMOD/ groups. Results are corrected for protein loading using GAPDH levels. (A, B). Kidney KIM-1 mRNA levels measured by real-time PCR (n = 3 for sham groups; n = 5 for UUO groups) were significantly higher on UUO days 14 and 21 (C). Representative day 14 immunofluorescence photomicrographs illustrate more intense KIM-1 staining in the UMOD/ kidney (D). KIM-1 staining was concentrated along the apical membrane with lesser amounts in tubular lumina; the latter more evident in the UMOD+/+ mice. Brightly stained ribbon-like KIM-1 + bands, frequently observed along the apical membrane in the UMOD-/- mice, were much less common in the UMOD+/+ mice. Dual staining for UMOD (green) and KIM-1 (red) in a UMOD+/+ day 14 kidney illustrates an area of tubular intraluminal staining for both proteins (yellow; highlighted by the arrow) (E). Dual staining for UMOD (green) and the proximal tubular apical membrane protein LRP2 (red) in a UMOD+/+ day 14 kidney illustrates the presence of luminal UMOD deposits surrounded by LRP2 + tubules (arrows) (F). Bar graphs represent means +1SEM; * indicates a P value < 0.05. Photomicrograph magnification: 940. Scale bars: 25 lm.

Article Snippet: Primary antibodies used in the study were: sheep-anti mouse UMOD, goat-anti-mouse KIM-1, mouse-antihuman KIM-1, goat-anti mouse NGAL (R&D Systems Inc, Minneapolis, MN, USA); sheep-anti-human UMOD, rat anti-mouse F4/80 (AbD Serotec/Bio-Rad, Mississauga, ON, Canada); mouse anti-mouse aSMA, rabbit antihuman fibronectin (Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-mouse/human/rat TRPV5 (Alomone Labs, Jerusalem, Israel); mouse anti-human PCNA (eBioscience/ Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-mouse/human/monkey megalin/LRP-2 (Abcam, Toronto, ON, Canada); rabbit-anti-mouse Laminin (Millipore/Sigma), mouse-anti-mammalian acetylated a-Tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-mouse GAPDH (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Marker, Western Blot, Real-time Polymerase Chain Reaction, Staining, Membrane

Journal: Physiological reports

Article Title: Uromodulin deficiency alters tubular injury and interstitial inflammation but not fibrosis in experimental obstructive nephropathy.

doi: 10.14814/phy2.13654

Figure Lengend Snippet: Figure 4. Effect of UMOD on other tubular injury markers in UUO nephropathy. Kidney NGAL protein levels, measured by western blotting, were elevated after UUO (n = 5 for sham groups; n = 7 for UUO groups) at all time-points (days 7, 14, and 21); differences between the genotypes were only found on day 21, when they were significantly higher in the UMOD+/+ group (A, B). Kidney TRPV5 mRNA levels are markedly upregulated after UUO (n = 3 for sham groups; n = 5 for UUO groups); at all time-points (days 7, 14, and 21) mRNA levels were significantly higher in the UMOD+/+ groups (C). By dual staining fluorescence microscopy, TRPV5 (red) was almost exclusively found in tubules with intraluminal UMOD deposits (green), as shown for a day 14 UUO UMOD+/+ kidney (D). Bar graphs represent means +1SEM; * indicates P value < 0.05. Photomicrograph magnification: 940. Scale bar: 25 lm.

Article Snippet: Primary antibodies used in the study were: sheep-anti mouse UMOD, goat-anti-mouse KIM-1, mouse-antihuman KIM-1, goat-anti mouse NGAL (R&D Systems Inc, Minneapolis, MN, USA); sheep-anti-human UMOD, rat anti-mouse F4/80 (AbD Serotec/Bio-Rad, Mississauga, ON, Canada); mouse anti-mouse aSMA, rabbit antihuman fibronectin (Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-mouse/human/rat TRPV5 (Alomone Labs, Jerusalem, Israel); mouse anti-human PCNA (eBioscience/ Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-mouse/human/monkey megalin/LRP-2 (Abcam, Toronto, ON, Canada); rabbit-anti-mouse Laminin (Millipore/Sigma), mouse-anti-mammalian acetylated a-Tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-mouse GAPDH (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Staining, Microscopy

Journal: Physiological reports

Article Title: Uromodulin deficiency alters tubular injury and interstitial inflammation but not fibrosis in experimental obstructive nephropathy.

doi: 10.14814/phy2.13654

Figure Lengend Snippet: Figure 9. UMOD reduces tubular cell KIM-1 levels in vitro. Preliminary studies to maximize the exposure of KIM-1 producing 769-P cells to UMOD demonstrate that both 769-P and 293 cells transiently transfected with a pcDNA3.1 vector expressing human UMOD (pcDNA3.1-UMOD) produced readily detectable UMOD protein levels within cells and their conditioned medium compared with cells transfected with a pcDNA3.1 vector expressing a noncoding control vector (pcDNA3.1-C) (A). Under transwell culture conditions, 769-P cells transfected with pcDNA3.1-UMOD (769-P- UMOD) and harvested from the upper chamber after exposure to UMOD from both the upper and lower chambers for 48 h, expressed significantly lower KIM-1 protein levels compared with cells transfected with pcDNA3.1-C (769-P-C) and not exposed to UMOD (B). Lower chambers contained 293 cells transfected with pcDNA3.1-C (293-C) or pcDNA3.1-UMOD (293-UMOD). Results are western blot data corrected for GAPDH protein levels (n = 3). KIM- 1 mRNA levels in 769-P cells cultured under the same conditions (n = 3) were similar between control transfected and UMOD transfected cells (C). * indicates P value < 0.05.

Article Snippet: Primary antibodies used in the study were: sheep-anti mouse UMOD, goat-anti-mouse KIM-1, mouse-antihuman KIM-1, goat-anti mouse NGAL (R&D Systems Inc, Minneapolis, MN, USA); sheep-anti-human UMOD, rat anti-mouse F4/80 (AbD Serotec/Bio-Rad, Mississauga, ON, Canada); mouse anti-mouse aSMA, rabbit antihuman fibronectin (Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-mouse/human/rat TRPV5 (Alomone Labs, Jerusalem, Israel); mouse anti-human PCNA (eBioscience/ Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-mouse/human/monkey megalin/LRP-2 (Abcam, Toronto, ON, Canada); rabbit-anti-mouse Laminin (Millipore/Sigma), mouse-anti-mammalian acetylated a-Tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-mouse GAPDH (Cell Signaling Technology, Danvers, MA, USA).

Techniques: In Vitro, Transfection, Plasmid Preparation, Expressing, Produced, Control, Western Blot, Cell Culture