immunodetection  (OriGene)

Journal: Antioxidants

Article Title: A Preliminary Study on the Effect of Hydrogen Gas on Alleviating Early CCl 4 -Induced Chronic Liver Injury in Rats

doi: 10.3390/antiox10121933

Figure Lengend Snippet: Changes in the expression of uncoupling protein 2 (UCP2) were observed by immunohistochemistry. ( a , b ) The expression of UCP2 in liver tissue of control group rats. A small number of UCP2-positive cells were brownish yellow in the cytoplasm. ( c , d ) The expression of UCP2 in liver tissue of CCl 4 group rats. A small number of UCP2 positive cells were brownish yellow in the cytoplasm. ( e , f ) The expression of UCP2 in liver tissue of CCl 4 + H 2 group rats. UCP2 was diffusely expressed in liver cells, and the staining sites were mostly located on the cell membrane. ( g , h ) The expression of UCP2 in liver tissue of H 2 group rats. The number of UCP2 positive cells with brownish-yellow staining in the cytoplasm increased significantly. ( a , c , e , g ) Fields of vision at 40× magnification. ( b , d , f , h ) Higher magnifications of the area outlined in ( a , c , e , g ). The long scale bar refers to 100 μm. The short scale bar refers to 50 μm.

Article Snippet: The membrane was incubated with antibodies of goat anti-UCP2 (bs-20750R, Bioss, Beijing, China) and mouse anti-β actin (A2228, Sigma, St. Louis, MO, USA) overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Antioxidants

Article Title: A Preliminary Study on the Effect of Hydrogen Gas on Alleviating Early CCl 4 -Induced Chronic Liver Injury in Rats

doi: 10.3390/antiox10121933

Figure Lengend Snippet: UCP2-positive area rate. N = 8, * p < 0.05.

Article Snippet: The membrane was incubated with antibodies of goat anti-UCP2 (bs-20750R, Bioss, Beijing, China) and mouse anti-β actin (A2228, Sigma, St. Louis, MO, USA) overnight at 4 °C.

Techniques:

Journal: Antioxidants

Article Title: A Preliminary Study on the Effect of Hydrogen Gas on Alleviating Early CCl 4 -Induced Chronic Liver Injury in Rats

doi: 10.3390/antiox10121933

Figure Lengend Snippet: Expression changes of UCP2 (Western blotting). ( a ) Representative western blotting image of UCP2. ( b ) Statistical results of western blotting. N = 8, * p < 0.05.

Article Snippet: The membrane was incubated with antibodies of goat anti-UCP2 (bs-20750R, Bioss, Beijing, China) and mouse anti-β actin (A2228, Sigma, St. Louis, MO, USA) overnight at 4 °C.

Techniques: Expressing, Western Blot

Journal: Nutrients

Article Title: A Novel Nutraceuticals Mixture Improves Liver Steatosis by Preventing Oxidative Stress and Mitochondrial Dysfunction in a NAFLD Model

doi: 10.3390/nu13020652

Figure Lengend Snippet: Primers list.

Article Snippet: In total, 40 μg samples of proteins from liver homogenates were loaded in a 10% SDS-PAGE and transferred to a nitrocellulose membrane, blocked for 1.5 h using 5% non-fat dry milk in TBS-t and incubated over night at 4 °C with primary UCP2 antibody (goat polyclonal UCP2 purchased from Santa cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques:

Journal: Nutrients

Article Title: A Novel Nutraceuticals Mixture Improves Liver Steatosis by Preventing Oxidative Stress and Mitochondrial Dysfunction in a NAFLD Model

doi: 10.3390/nu13020652

Figure Lengend Snippet: Flinax up-regulates uncoupling proteins. ( A ) mRNA levels of UCP2 fold over HFHCD+CD (HFHCD+CD = n7; HFHCD+CD&Flinax = n6); ( B ) Representative picture of western blot analysis of UCP-2; ( C ) mRNA levels of UCP three-fold over Ctrl (HFHCD+CD=n6; HFHCD+CD&Flinax = n6). Data are expressed in mean ± SEM; * p < 0.05 according to the Student’s t test. CD, chow diet; HFHCD, high-fat, high-cholesterol diet; UCP2, uncoupling protein 2; UCP3, uncoupling protein 3.

Article Snippet: In total, 40 μg samples of proteins from liver homogenates were loaded in a 10% SDS-PAGE and transferred to a nitrocellulose membrane, blocked for 1.5 h using 5% non-fat dry milk in TBS-t and incubated over night at 4 °C with primary UCP2 antibody (goat polyclonal UCP2 purchased from Santa cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques: Western Blot

Journal: JCI Insight

Article Title: Endothelial mitochondria determine rapid barrier failure in chemical lung injury

doi: 10.1172/jci.insight.124329

Figure Lengend Snippet: (A) Confocal image shows endothelial immunofluorescence in lung capillaries (cap) at low magnification. The other image shows the region of interest marked by the rectangle at high magnification. Capillaries were microinfused with 4% paraformaldehyde and 0.2% Triton X-100 (45 minutes), followed by Alexa Fluor 633–labeled anti–UCP2 goat polyclonal antibody (120 ng/ml, 30 minutes). Capillaries were buffer washed (5 minutes). n = 4 lungs. Scale bar: 20 μm. (B–E) Data are for analyses carried out 48 hours after tail vein injection of indicated siRNA. (B) Gel and bars show immunoblotting (IB) and densitometry of mitochondria isolated from lung homogenates. The antibodies were UCP2 mouse monoclonal antibody and voltage-dependent anion channel (VDAC) rabbit polyclonal antibody. Lanes were run on the same gel. Vertical line indicates the lanes are not contiguous. Results were identical for IB using UCP2 goat polyclonal antibody (data not shown). SI, siUCP2; SC, scRNA. *P < 0.05 versus scRNA. (C) Bars show effects of indicated treatments following alveolar HCl or microvascular H2O2 injections. KO, endothelial cell–specific UCP2-KO; LM, littermate control. For each pair, *P < 0.05 versus left bar. (D) Confocal images show dextran distribution at indicated locations. The endothelial cytosol was loaded with CR. FITC-D70 was infused in vessels at baseline and then again after injection of alveolar HCl. Scale bar: 20 μm. (E) Bars quantify FITC-D70–filled alveoli (edematous alveoli) following the indicated alveolar injections. Equal numbers of alveoli were injected in each group. Alveoli with greater than 50% luminal area filled with FITC-D70 were defined as edematous. UN, untreated. *P < 0.05 versus PBS. Data are shown as mean ± SEM for the number of injections indicated by dots. n, number of lungs. One-way ANOVA with post hoc Bonferroni’s test was used to determine statistical differences between groups.

Article Snippet: UCP2 goat polyclonal antibody (catalog sc-6525), UCP2 mouse monoclonal antibody (catalog sc-390189), and VDAC mouse monoclonal antibody (catalog sc-390996) were purchased from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Labeling, Injection, Western Blot, Isolation, Control

Journal: JCI Insight

Article Title: Endothelial mitochondria determine rapid barrier failure in chemical lung injury

doi: 10.1172/jci.insight.124329

Figure Lengend Snippet: (A) HCl contact with the alveolar epithelium causes pore formation and NOX2-induced diffusion of H2O2 to the capillary endothelium (1). (B) H2O2 activates endothelial UCP2, causing mitochondrial proton entry and depolarization. (C) Loss of mitochondrial potential leads to increases in cytosolic Ca2+, which depolymerizes F-actin by a calcineurin-cofilin mechanism. F-actin decrease causes endothelial hyperpermeability.

Article Snippet: UCP2 goat polyclonal antibody (catalog sc-6525), UCP2 mouse monoclonal antibody (catalog sc-390189), and VDAC mouse monoclonal antibody (catalog sc-390996) were purchased from Santa Cruz Biotechnology.

Techniques: Diffusion-based Assay

Journal: Redox Biology

Article Title: Upregulation of UCP2 in beta-cells confers partial protection against both oxidative stress and glucotoxicity

doi: 10.1016/j.redox.2017.07.012

Figure Lengend Snippet: UCP2 over-expression in ß-cells of islets exposed to glucotoxic conditions. WT and RIP- UCP2 islets were collected for analysis 7 days after culture at either 11 mM glucose (11 mM) or 30 mM glucotoxic condition (30 mM). (A) UCP2 protein levels, normalized to ß-actin, were assessed by immunoblotting. Bar-graph shows means ± SE of 3 independent experiments, p < 0.05 versus WT. (B) binocular observation of islets at the end of the culture period.

Article Snippet: Membranes were then incubated overnight at 4 °C with different antibodies: goat anti-human polyclonal antibody to UCP2 (1:1000, #6527, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-actin (1:5000, #4700, Sigma-Aldrich) in PBS containing 3% BSA and 0.05% Tween-20.

Techniques: Over Expression, Western Blot

Journal: Redox Biology

Article Title: Upregulation of UCP2 in beta-cells confers partial protection against both oxidative stress and glucotoxicity

doi: 10.1016/j.redox.2017.07.012

Figure Lengend Snippet: Plasma parameters following a glucose challenge and weight of RIP- UCP2 mice.

Article Snippet: Membranes were then incubated overnight at 4 °C with different antibodies: goat anti-human polyclonal antibody to UCP2 (1:1000, #6527, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-actin (1:5000, #4700, Sigma-Aldrich) in PBS containing 3% BSA and 0.05% Tween-20.

Techniques: Clinical Proteomics

Journal: Redox Biology

Article Title: Upregulation of UCP2 in beta-cells confers partial protection against both oxidative stress and glucotoxicity

doi: 10.1016/j.redox.2017.07.012

Figure Lengend Snippet: Effects of UCP2 over-expression on mitochondrial membrane potential in islets exposed to glucotoxic conditions. At the end of the 7-day culture period at either standard 11 mM glucose (11) or glucotoxic condition (30), WT and RIP- UCP2 islets were assayed for mitochondrial membrane potential. (A) WT and RIP- UCP2 islets from the standard 11 culture conditions were monitored first at low 2.8 mM Glc followed by acute stimulation with 22.8 mM Glc (Glucose). (B) WT and RIP- UCP2 islets from either standard 11 or glucotoxic 30 culture condition were monitored at low 2.8 mM Glc before complete depolarization of the mitochondrial membrane induced by the addition of 1 µM FCCP. (C, D) WT (C) and RIP- UCP2 (D) islets from either standard 11 or glucotoxic 30 culture condition were monitored first at low 2.8 mM Glc followed by acute stimulation with 22.8 mM Glc (Glucose). Values are means from 3 independent experiments (n = 11–14 islets); * p < 0.05 wt 30 versus WT 11, # p < 0.05 RIP- UCP2 30 versus RIP- UCP2 11.

Article Snippet: Membranes were then incubated overnight at 4 °C with different antibodies: goat anti-human polyclonal antibody to UCP2 (1:1000, #6527, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-actin (1:5000, #4700, Sigma-Aldrich) in PBS containing 3% BSA and 0.05% Tween-20.

Techniques: Over Expression, Membrane

Journal: Redox Biology

Article Title: Upregulation of UCP2 in beta-cells confers partial protection against both oxidative stress and glucotoxicity

doi: 10.1016/j.redox.2017.07.012

Figure Lengend Snippet: Effects of UCP2 over-expression on cellular ATP levels in islets exposed to glucotoxic conditions. At the end of the 7-day culture period at either standard glucose (11 mM) or glucotoxic condition (30 mM), WT and RIP- UCP2 islets transduced with Ad-ATEAM were pre-incubated for 1 h at 2.8 mM glucose before ATP monitoring by FRET, first at low glucose (2.8 mM) before acute stimulation with 22.8 mM (Glucose). Overall mitochondrial contribution to ATP levels was determined by further addition of 2 mM of the mitochondrial poison azide. (A) Signals were acquired every 15 s and kinetic data were normalized to the azide response for WT (orange lines) and RIP- UCP2 (blue lines) islets cultured in standard (11, dashed lines) or glucotoxic (30, solid lines) conditions. (B) Corresponding quantifications of glucose-stimulated ATP rise shown as fold changes over basal levels. (A) values are means ± SE; * p < 0.05 wt 30 versus WT 11 (orange*) and RIP- UCP2 30 versus RIP- UCP2 11 (blue*), # p < 0.05 RIP- UCP2 30 versus WT 30; (B) * p < 0.05 RIP- UCP2 30 versus RIP- UCP2 11; # p < 0.05 RIP- UCP2 11 versus WT 11. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Membranes were then incubated overnight at 4 °C with different antibodies: goat anti-human polyclonal antibody to UCP2 (1:1000, #6527, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-actin (1:5000, #4700, Sigma-Aldrich) in PBS containing 3% BSA and 0.05% Tween-20.

Techniques: Over Expression, Transduction, Incubation, Cell Culture

Journal: Redox Biology

Article Title: Upregulation of UCP2 in beta-cells confers partial protection against both oxidative stress and glucotoxicity

doi: 10.1016/j.redox.2017.07.012

Figure Lengend Snippet: Effects of UCP2 over-expression on cellular [Ca 2+ ] changes in islets exposed to glucotoxic conditions. At the end of the 7-day culture period at either standard glucose (11 mM) or glucotoxic condition (30 mM), cellular [Ca 2+ ] changes were monitored in WT and RIP- UCP2 as ratiometric measurements of Fura-2 fluorescence. Islets were first kept at low 2.8 mM glucose before raising glucose to 22.8 mM (Glucose), as indicated. Finally, 1 µM thapsigargin was added to release Ca 2+ from the endoplasmic reticulum. Signals were acquired every 10 s. Data were calculated as 340/380 ratio from an average of islets from 3 mice in each condition (A) and then normalized to low glucose levels (B). Values are means ± SE; * p < 0.05 wt 30 versus WT 11 (orange*) and RIP- UCP2 30 versus RIP- UCP2 11 (blue*), # p < 0.05 RIP- UCP2 11 versus WT 11. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Membranes were then incubated overnight at 4 °C with different antibodies: goat anti-human polyclonal antibody to UCP2 (1:1000, #6527, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-actin (1:5000, #4700, Sigma-Aldrich) in PBS containing 3% BSA and 0.05% Tween-20.

Techniques: Over Expression, Fluorescence

Journal: Redox Biology

Article Title: Upregulation of UCP2 in beta-cells confers partial protection against both oxidative stress and glucotoxicity

doi: 10.1016/j.redox.2017.07.012

Figure Lengend Snippet: Effects of UCP2 over-expression on insulin secretion and cell death in islets exposed to glucotoxic conditions. (A) Glucose stimulated insulin secretion from WT and RIP- UCP2 islets was tested at the end of the 7-day culture period at either standard glucose (11 mM Glc) or glucotoxic condition (30 mM Glc). Both WT and RIP- UCP2 islets were pre-incubated at 2.8 mM glucose for 1hr and then insulin release was measured from islets kept at 2.8 mM glucose (Low) or stimulated by 22.8 mM glucose (Stim) for 1hr. * p < 0.05 versus corresponding Low of the same genotype and the same culture condition, # p < 0.05 versus corresponding WT in the same culture condition, & p < 0.05 versus corresponding secretion assay condition from the 11 mM Glc culture islets. (B) Islet cell apoptosis was determined in cell lysates by quantification of cytoplasmic nucleosomes in WT and RIP- UCP2 islets collected at the end of the 7-day culture period. ** p < 0.01, * p < 0.05 versus corresponding genotype of 11 mM Glc; # p < 0.05 versus WT of 30 mM Glc.

Article Snippet: Membranes were then incubated overnight at 4 °C with different antibodies: goat anti-human polyclonal antibody to UCP2 (1:1000, #6527, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-actin (1:5000, #4700, Sigma-Aldrich) in PBS containing 3% BSA and 0.05% Tween-20.

Techniques: Over Expression, Incubation

Journal: Redox Biology

Article Title: Upregulation of UCP2 in beta-cells confers partial protection against both oxidative stress and glucotoxicity

doi: 10.1016/j.redox.2017.07.012

Figure Lengend Snippet: Effects of UCP2 over-expression and of oxidative stress on UCP2 levels and insulin secretion. Isolated WT and RIP- UCP2 islets were subjected to transient oxidative stress (200 µM H 2 O 2 for 10 min). Then, islets were either collected immediately (Acute ox-stress, A, C) or after a 3-day recovery period (Post ox-stress 3 days, B, C). (A, B) UCP2 protein levels, normalized to ß-actin, were assessed by immunoblotting. Bar-graphs show averages of 3 individual experiments. (C) Insulin secretion over a 1 h incubation at either 2.8 mM glucose (Low) or stimulatory 22.8 mM glucose (Stim) was determined both directly after the 10 min transient ox-stress (Acute stress) and 3 days after H 2 O 2 exposure (3 days after stress), and compared with islets not exposed to H 2 O 2 (No stress). * p < 0.05 versus corresponding Low of the same genotype in the same condition; ## p < 0.01 versus corresponding condition in No stress; &, p < 0.05 versus corresponding WT of the same stress.

Article Snippet: Membranes were then incubated overnight at 4 °C with different antibodies: goat anti-human polyclonal antibody to UCP2 (1:1000, #6527, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-actin (1:5000, #4700, Sigma-Aldrich) in PBS containing 3% BSA and 0.05% Tween-20.

Techniques: Over Expression, Isolation, Western Blot, Incubation

Journal: Redox Biology

Article Title: Upregulation of UCP2 in beta-cells confers partial protection against both oxidative stress and glucotoxicity

doi: 10.1016/j.redox.2017.07.012

Figure Lengend Snippet: Effects of UCP2 over-expression on superoxide generation in islets exposed to glucotoxic conditions. At the end of the 7-day culture period at either standard glucose (11 mM Glc) or glucotoxic condition (30 mM Glc), superoxide generation was measured in WT and RIP- UCP2 islets either immediately at the end of the 7-day culture (A) or following further glucose challenge (B). (A) superoxide levels from control (11 mM Glc) and glucotoxic (30 mM Glc) conditions in both WT and RIP- UCP2 islets. * p < 0.05 versus WT from the 11 mM Glc culture condition. (B) superoxide levels were measured in WT and RIP- UCP2 islets following further acute incubation at 2.8 mM glucose (Low) and 22.8 mM glucose (Stim) after 1hr pre-incubation at 2.8 mM glucose. ** p < 0.01, * p < 0.05 versus corresponding Low of the same genotype.

Article Snippet: Membranes were then incubated overnight at 4 °C with different antibodies: goat anti-human polyclonal antibody to UCP2 (1:1000, #6527, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-actin (1:5000, #4700, Sigma-Aldrich) in PBS containing 3% BSA and 0.05% Tween-20.

Techniques: Over Expression, Control, Incubation