monoclonal anti beta actin  (Millipore)


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    Name:
    Monoclonal Anti beta Actin antibody
    Description:
    Actin is a highly conserved protein that is a major component of both the cytoskeletal and contractile structures in all cell types It varies in amount being related to the type of differentiation and to the functional state of cells and tissues Actin can be found in two different forms of aggregation the globular or the fibrillar state and at least six distinct isoforms occur in vertebrates The actins exhibit over 90 sequence homology but each isoform has a unique NH2 terminal sequence The isoforms are comprised of three alpha actins skeletal cardiac smooth one beta actin beta non muscle and two gamma actins gamma smooth muscle and gamma non muscle Recognizes an epitope located on the N terminal end of the beta isoform of actin It specifically labels beta actin in a wide variety of tissues and species In immunofluorescent staining of chicken gizzard ultrathin cryosections the antibody labels the dense bodies the longitudinal channels linking consecutive dense bodies that are also occupied by desmin and the membrane associated dense plaque The antibody does not react with adult cardiac or skeletal muscle besides traces due to contamination of the sample with non muscle cells or with beta actin expressing cells in Dictyostelium discoideum amoeba The epitope recognized by the antibody is resistant to formalin fixation and paraffin embedding B5 methacarn ethanol or Bouin s solutions may be also used as fixatives The antibody can be used as probes for beta actin as an internal control in immunoblotting
    Catalog Number:
    A5316
    Molecular Weight:
    antigen mol wt 42 kDa
    Price:
    None
    Applications:
    Mouse Monoclonal Anti-β-Actin antibody has been used for western blot analyses. The antibody can also be used for immunohistochemistry, indirect immunofluorescence (1:1,000) using cultured human or chicken fibroblasts, and indirect ELISA.Western blot analysis of MDCK cell lysates were performed using monoclonal anti-actin antibody as a primary antibody.Monoclonal mouse anti-actin antibody was used as a loading control for western blot analysis of immunoprecipitated proteins from rat dorsal root ganglion cocultures.
    Host:
    mouse
    Conjugate:
    unconjugated
    Immunogen:
    slightly modified β-cytoplasmic actin N-terminal peptide, Ac-Asp-Asp-Asp-Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly-Ser-Gly-Lys, conjugated to KLH.
    Category:
    Antibodies
    Isotype:
    IgG2a
    Source:
    goat
    Reactivity:
    human
    Score:
    85
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    Structured Review

    Millipore monoclonal anti beta actin
    Monoclonal Anti beta Actin antibody
    Actin is a highly conserved protein that is a major component of both the cytoskeletal and contractile structures in all cell types It varies in amount being related to the type of differentiation and to the functional state of cells and tissues Actin can be found in two different forms of aggregation the globular or the fibrillar state and at least six distinct isoforms occur in vertebrates The actins exhibit over 90 sequence homology but each isoform has a unique NH2 terminal sequence The isoforms are comprised of three alpha actins skeletal cardiac smooth one beta actin beta non muscle and two gamma actins gamma smooth muscle and gamma non muscle Recognizes an epitope located on the N terminal end of the beta isoform of actin It specifically labels beta actin in a wide variety of tissues and species In immunofluorescent staining of chicken gizzard ultrathin cryosections the antibody labels the dense bodies the longitudinal channels linking consecutive dense bodies that are also occupied by desmin and the membrane associated dense plaque The antibody does not react with adult cardiac or skeletal muscle besides traces due to contamination of the sample with non muscle cells or with beta actin expressing cells in Dictyostelium discoideum amoeba The epitope recognized by the antibody is resistant to formalin fixation and paraffin embedding B5 methacarn ethanol or Bouin s solutions may be also used as fixatives The antibody can be used as probes for beta actin as an internal control in immunoblotting
    https://www.bioz.com/result/monoclonal anti beta actin/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti beta actin - by Bioz Stars, 2019-12
    99/100 stars

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    Clone Assay:

    Article Title: LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication
    Article Snippet: hATG5 and hATG16L1 sequences were cloned into peGFP-C1 plasmid (Clontech) to form pGFP-ATG5 and pGFP-ATG16L1, respectively. .. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5 (used for western blot), mouse monoclonal anti-Flag, and mouse monoclonal anti-β-actin antibodies were purchased from Sigma Aldrich (USA).

    Centrifugation:

    Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line
    Article Snippet: PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich). .. PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich).

    Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS
    Article Snippet: The β-actin antibody was from Sigma-Aldrich (cat. no. A2228; 1:2,000, Merck KGaA, Darmstadt, Germany), antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3)-I/II (cat. no. 3868; 1:1,000), cleaved caspase-3 (cas3; cat. no. 9661; 1:500) and p62 (cat. no. 5114; 1:1,000) were from Cell Signaling Technology, Inc., the p53 (cat. no. sc-6243; 1:1,000) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the caspase-8 (cas8; cat. no. 551242; 1:1,000) antibody was from BD Biosciences (Franklin Lakes, NJ, USA). .. The β-actin antibody was from Sigma-Aldrich (cat. no. A2228; 1:2,000, Merck KGaA, Darmstadt, Germany), antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3)-I/II (cat. no. 3868; 1:1,000), cleaved caspase-3 (cas3; cat. no. 9661; 1:500) and p62 (cat. no. 5114; 1:1,000) were from Cell Signaling Technology, Inc., the p53 (cat. no. sc-6243; 1:1,000) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the caspase-8 (cas8; cat. no. 551242; 1:1,000) antibody was from BD Biosciences (Franklin Lakes, NJ, USA).

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251). .. Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251).

    Article Title: Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells
    Article Snippet: The cellular debris was removed by centrifugation (8,000×g for 15 min) at 4°C, and the cell lysate was carefully transferred to a microcentrifuge tube. .. The membranes were blocked at 4°C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for 8 h. Blots were incubated with the appropriate antibodies overnight at 4°C: anti-CREB (1∶1000), anti-phospho-CREB (Ser-133) (1∶1000), anti-ERK (1∶1000 ), anti-phospho-ERK (1∶1000 ) (Cell Signaling Technology, Inc.), and Monoclonal anti-β actin (1∶8000) (Sigma-Aldrich Co.).

    Autoradiography:

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251). .. Specific antibody binding was revealed using the Western Lightning-ECL chemiluminiscence system (PerkinElmer, Waltham, MA, USA), according to the manufacturer’s recommendations.

    Blocking Assay:

    Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line
    Article Snippet: PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich). .. PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich).

    Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
    Article Snippet: Membranes were blocked in blocking buffer (5% dry milk in PBS with 0.01% Tween) for one hour and incubated overnight at 4 °C with respective primary antibodies diluted in PBS with 5% dry milk and 0.01% Tween. .. The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000).

    Article Title: Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells
    Article Snippet: Cell lysate (30 µg) was separated on 10% SDS-PAGE and transferred onto a PVDF membrane (PerkinElmer, Boston, MA, USA) at 25 volts overnight at 4°C. .. The membranes were blocked at 4°C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for 8 h. Blots were incubated with the appropriate antibodies overnight at 4°C: anti-CREB (1∶1000), anti-phospho-CREB (Ser-133) (1∶1000), anti-ERK (1∶1000 ), anti-phospho-ERK (1∶1000 ) (Cell Signaling Technology, Inc.), and Monoclonal anti-β actin (1∶8000) (Sigma-Aldrich Co.). .. After three washes with PBST, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1∶10,000) (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. The blots were washed with PBST and the proteins were detected by Western Lightning™ Chemiluminescence Reagent Plus (PerkinElmer) according to the manufacturer’s instructions, and the chemiluminescence signal was visualized with Amersham Hyperfilm™ ECL (GE Healthcare, Buckinghamshire, UK).

    Electrophoresis:

    Article Title: Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor
    Article Snippet: Supernatants from TIMP-3/HEK or HEK293 cells were loaded onto an acrylamide gel for electrophoresis. .. After electrophoretic separation, proteins were blotted onto a PVDF membrane using the Trans-Blot Turbo transfer system (Biorad) and detected by the following antibodies: anti-TIMP-3 (AB6000, Millipore), anti-TIMP-1 (generated as previously described ), anti-TIMP-2 (generated as previously described ), anti-SPARC (number 5031, Thermo Fisher), anti-MIF (clone FL-115, Santa Cruz) anti-APP (clone 22C11, Millipore), anti-MMP-1 (clone 2A7.2, Millipore) anti-actin (A5316, Sigma Aldrich).

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: After electrophoresis the proteins were electrotransferred onto nitrocellulose membranes (Whatman, Dassel, Germany) for 1.5 h at 100 mV using an electrophoretic transfer system (Mini-Trans-blot Electrophoretic Transfer Cell). .. Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251).

    Modification:

    Article Title: Urinary exosomal expression of activator of G protein signaling 3 in polycystic kidney disease
    Article Snippet: Mouse anti-β-actin (1:4000; cat #A5441, Sigma, St. Louis, MO) was used as a loading control. .. Rat brain lysates were used as a positive control, since AGS3 is enriched in brain tissue [ ].

    Incubation:

    Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells
    Article Snippet: The whole-cell proteins (35 μ g/lane) were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane. .. Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or anti– β -actin antibody (1:5000 dilution, catalog #A5441; Sigma-Aldrich) overnight at 4°C. .. The membranes were subsequently incubated with the secondary horseradish peroxidase–labeled anti-mouse (1:3000 dilution, catalog #7076; Cell Signaling Technology, Danvers, MA) or anti-rabbit IgG antibodies (1:10,000 dilution, catalog #111035003); Jackson ImmunoResearch, West Grove, PA) for 2 hours, followed with Clarity Western ECL substrates.

    Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line
    Article Snippet: PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich). .. PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich).

    Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
    Article Snippet: Membranes were blocked in blocking buffer (5% dry milk in PBS with 0.01% Tween) for one hour and incubated overnight at 4 °C with respective primary antibodies diluted in PBS with 5% dry milk and 0.01% Tween. .. The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000).

    Article Title: Thyroxin Protects White Matter from Hypoxic-Ischemic Insult in the Immature Sprague–Dawley Rat Brain by Regulating Periventricular White Matter and Cortex BDNF and CREB Pathways
    Article Snippet: We detected proteins of interest with a chemiluminescence ECL system (GE Healthcare, Chicago, IL, USA) using secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA). .. The blots were stripped with buffer (2.5% SDS, 0.7% 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) and incubated with the β-actin antibody (catalog #A5316, Sigma), followed by a secondary antibody and visualized with the chemiluminescence ECL system. .. The blots from each experiment were densitometrically analyzed using Image J. OD values, which were normalized to β-actin, and graphs are presented as “adjusted OD”.

    Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS
    Article Snippet: Membranes were blocked with 5% non-fat dried milk at 25°C for 1 h, followed by incubation with primary antibodies overnight at 4°C. .. The β-actin antibody was from Sigma-Aldrich (cat. no. A2228; 1:2,000, Merck KGaA, Darmstadt, Germany), antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3)-I/II (cat. no. 3868; 1:1,000), cleaved caspase-3 (cas3; cat. no. 9661; 1:500) and p62 (cat. no. 5114; 1:1,000) were from Cell Signaling Technology, Inc., the p53 (cat. no. sc-6243; 1:1,000) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the caspase-8 (cas8; cat. no. 551242; 1:1,000) antibody was from BD Biosciences (Franklin Lakes, NJ, USA).

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: After electrophoresis the proteins were electrotransferred onto nitrocellulose membranes (Whatman, Dassel, Germany) for 1.5 h at 100 mV using an electrophoretic transfer system (Mini-Trans-blot Electrophoretic Transfer Cell). .. Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251). .. The membranes were washed with PBS-T and incubated for 1 h at room temperature with the corresponding secondary antibodies: peroxidase-conjugated goat anti-mouse IgG (H + L, 1:1,000; Thermo Scientific no. 32430) or goat anti-rabbit IgG-HRP (1:5,000; Santa Cruz Biotechnology no. sc-2004).

    Article Title: Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells
    Article Snippet: Proteins were extracted using lysis buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, pro­tease and phosphatase inhibitor cocktail (Sigma)), and concentrations determined using a BCA Protein Assay Kit (Thermo Scientific). .. Samples were separated by SDS-PAGE and proteins transferred on to polyvinylidene difluoride membranes, blocked in 5% milk in Tris-buffered saline and 0.05% Tween 20, incubated with primary antibodies against myocardin (Sigma M8948; 1∶1000 dilution) and β-actin (Sigma A2228; 1∶10,000 dilution) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Dako). .. Signals were detected using ECL Western Blotting Detection Reagents (GE Healthcare Life Sciences).

    Article Title: Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells
    Article Snippet: Cell lysate (30 µg) was separated on 10% SDS-PAGE and transferred onto a PVDF membrane (PerkinElmer, Boston, MA, USA) at 25 volts overnight at 4°C. .. The membranes were blocked at 4°C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for 8 h. Blots were incubated with the appropriate antibodies overnight at 4°C: anti-CREB (1∶1000), anti-phospho-CREB (Ser-133) (1∶1000), anti-ERK (1∶1000 ), anti-phospho-ERK (1∶1000 ) (Cell Signaling Technology, Inc.), and Monoclonal anti-β actin (1∶8000) (Sigma-Aldrich Co.). .. After three washes with PBST, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1∶10,000) (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. The blots were washed with PBST and the proteins were detected by Western Lightning™ Chemiluminescence Reagent Plus (PerkinElmer) according to the manufacturer’s instructions, and the chemiluminescence signal was visualized with Amersham Hyperfilm™ ECL (GE Healthcare, Buckinghamshire, UK).

    Stripping Membranes:

    Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line
    Article Snippet: PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich). .. Chemiluminescence was detected using the ImageQuant™ LAS 4000 Biomolecular imager (28-9558-10, GE Healthcare Life Sciences) with associated software.

    Significance Assay:

    Article Title: Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor
    Article Snippet: After electrophoretic separation, proteins were blotted onto a PVDF membrane using the Trans-Blot Turbo transfer system (Biorad) and detected by the following antibodies: anti-TIMP-3 (AB6000, Millipore), anti-TIMP-1 (generated as previously described ), anti-TIMP-2 (generated as previously described ), anti-SPARC (number 5031, Thermo Fisher), anti-MIF (clone FL-115, Santa Cruz) anti-APP (clone 22C11, Millipore), anti-MMP-1 (clone 2A7.2, Millipore) anti-actin (A5316, Sigma Aldrich). .. Bands corresponding to each protein were quantified by using Multi Gauge software (Fujifilm) and normalized to the mean of the original non-normalized control values (HEK293 cells).

    Expressing:

    Article Title: Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study
    Article Snippet: Paragraph title: Intracellular protein expression ... To control for protein loading, each membrane was re-probed with a mouse monoclonal antibody to β-actin (AC-74; Sigma).

    BIA-KA:

    Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells
    Article Snippet: Proteins were extracted from harvested cells using radioimmunoprecipitation lysis buffer supplemented with complete protease inhibitors, and protein concentrations were determined using the BCA Protein Assay Kit. .. Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or anti– β -actin antibody (1:5000 dilution, catalog #A5441; Sigma-Aldrich) overnight at 4°C.

    Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line
    Article Snippet: Protein concentration was determined using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific). .. PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich).

    Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
    Article Snippet: The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000). .. The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000).

    Article Title: Thyroxin Protects White Matter from Hypoxic-Ischemic Insult in the Immature Sprague–Dawley Rat Brain by Regulating Periventricular White Matter and Cortex BDNF and CREB Pathways
    Article Snippet: Protein concentrations of supernatant were determined using a BCA protein-assay kit (Pierce Kit #23227, Thermo Scientific, Waltham, MA, USA) with bovine-serum albumin to plot a standard curve. .. The blots were stripped with buffer (2.5% SDS, 0.7% 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) and incubated with the β-actin antibody (catalog #A5316, Sigma), followed by a secondary antibody and visualized with the chemiluminescence ECL system.

    Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS
    Article Snippet: The protein concentration was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). .. The β-actin antibody was from Sigma-Aldrich (cat. no. A2228; 1:2,000, Merck KGaA, Darmstadt, Germany), antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3)-I/II (cat. no. 3868; 1:1,000), cleaved caspase-3 (cas3; cat. no. 9661; 1:500) and p62 (cat. no. 5114; 1:1,000) were from Cell Signaling Technology, Inc., the p53 (cat. no. sc-6243; 1:1,000) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the caspase-8 (cas8; cat. no. 551242; 1:1,000) antibody was from BD Biosciences (Franklin Lakes, NJ, USA).

    Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication
    Article Snippet: Protein concentration was determined using the Pierce BCA protein assay kit (Life Technologies), according to the manufacturer’s instructions. .. The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich).

    Article Title: Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells
    Article Snippet: Proteins were extracted using lysis buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, pro­tease and phosphatase inhibitor cocktail (Sigma)), and concentrations determined using a BCA Protein Assay Kit (Thermo Scientific). .. Samples were separated by SDS-PAGE and proteins transferred on to polyvinylidene difluoride membranes, blocked in 5% milk in Tris-buffered saline and 0.05% Tween 20, incubated with primary antibodies against myocardin (Sigma M8948; 1∶1000 dilution) and β-actin (Sigma A2228; 1∶10,000 dilution) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Dako).

    Acrylamide Gel Assay:

    Article Title: Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor
    Article Snippet: Supernatants from TIMP-3/HEK or HEK293 cells were loaded onto an acrylamide gel for electrophoresis. .. After electrophoretic separation, proteins were blotted onto a PVDF membrane using the Trans-Blot Turbo transfer system (Biorad) and detected by the following antibodies: anti-TIMP-3 (AB6000, Millipore), anti-TIMP-1 (generated as previously described ), anti-TIMP-2 (generated as previously described ), anti-SPARC (number 5031, Thermo Fisher), anti-MIF (clone FL-115, Santa Cruz) anti-APP (clone 22C11, Millipore), anti-MMP-1 (clone 2A7.2, Millipore) anti-actin (A5316, Sigma Aldrich).

    Western Blot:

    Article Title: P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand
    Article Snippet: Paragraph title: Western blotting ... Membranes were probed overnight at 4°C with antibodies against Fas (sc-736), FasL (sc-6237), P2X7 (sc-25698), High mobility group box 1 (HMGB1, sc-26351), caspase-8 (sc-7890) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); CD3 (Abcam Cambridge, UK, Catalog no: ab5690); FADD (Epitomics, Inc., Burlingame, CA, USA, Catalog no: 2988–1); and β-actin (Sigma Chemical, St Louis, MO, USA, Catalog no:A2228).

    Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells
    Article Snippet: Paragraph title: Protein Isolation and Western Blots. ... Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or anti– β -actin antibody (1:5000 dilution, catalog #A5441; Sigma-Aldrich) overnight at 4°C.

    Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line
    Article Snippet: Paragraph title: Western blot analysis ... PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich).

    Article Title: Urinary exosomal expression of activator of G protein signaling 3 in polycystic kidney disease
    Article Snippet: AGS3 protein detection was determined using standard Western blot techniques [ , , ]. .. Mouse anti-β-actin (1:4000; cat #A5441, Sigma, St. Louis, MO) was used as a loading control.

    Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
    Article Snippet: Paragraph title: 4.5. Protein Isolation and Western Blotting ... The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000).

    Article Title: Thyroxin Protects White Matter from Hypoxic-Ischemic Insult in the Immature Sprague–Dawley Rat Brain by Regulating Periventricular White Matter and Cortex BDNF and CREB Pathways
    Article Snippet: Paragraph title: 4.8. Western Blotting ... The blots were stripped with buffer (2.5% SDS, 0.7% 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) and incubated with the β-actin antibody (catalog #A5316, Sigma), followed by a secondary antibody and visualized with the chemiluminescence ECL system.

    Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS
    Article Snippet: Paragraph title: Western blot assay ... The β-actin antibody was from Sigma-Aldrich (cat. no. A2228; 1:2,000, Merck KGaA, Darmstadt, Germany), antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3)-I/II (cat. no. 3868; 1:1,000), cleaved caspase-3 (cas3; cat. no. 9661; 1:500) and p62 (cat. no. 5114; 1:1,000) were from Cell Signaling Technology, Inc., the p53 (cat. no. sc-6243; 1:1,000) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the caspase-8 (cas8; cat. no. 551242; 1:1,000) antibody was from BD Biosciences (Franklin Lakes, NJ, USA).

    Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication
    Article Snippet: Paragraph title: 2.1.6. Western blot analysis ... The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich).

    Article Title: Transcriptional coactivator PGC-1α contains a novel CBP80-binding motif that orchestrates efficient target gene expression
    Article Snippet: Paragraph title: Western blotting ... Proteins were detected using the following antibodies: anti-PGC-1β (Bethyl Laboratories, A302-273A), anti-PGC-1α (Novus, NBP1-04676), anti-Flag (Sigma-Aldrich, A8592), anti-β-actin (Sigma-Aldrich, A2228), anti-CBP80 (Bethyl Laboratories, A301-793A or, alternatively, ), anti-CBP20 (Santa Cruz Biotechnology, sc-48793), anti-PABPC1 (Abcam, ab21060), anti-eIF4E (Bethyl Laboratories, A301-153A), anti-α-Tubulin (Santa Cruz Biotechnology, sc-58666), anti-P62 (BabCO, mAb414), anti-vimentin (Santa Cruz Biotechnology, sc-6260), anti-pSer2-RNAPII (Abcam, ab5095), anti-GAPDH (Cell Signaling Technology, 2118), anti-NONO (Bethyl Laboratories, A300-582A), anti-myoglobin (Santa Cruz Biotechnology, sc25607), anti-MHC (Abcam, ab91506), or anti-calnexin (Enzo, SPA-865F).

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: Paragraph title: Western blotting ... Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251).

    Article Title: Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells
    Article Snippet: Paragraph title: Western Blot Analysis ... Samples were separated by SDS-PAGE and proteins transferred on to polyvinylidene difluoride membranes, blocked in 5% milk in Tris-buffered saline and 0.05% Tween 20, incubated with primary antibodies against myocardin (Sigma M8948; 1∶1000 dilution) and β-actin (Sigma A2228; 1∶10,000 dilution) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Dako).

    Article Title: Expression of Extracellular Superoxide Dismutase Protein in Diabetes
    Article Snippet: Paragraph title: Western blot analysis ... The membranes were stripped and reblotted with anti-actin antibody (Sigma, catalog number A5441, St Louis, MI, USA).

    Article Title: LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication
    Article Snippet: The Flag-tagged ATG12 (pATG12) and its dominant-negative derivative pATG12ΔG140 (ATG12-DN) constructs were kindly provided by Dr. Adi Kimchi (Israel) [ ]. .. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5 (used for western blot), mouse monoclonal anti-Flag, and mouse monoclonal anti-β-actin antibodies were purchased from Sigma Aldrich (USA). .. Mouse monoclonal anti-ATG5 (used for immunofluorescence) and anti-P62 antibodies were purchased from Abnova (Taiwan).

    Article Title: Herpes Simplex Virus Type 1 Single Strand DNA Binding Protein and Helicase/Primase Complex Disable Cellular ATR Signaling
    Article Snippet: Paragraph title: Western blot analysis ... Primary antibodies used include polyclonal rabbit anti-ATRIP 403 (rATRIP 403) (1∶3,000) , monoclonal mouse anti-ICP4 (1∶10,000; US Biologics), monoclonal mouse anti-β-actin (1∶15,000; Sigma), polyclonal goat anti-ATR N19 (1∶1,000; Santa Cruz), monoclonal mouse anti-Chk1 (1∶1,000; Santa Cruz), monoclonal mouse anti-HA (F7) (1∶3,000; Santa Cruz), monoclonal mouse anti-RPA32 (9H8) (1∶1,000; Genetex), monoclonal rabbit anti-phospho-Chk1 S345 (1∶5,000; Cell Signaling), polyclonal rabbit anti-phospho-RPA S33 (1∶3,000; Bethyl), and polyclonal rabbit anti-phospho-RPA S4/S8 (1∶3,000; Bethyl).

    Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1
    Article Snippet: Paragraph title: Distal lung protein extraction and Western blot analysis ... Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and mouse monoclonal anti-β-actin (dilution 1:1000) (Sigma).

    Article Title: Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells
    Article Snippet: Paragraph title: Western Blotting Analysis of CREB, Phospho-CREB, ERK, Phospho-ERK and β-actin Proteins ... The membranes were blocked at 4°C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for 8 h. Blots were incubated with the appropriate antibodies overnight at 4°C: anti-CREB (1∶1000), anti-phospho-CREB (Ser-133) (1∶1000), anti-ERK (1∶1000 ), anti-phospho-ERK (1∶1000 ) (Cell Signaling Technology, Inc.), and Monoclonal anti-β actin (1∶8000) (Sigma-Aldrich Co.).

    Protease Inhibitor:

    Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication
    Article Snippet: Briefly, protein extracts were prepared by cell lysis in RIPA buffer (Sigma-Aldrich) supplemented with a mammalian protease inhibitor cocktail (Sigma-Aldrich). .. The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich).

    Article Title: Herpes Simplex Virus Type 1 Single Strand DNA Binding Protein and Helicase/Primase Complex Disable Cellular ATR Signaling
    Article Snippet: Primary antibodies used include polyclonal rabbit anti-ATRIP 403 (rATRIP 403) (1∶3,000) , monoclonal mouse anti-ICP4 (1∶10,000; US Biologics), monoclonal mouse anti-β-actin (1∶15,000; Sigma), polyclonal goat anti-ATR N19 (1∶1,000; Santa Cruz), monoclonal mouse anti-Chk1 (1∶1,000; Santa Cruz), monoclonal mouse anti-HA (F7) (1∶3,000; Santa Cruz), monoclonal mouse anti-RPA32 (9H8) (1∶1,000; Genetex), monoclonal rabbit anti-phospho-Chk1 S345 (1∶5,000; Cell Signaling), polyclonal rabbit anti-phospho-RPA S33 (1∶3,000; Bethyl), and polyclonal rabbit anti-phospho-RPA S4/S8 (1∶3,000; Bethyl). .. Primary antibodies used include polyclonal rabbit anti-ATRIP 403 (rATRIP 403) (1∶3,000) , monoclonal mouse anti-ICP4 (1∶10,000; US Biologics), monoclonal mouse anti-β-actin (1∶15,000; Sigma), polyclonal goat anti-ATR N19 (1∶1,000; Santa Cruz), monoclonal mouse anti-Chk1 (1∶1,000; Santa Cruz), monoclonal mouse anti-HA (F7) (1∶3,000; Santa Cruz), monoclonal mouse anti-RPA32 (9H8) (1∶1,000; Genetex), monoclonal rabbit anti-phospho-Chk1 S345 (1∶5,000; Cell Signaling), polyclonal rabbit anti-phospho-RPA S33 (1∶3,000; Bethyl), and polyclonal rabbit anti-phospho-RPA S4/S8 (1∶3,000; Bethyl).

    Cell Culture:

    Article Title: Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study
    Article Snippet: Proteins from normal blood MNC cultured with cisplatin and phytohemagglutinin (PHA), and untreated MNC probed with antibodies on the same membrane served as positive and negative controls respectively. .. To control for protein loading, each membrane was re-probed with a mouse monoclonal antibody to β-actin (AC-74; Sigma).

    Generated:

    Article Title: Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor
    Article Snippet: Supernatants from TIMP-3/HEK or HEK293 cells were loaded onto an acrylamide gel for electrophoresis. .. After electrophoretic separation, proteins were blotted onto a PVDF membrane using the Trans-Blot Turbo transfer system (Biorad) and detected by the following antibodies: anti-TIMP-3 (AB6000, Millipore), anti-TIMP-1 (generated as previously described ), anti-TIMP-2 (generated as previously described ), anti-SPARC (number 5031, Thermo Fisher), anti-MIF (clone FL-115, Santa Cruz) anti-APP (clone 22C11, Millipore), anti-MMP-1 (clone 2A7.2, Millipore) anti-actin (A5316, Sigma Aldrich). .. Bands corresponding to each protein were quantified by using Multi Gauge software (Fujifilm) and normalized to the mean of the original non-normalized control values (HEK293 cells).

    other:

    Article Title: Insulin-like growth factor receptor signaling in breast tumor epithelium protects cells from endoplasmic reticulum stress and regulates the tumor microenvironment
    Article Snippet: Mouse monoclonal anti-β-actin (A5441) was purchased from Sigma Aldrich.

    Imaging:

    Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells
    Article Snippet: Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or anti– β -actin antibody (1:5000 dilution, catalog #A5441; Sigma-Aldrich) overnight at 4°C. .. The membranes were subsequently incubated with the secondary horseradish peroxidase–labeled anti-mouse (1:3000 dilution, catalog #7076; Cell Signaling Technology, Danvers, MA) or anti-rabbit IgG antibodies (1:10,000 dilution, catalog #111035003); Jackson ImmunoResearch, West Grove, PA) for 2 hours, followed with Clarity Western ECL substrates.

    Protein Concentration:

    Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line
    Article Snippet: Protein concentration was determined using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific). .. PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich).

    Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS
    Article Snippet: The protein concentration was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). .. The β-actin antibody was from Sigma-Aldrich (cat. no. A2228; 1:2,000, Merck KGaA, Darmstadt, Germany), antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3)-I/II (cat. no. 3868; 1:1,000), cleaved caspase-3 (cas3; cat. no. 9661; 1:500) and p62 (cat. no. 5114; 1:1,000) were from Cell Signaling Technology, Inc., the p53 (cat. no. sc-6243; 1:1,000) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the caspase-8 (cas8; cat. no. 551242; 1:1,000) antibody was from BD Biosciences (Franklin Lakes, NJ, USA).

    Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication
    Article Snippet: Protein concentration was determined using the Pierce BCA protein assay kit (Life Technologies), according to the manufacturer’s instructions. .. The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich).

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: The protein concentration of each sample was determined using the DC Protein Assay kit (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. .. Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251).

    Article Title: Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells
    Article Snippet: The protein concentration was measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as the standard. .. The membranes were blocked at 4°C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for 8 h. Blots were incubated with the appropriate antibodies overnight at 4°C: anti-CREB (1∶1000), anti-phospho-CREB (Ser-133) (1∶1000), anti-ERK (1∶1000 ), anti-phospho-ERK (1∶1000 ) (Cell Signaling Technology, Inc.), and Monoclonal anti-β actin (1∶8000) (Sigma-Aldrich Co.).

    Binding Assay:

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251). .. The membranes were washed with PBS-T and incubated for 1 h at room temperature with the corresponding secondary antibodies: peroxidase-conjugated goat anti-mouse IgG (H + L, 1:1,000; Thermo Scientific no. 32430) or goat anti-rabbit IgG-HRP (1:5,000; Santa Cruz Biotechnology no. sc-2004).

    Molecular Weight:

    Article Title: Thyroxin Protects White Matter from Hypoxic-Ischemic Insult in the Immature Sprague–Dawley Rat Brain by Regulating Periventricular White Matter and Cortex BDNF and CREB Pathways
    Article Snippet: After denaturing in Laemmli buffer (catalog #161-0737, Bio-Rad, Hercules, CA, USA), equal amounts of protein (10–20 μg) were loaded onto 4–15% or 4–20% gradient precast gels (Bio-Rad), depending on the molecular weight of the target protein. .. The blots were stripped with buffer (2.5% SDS, 0.7% 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) and incubated with the β-actin antibody (catalog #A5316, Sigma), followed by a secondary antibody and visualized with the chemiluminescence ECL system.

    DC Protein Assay:

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: The protein concentration of each sample was determined using the DC Protein Assay kit (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. .. Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251).

    Isolation:

    Article Title: P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand
    Article Snippet: Proteins were extracted, and the soluble cytosolic fraction was isolated according to our previous study [ ]. .. Membranes were probed overnight at 4°C with antibodies against Fas (sc-736), FasL (sc-6237), P2X7 (sc-25698), High mobility group box 1 (HMGB1, sc-26351), caspase-8 (sc-7890) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); CD3 (Abcam Cambridge, UK, Catalog no: ab5690); FADD (Epitomics, Inc., Burlingame, CA, USA, Catalog no: 2988–1); and β-actin (Sigma Chemical, St Louis, MO, USA, Catalog no:A2228).

    Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells
    Article Snippet: Paragraph title: Protein Isolation and Western Blots. ... Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or anti– β -actin antibody (1:5000 dilution, catalog #A5441; Sigma-Aldrich) overnight at 4°C.

    Article Title: Urinary exosomal expression of activator of G protein signaling 3 in polycystic kidney disease
    Article Snippet: SD and PCK rat kidney lysates at 8, 16, and 24 weeks of age, and human and rat urine exosome protein lysates were isolated using 1X RIPA buffer containing protease (Roche) and phosphatase inhibitors (Pierce, Rockford, IL). .. Mouse anti-β-actin (1:4000; cat #A5441, Sigma, St. Louis, MO) was used as a loading control.

    Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
    Article Snippet: Paragraph title: 4.5. Protein Isolation and Western Blotting ... The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000).

    Protein Extraction:

    Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1
    Article Snippet: Paragraph title: Distal lung protein extraction and Western blot analysis ... Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and mouse monoclonal anti-β-actin (dilution 1:1000) (Sigma).

    Construct:

    Article Title: LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication
    Article Snippet: The Flag-tagged ATG12 (pATG12) and its dominant-negative derivative pATG12ΔG140 (ATG12-DN) constructs were kindly provided by Dr. Adi Kimchi (Israel) [ ]. .. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5 (used for western blot), mouse monoclonal anti-Flag, and mouse monoclonal anti-β-actin antibodies were purchased from Sigma Aldrich (USA).

    Polyacrylamide Gel Electrophoresis:

    Article Title: P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand
    Article Snippet: Samples containing equivalent amounts of protein were loaded onto 10% to 15% acrylamide denaturing gels (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE). .. Membranes were probed overnight at 4°C with antibodies against Fas (sc-736), FasL (sc-6237), P2X7 (sc-25698), High mobility group box 1 (HMGB1, sc-26351), caspase-8 (sc-7890) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); CD3 (Abcam Cambridge, UK, Catalog no: ab5690); FADD (Epitomics, Inc., Burlingame, CA, USA, Catalog no: 2988–1); and β-actin (Sigma Chemical, St Louis, MO, USA, Catalog no:A2228).

    Staining:

    Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication
    Article Snippet: The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich). .. The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich).

    SDS Page:

    Article Title: P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand
    Article Snippet: Samples containing equivalent amounts of protein were loaded onto 10% to 15% acrylamide denaturing gels (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE). .. Membranes were probed overnight at 4°C with antibodies against Fas (sc-736), FasL (sc-6237), P2X7 (sc-25698), High mobility group box 1 (HMGB1, sc-26351), caspase-8 (sc-7890) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); CD3 (Abcam Cambridge, UK, Catalog no: ab5690); FADD (Epitomics, Inc., Burlingame, CA, USA, Catalog no: 2988–1); and β-actin (Sigma Chemical, St Louis, MO, USA, Catalog no:A2228).

    Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells
    Article Snippet: The whole-cell proteins (35 μ g/lane) were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane. .. Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or anti– β -actin antibody (1:5000 dilution, catalog #A5441; Sigma-Aldrich) overnight at 4°C.

    Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line
    Article Snippet: Approximately 50 μg of lysate was fractioned in 15% SDS-PAGE gels that were subsequently transferred to PVDF Immobilon-P membranes (IPVH00010, Millipore-Merck). .. PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich).

    Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
    Article Snippet: Twenty micrograms (20 μg) of protein lysates were separated in 1.5 mm thick 12% SDS-PAGE gels. .. The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000).

    Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS
    Article Snippet: Proteins (30 µ g) were separated on 10% SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes. .. The β-actin antibody was from Sigma-Aldrich (cat. no. A2228; 1:2,000, Merck KGaA, Darmstadt, Germany), antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3)-I/II (cat. no. 3868; 1:1,000), cleaved caspase-3 (cas3; cat. no. 9661; 1:500) and p62 (cat. no. 5114; 1:1,000) were from Cell Signaling Technology, Inc., the p53 (cat. no. sc-6243; 1:1,000) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the caspase-8 (cas8; cat. no. 551242; 1:1,000) antibody was from BD Biosciences (Franklin Lakes, NJ, USA).

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: The proteins were resolved by SDS-PAGE (8%) using a Mini-Protean system (Bio-Rad) and depending on the epitope to be analyzed, 30–50 μg of total protein was loaded into each lane in loading buffer containing: 0.062 M Tris (pH 6.8), 10% glycerol, 5% β-mercaptoethanol, 7.5 mM EDTA, 2% SDS, and 0.002% bromophenol blue. .. Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251).

    Article Title: Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells
    Article Snippet: Proteins were extracted using lysis buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, pro­tease and phosphatase inhibitor cocktail (Sigma)), and concentrations determined using a BCA Protein Assay Kit (Thermo Scientific). .. Samples were separated by SDS-PAGE and proteins transferred on to polyvinylidene difluoride membranes, blocked in 5% milk in Tris-buffered saline and 0.05% Tween 20, incubated with primary antibodies against myocardin (Sigma M8948; 1∶1000 dilution) and β-actin (Sigma A2228; 1∶10,000 dilution) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Dako). .. Signals were detected using ECL Western Blotting Detection Reagents (GE Healthcare Life Sciences).

    Article Title: Expression of Extracellular Superoxide Dismutase Protein in Diabetes
    Article Snippet: Proteins from tissue were separated by SDS-PAGE using NuPAGE 4% to 12% bis-Tris gels (Invitrogen, Carlsbad, CA, USA, NP0335Box) and then transferred to Immobilon-P membrane. .. The membranes were stripped and reblotted with anti-actin antibody (Sigma, catalog number A5441, St Louis, MI, USA).

    Article Title: Herpes Simplex Virus Type 1 Single Strand DNA Binding Protein and Helicase/Primase Complex Disable Cellular ATR Signaling
    Article Snippet: Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. .. Primary antibodies used include polyclonal rabbit anti-ATRIP 403 (rATRIP 403) (1∶3,000) , monoclonal mouse anti-ICP4 (1∶10,000; US Biologics), monoclonal mouse anti-β-actin (1∶15,000; Sigma), polyclonal goat anti-ATR N19 (1∶1,000; Santa Cruz), monoclonal mouse anti-Chk1 (1∶1,000; Santa Cruz), monoclonal mouse anti-HA (F7) (1∶3,000; Santa Cruz), monoclonal mouse anti-RPA32 (9H8) (1∶1,000; Genetex), monoclonal rabbit anti-phospho-Chk1 S345 (1∶5,000; Cell Signaling), polyclonal rabbit anti-phospho-RPA S33 (1∶3,000; Bethyl), and polyclonal rabbit anti-phospho-RPA S4/S8 (1∶3,000; Bethyl).

    Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1
    Article Snippet: Lungs were removed from thorax and homogenized for 3 min in ice-cold lysis RIPA buffer (pH 8) containing 20 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate and protease inhibitors. .. Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and mouse monoclonal anti-β-actin (dilution 1:1000) (Sigma). .. The anti-rabbit IgG secondary antibody (Amersham Pharmacia Biotech, UK) was used at dilution 1:5000 and the anti-mouse IgG (Sigma) at the dilution 1: 10,000.

    Article Title: Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells
    Article Snippet: Cell lysate (30 µg) was separated on 10% SDS-PAGE and transferred onto a PVDF membrane (PerkinElmer, Boston, MA, USA) at 25 volts overnight at 4°C. .. The membranes were blocked at 4°C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for 8 h. Blots were incubated with the appropriate antibodies overnight at 4°C: anti-CREB (1∶1000), anti-phospho-CREB (Ser-133) (1∶1000), anti-ERK (1∶1000 ), anti-phospho-ERK (1∶1000 ) (Cell Signaling Technology, Inc.), and Monoclonal anti-β actin (1∶8000) (Sigma-Aldrich Co.).

    Plasmid Preparation:

    Article Title: LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication
    Article Snippet: hATG5 and hATG16L1 sequences were cloned into peGFP-C1 plasmid (Clontech) to form pGFP-ATG5 and pGFP-ATG16L1, respectively. .. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5 (used for western blot), mouse monoclonal anti-Flag, and mouse monoclonal anti-β-actin antibodies were purchased from Sigma Aldrich (USA).

    Software:

    Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells
    Article Snippet: Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or anti– β -actin antibody (1:5000 dilution, catalog #A5441; Sigma-Aldrich) overnight at 4°C. .. The membranes were subsequently incubated with the secondary horseradish peroxidase–labeled anti-mouse (1:3000 dilution, catalog #7076; Cell Signaling Technology, Danvers, MA) or anti-rabbit IgG antibodies (1:10,000 dilution, catalog #111035003); Jackson ImmunoResearch, West Grove, PA) for 2 hours, followed with Clarity Western ECL substrates.

    Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line
    Article Snippet: PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 (A5441, Sigma-Aldrich). .. Antibody binding was revealed using peroxidase conjugated secondary antibodies donkey anti-rabbit (1:10,000 dilution, 711-035-152, Jackson) or donkey anti-mouse (1:10,000 dilution, 715-035-150, Jackson) with enhanced luminol-based chemiluminescent (ECL) Western Blotting Substrate ( , Thermo Scientific).

    Article Title: Urinary exosomal expression of activator of G protein signaling 3 in polycystic kidney disease
    Article Snippet: Mouse anti-β-actin (1:4000; cat #A5441, Sigma, St. Louis, MO) was used as a loading control. .. Rat brain lysates were used as a positive control, since AGS3 is enriched in brain tissue [ ].

    Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
    Article Snippet: The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000). .. The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000).

    Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication
    Article Snippet: The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich). .. Protein bands were detected using Immobilon Western chemiluminescent HRP substrate (Millipore) in a ChemiDoc XRS+ instrument (Bio-Rad).

    Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia
    Article Snippet: Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), β-actin (1:1,000; clone AC-15, Sigma no. A5441), GSK3 (1:1,000; Biosource no. 44–610), phospho-GSK3Ser21/9 (1:1,000; Cell Signaling no. 9331), SAPK-JNK (1:1,000; Cell Signaling no. 9252), pSAPK-JNKThr183/Tyr185 (1:1,000; Cell Signaling no. 9251). .. Autoradiography was performed on AGFA RP2 plus films, using different exposure times depending on the primary antibody used.

    Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1
    Article Snippet: Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and mouse monoclonal anti-β-actin (dilution 1:1000) (Sigma). .. Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and mouse monoclonal anti-β-actin (dilution 1:1000) (Sigma).

    Dominant Negative Mutation:

    Article Title: LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication
    Article Snippet: The Flag-tagged ATG12 (pATG12) and its dominant-negative derivative pATG12ΔG140 (ATG12-DN) constructs were kindly provided by Dr. Adi Kimchi (Israel) [ ]. .. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5 (used for western blot), mouse monoclonal anti-Flag, and mouse monoclonal anti-β-actin antibodies were purchased from Sigma Aldrich (USA).

    In Vitro:

    Article Title: Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study
    Article Snippet: To control for protein loading, each membrane was re-probed with a mouse monoclonal antibody to β-actin (AC-74; Sigma). .. Immunoreactive bands for p53 were quantified by densitometry in a linear range using calibrated office scanner and ImageJ software, and the ratio of the immunoreactive p53 protein band and β-actin was calculated.

    Radio Immunoprecipitation:

    Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS
    Article Snippet: Briefly, radioimmunoprecipitation assay buffer (Qiagen, Inc., Valencia, CA, USA) was used to extract total proteins from Huh7 cells. .. The β-actin antibody was from Sigma-Aldrich (cat. no. A2228; 1:2,000, Merck KGaA, Darmstadt, Germany), antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3)-I/II (cat. no. 3868; 1:1,000), cleaved caspase-3 (cas3; cat. no. 9661; 1:500) and p62 (cat. no. 5114; 1:1,000) were from Cell Signaling Technology, Inc., the p53 (cat. no. sc-6243; 1:1,000) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the caspase-8 (cas8; cat. no. 551242; 1:1,000) antibody was from BD Biosciences (Franklin Lakes, NJ, USA).

    Concentration Assay:

    Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology
    Article Snippet: The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000). .. The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000).

    Lysis:

    Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells
    Article Snippet: Proteins were extracted from harvested cells using radioimmunoprecipitation lysis buffer supplemented with complete protease inhibitors, and protein concentrations were determined using the BCA Protein Assay Kit. .. Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or anti– β -actin antibody (1:5000 dilution, catalog #A5441; Sigma-Aldrich) overnight at 4°C.

    Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication
    Article Snippet: Briefly, protein extracts were prepared by cell lysis in RIPA buffer (Sigma-Aldrich) supplemented with a mammalian protease inhibitor cocktail (Sigma-Aldrich). .. The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich).

    Article Title: Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells
    Article Snippet: Proteins were extracted using lysis buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, pro­tease and phosphatase inhibitor cocktail (Sigma)), and concentrations determined using a BCA Protein Assay Kit (Thermo Scientific). .. Samples were separated by SDS-PAGE and proteins transferred on to polyvinylidene difluoride membranes, blocked in 5% milk in Tris-buffered saline and 0.05% Tween 20, incubated with primary antibodies against myocardin (Sigma M8948; 1∶1000 dilution) and β-actin (Sigma A2228; 1∶10,000 dilution) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Dako).

    Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1
    Article Snippet: Lungs were removed from thorax and homogenized for 3 min in ice-cold lysis RIPA buffer (pH 8) containing 20 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate and protease inhibitors. .. Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and mouse monoclonal anti-β-actin (dilution 1:1000) (Sigma).

    T-Test:

    Article Title: Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor
    Article Snippet: After electrophoretic separation, proteins were blotted onto a PVDF membrane using the Trans-Blot Turbo transfer system (Biorad) and detected by the following antibodies: anti-TIMP-3 (AB6000, Millipore), anti-TIMP-1 (generated as previously described ), anti-TIMP-2 (generated as previously described ), anti-SPARC (number 5031, Thermo Fisher), anti-MIF (clone FL-115, Santa Cruz) anti-APP (clone 22C11, Millipore), anti-MMP-1 (clone 2A7.2, Millipore) anti-actin (A5316, Sigma Aldrich). .. Bands corresponding to each protein were quantified by using Multi Gauge software (Fujifilm) and normalized to the mean of the original non-normalized control values (HEK293 cells).

    Activity Assay:

    Article Title: Reduced PKC α Activity Induces Senescent Phenotype in Erythrocytes
    Article Snippet: PMA (P 8139), 4α -phorbol 12,13-didecanoate (4α PDD) (P 8014), phenyl methyl sulfonyl fluoride (PMSF) (P 7626), anti-band 3 N-terminus monoclonal antibody (B 9277), and anti-β -actin antibody (A5316) were purchased from Sigma. .. PMA (P 8139), 4α -phorbol 12,13-didecanoate (4α PDD) (P 8014), phenyl methyl sulfonyl fluoride (PMSF) (P 7626), anti-band 3 N-terminus monoclonal antibody (B 9277), and anti-β -actin antibody (A5316) were purchased from Sigma.

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    Millipore monoclonal rabbit beta actin antibody
    Western blot of <t>beta-actin.</t> Comparative analysis of RBC ghosts from healthy controls and RTT patients using four antibodies, which recognize different beta-actin amino acidic sequences (A: full length, B: N-terminal, C: C-terminal and D: C-terminal, amino acidic sequence not specified), confirm a beta-actin decrease in RTT patients.
    Monoclonal Rabbit Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal rabbit beta actin antibody/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monoclonal rabbit beta actin antibody - by Bioz Stars, 2019-12
    99/100 stars
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    99
    Millipore mouse anti β actin antibody
    p17 interferes with the formation of the CDK2/cyclin A2 complex, which impedes Akt phosphorylation. ( A ) Levels of CDK2, cyclin A2, p-Akt (S473), p-GSK3α (S21), p-GSK3β (S9), and p-Rb (S249) in ARV-infected and p17-transfected Vero cells were examined. Cells were collected at the indicated points, and whole cell lysates were harvested for Western blot assays. p17 (1–118)-transfected and mock-infected cells were used as negative controls. <t>β-actin</t> was included as a loading control. ( B ) The level of CDK2 was examined in Vero cells without treatment or pretreated with MG132 followed by mock infection, ARV infection, and p17 transfection, respectively. Levels of CDK 2 mRNA in ARV-infected and pcDNA3.1-flag-p17-transfected Vero cells were analyzed by semi-quantitative RT-PCR. Mock infection (cells alone) was used as a negative control. The graph represents the mean ± SD calculated from three independent experiments. ( C ) The amount of CDK2 and cyclin A2 association were examined in either ARV-infected or p17-transfected Vero cells. ( D ) An in vitro GST pull-down assay was carried out. Elution fractions were boiled and examined by Western blot analysis. 30% total input of TrxA-His-17 or TrxA-His-17(1–118) mutant represented the internal loading control. ( E ) To confirm whether CDK2 phosphorylates Akt, knockdown of CDK2 with an shRNA and overexpression of CDK2 in p17-transfected cells were carried out, followed by Western blot analysis with indicated antibodies. For negative controls, cells were transfected as indicated. ( F ) To test whether insulin and CDK2 overexpression counteract the inhibitory effect of p17 on mTORC2 complex association, Vero cells were pretreated with insulin (0.2 μm) or transfected with pCI-neo-CDK2 plasmid for 3 hours, respectively, followed by transfection with pcDNA3.1-Flag-p17 for 18 hours. Vero cells were collected and washed twice in phosphate-buffered saline (PBS) and scraped in 200 μl of CHAPS lysis buffer. ( G ) To determine the effects of Akt and CDK2 on ARV replication, individual 24-well plates of Vero cells were infected with ARV at an MOI of 5 for 6 hours, followed by transfection with Akt and CDK2 shRNAs or the pCI-neo-CDK2 plasmid for 24 hours, respectively. The ARV-infected cell supernatant was collected at 24 hpi for determining virus titer. All the data shown represent the mean ± SD calculated from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The uncropped blots with molecular weights are shown in Figs S7 and S8 .
    Mouse Anti β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti β actin antibody/product/Millipore
    Average 99 stars, based on 258 article reviews
    Price from $9.99 to $1999.99
    mouse anti β actin antibody - by Bioz Stars, 2019-12
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    99
    Millipore anti αsma antibody
    Proliferation potential of Tcf21 lineage–traced fibroblasts residing in stable scar. ( A ) Experimental scheme whereby Tcf21 MCM/+ ; R26 EGFP mice previously treated with tamoxifen were subjected to MI and then treated with Ang II/PE through osmotic pump 4 weeks after MI. Mice were treated with EdU through daily i.p. injections for 6 days starting at day 2 after pump implantation, and hearts were harvested 4 hours after the last EdU injection for IHC analysis. ( B and C ) Quantification ( B ) and representative IHC images from 3 hearts analyzed ( C ) of EdU + (white) and Ki67 + (red) Tcf21 lineage–traced (EGFP + ) fibroblasts in the infarct region and septum of hearts from Tcf21 MCM/+ ; R26 EGFP mice that received treatment as shown in A . Nuclei are shown with DAPI (blue). Scale bars: 20 μm. ( D and E ) Quantification ( D ) and representative immunocytochemistry from 3 separate experiments ( E ) of EdU + (white) Tcf21 lineage–traced (EGFP + ) fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks later. EdU was given for 6 hours with and without TGF-β stimulation. Scale bars: 200 μm. ( F ) Representative immunocytochemistry images from 3 separate experiments showing <t>αSMA</t> stress fibers (red) in Tcf21 lineage–traced fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks after MI. Cells were also treated with TGF-β for 3 days. Scale bars: 10 μm. ( B and D ) Data are shown as mean ± SD ( n = 3). ** P
    Anti αsma Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti αsma antibody - by Bioz Stars, 2019-12
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    Image Search Results


    Western blot of beta-actin. Comparative analysis of RBC ghosts from healthy controls and RTT patients using four antibodies, which recognize different beta-actin amino acidic sequences (A: full length, B: N-terminal, C: C-terminal and D: C-terminal, amino acidic sequence not specified), confirm a beta-actin decrease in RTT patients.

    Journal: PLoS ONE

    Article Title: Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization

    doi: 10.1371/journal.pone.0093181

    Figure Lengend Snippet: Western blot of beta-actin. Comparative analysis of RBC ghosts from healthy controls and RTT patients using four antibodies, which recognize different beta-actin amino acidic sequences (A: full length, B: N-terminal, C: C-terminal and D: C-terminal, amino acidic sequence not specified), confirm a beta-actin decrease in RTT patients.

    Article Snippet: Cells were blocked with 1% BSA for 30 min at RT and incubated overnight at 4°C with a monoclonal rabbit beta-actin antibody (Millipore Corporation, Billerica, MA, USA) diluted 1∶50 in PBS.

    Techniques: Western Blot, Sequencing

    4-HNE/beta-actin adducts in RBC membranes. (A) SDS-PAGE (silver staining) of immunoprecipitated beta-actin from RBC ghosts of healthy control subjects (1–4) and RTT patients (5–8). (B) Western blot analysis of 4-HNE on immunoprecipitated beta-actin from RBC ghosts of healthy control subjects (1–4) and RTT patients (5–8). Immunoprecipitation (IP) with normal rabbit IgG served as a negative control. (C) 2-DE/Western blot analysis of 4-HNE on beta-actin from RBC ghosts proteome.

    Journal: PLoS ONE

    Article Title: Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization

    doi: 10.1371/journal.pone.0093181

    Figure Lengend Snippet: 4-HNE/beta-actin adducts in RBC membranes. (A) SDS-PAGE (silver staining) of immunoprecipitated beta-actin from RBC ghosts of healthy control subjects (1–4) and RTT patients (5–8). (B) Western blot analysis of 4-HNE on immunoprecipitated beta-actin from RBC ghosts of healthy control subjects (1–4) and RTT patients (5–8). Immunoprecipitation (IP) with normal rabbit IgG served as a negative control. (C) 2-DE/Western blot analysis of 4-HNE on beta-actin from RBC ghosts proteome.

    Article Snippet: Cells were blocked with 1% BSA for 30 min at RT and incubated overnight at 4°C with a monoclonal rabbit beta-actin antibody (Millipore Corporation, Billerica, MA, USA) diluted 1∶50 in PBS.

    Techniques: SDS Page, Silver Staining, Immunoprecipitation, Western Blot, Negative Control

    Beta-actin expression in RBC membranes. SDS-PAGE comparative analysis of RBC ghosts from healthy control subjects and RTT patients. Visible reduction of intensity for beta-actin (band 6) is present in RTT patients.

    Journal: PLoS ONE

    Article Title: Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization

    doi: 10.1371/journal.pone.0093181

    Figure Lengend Snippet: Beta-actin expression in RBC membranes. SDS-PAGE comparative analysis of RBC ghosts from healthy control subjects and RTT patients. Visible reduction of intensity for beta-actin (band 6) is present in RTT patients.

    Article Snippet: Cells were blocked with 1% BSA for 30 min at RT and incubated overnight at 4°C with a monoclonal rabbit beta-actin antibody (Millipore Corporation, Billerica, MA, USA) diluted 1∶50 in PBS.

    Techniques: Expressing, SDS Page

    Potential binding sites for 4-HNE in the beta-actin amino acid sequence. The potential binding sites for 4-HNE are highlighted. Red color for 6 cysteine (C), yellow color for 8 hystidine (H) and green color for 19 lysine (K) residues. Blue double lines indicate sub-domain 1; orange lines indicate sub-domain 2; black lines indicate sub-domain 3 and purple lines indicate sub-domain 4 (primary sequence extracted from the ExPASy: SIB Bioinformatics Resource Portal, http://www.expasy.org/ ).

    Journal: PLoS ONE

    Article Title: Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization

    doi: 10.1371/journal.pone.0093181

    Figure Lengend Snippet: Potential binding sites for 4-HNE in the beta-actin amino acid sequence. The potential binding sites for 4-HNE are highlighted. Red color for 6 cysteine (C), yellow color for 8 hystidine (H) and green color for 19 lysine (K) residues. Blue double lines indicate sub-domain 1; orange lines indicate sub-domain 2; black lines indicate sub-domain 3 and purple lines indicate sub-domain 4 (primary sequence extracted from the ExPASy: SIB Bioinformatics Resource Portal, http://www.expasy.org/ ).

    Article Snippet: Cells were blocked with 1% BSA for 30 min at RT and incubated overnight at 4°C with a monoclonal rabbit beta-actin antibody (Millipore Corporation, Billerica, MA, USA) diluted 1∶50 in PBS.

    Techniques: Binding Assay, Sequencing

    2-DE analysis of beta-actin isoforms. (A) Comparative analysis between RTT patients (right panel) and healthy controls (left panel). Arrows indicate the identified beta-actin isoforms which appear to be decrease in RTT patients. The top right panels represent the beta-actin differentially expressed spots on 3D view. (B) Quantitative analysis of the identified beta-actin spots changes in RTT patients as compared to control expression levels. Molecular mass and pI markers are indicated along the gels.

    Journal: PLoS ONE

    Article Title: Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization

    doi: 10.1371/journal.pone.0093181

    Figure Lengend Snippet: 2-DE analysis of beta-actin isoforms. (A) Comparative analysis between RTT patients (right panel) and healthy controls (left panel). Arrows indicate the identified beta-actin isoforms which appear to be decrease in RTT patients. The top right panels represent the beta-actin differentially expressed spots on 3D view. (B) Quantitative analysis of the identified beta-actin spots changes in RTT patients as compared to control expression levels. Molecular mass and pI markers are indicated along the gels.

    Article Snippet: Cells were blocked with 1% BSA for 30 min at RT and incubated overnight at 4°C with a monoclonal rabbit beta-actin antibody (Millipore Corporation, Billerica, MA, USA) diluted 1∶50 in PBS.

    Techniques: Expressing

    Confocal microscopy analysis of beta-actin distribution in RBCs. (A) Comparative analysis shows beta-actin expression and distribution differences between RBCs from RTT patients (right panel) and healthy control subjects (left panel) at confocal microscopy. Small panels represent, with flat and 3D views, two typical erythrocytes in which beta-actin signal is differentially distributed. Scale bar 25 μm. (B) Quantitative analysis of beta-actin signal differences in RBCs from healthy control (C) and RTT patients (RTT).

    Journal: PLoS ONE

    Article Title: Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization

    doi: 10.1371/journal.pone.0093181

    Figure Lengend Snippet: Confocal microscopy analysis of beta-actin distribution in RBCs. (A) Comparative analysis shows beta-actin expression and distribution differences between RBCs from RTT patients (right panel) and healthy control subjects (left panel) at confocal microscopy. Small panels represent, with flat and 3D views, two typical erythrocytes in which beta-actin signal is differentially distributed. Scale bar 25 μm. (B) Quantitative analysis of beta-actin signal differences in RBCs from healthy control (C) and RTT patients (RTT).

    Article Snippet: Cells were blocked with 1% BSA for 30 min at RT and incubated overnight at 4°C with a monoclonal rabbit beta-actin antibody (Millipore Corporation, Billerica, MA, USA) diluted 1∶50 in PBS.

    Techniques: Confocal Microscopy, Expressing

    p17 interferes with the formation of the CDK2/cyclin A2 complex, which impedes Akt phosphorylation. ( A ) Levels of CDK2, cyclin A2, p-Akt (S473), p-GSK3α (S21), p-GSK3β (S9), and p-Rb (S249) in ARV-infected and p17-transfected Vero cells were examined. Cells were collected at the indicated points, and whole cell lysates were harvested for Western blot assays. p17 (1–118)-transfected and mock-infected cells were used as negative controls. β-actin was included as a loading control. ( B ) The level of CDK2 was examined in Vero cells without treatment or pretreated with MG132 followed by mock infection, ARV infection, and p17 transfection, respectively. Levels of CDK 2 mRNA in ARV-infected and pcDNA3.1-flag-p17-transfected Vero cells were analyzed by semi-quantitative RT-PCR. Mock infection (cells alone) was used as a negative control. The graph represents the mean ± SD calculated from three independent experiments. ( C ) The amount of CDK2 and cyclin A2 association were examined in either ARV-infected or p17-transfected Vero cells. ( D ) An in vitro GST pull-down assay was carried out. Elution fractions were boiled and examined by Western blot analysis. 30% total input of TrxA-His-17 or TrxA-His-17(1–118) mutant represented the internal loading control. ( E ) To confirm whether CDK2 phosphorylates Akt, knockdown of CDK2 with an shRNA and overexpression of CDK2 in p17-transfected cells were carried out, followed by Western blot analysis with indicated antibodies. For negative controls, cells were transfected as indicated. ( F ) To test whether insulin and CDK2 overexpression counteract the inhibitory effect of p17 on mTORC2 complex association, Vero cells were pretreated with insulin (0.2 μm) or transfected with pCI-neo-CDK2 plasmid for 3 hours, respectively, followed by transfection with pcDNA3.1-Flag-p17 for 18 hours. Vero cells were collected and washed twice in phosphate-buffered saline (PBS) and scraped in 200 μl of CHAPS lysis buffer. ( G ) To determine the effects of Akt and CDK2 on ARV replication, individual 24-well plates of Vero cells were infected with ARV at an MOI of 5 for 6 hours, followed by transfection with Akt and CDK2 shRNAs or the pCI-neo-CDK2 plasmid for 24 hours, respectively. The ARV-infected cell supernatant was collected at 24 hpi for determining virus titer. All the data shown represent the mean ± SD calculated from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The uncropped blots with molecular weights are shown in Figs S7 and S8 .

    Journal: Scientific Reports

    Article Title: Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6

    doi: 10.1038/s41598-017-05510-x

    Figure Lengend Snippet: p17 interferes with the formation of the CDK2/cyclin A2 complex, which impedes Akt phosphorylation. ( A ) Levels of CDK2, cyclin A2, p-Akt (S473), p-GSK3α (S21), p-GSK3β (S9), and p-Rb (S249) in ARV-infected and p17-transfected Vero cells were examined. Cells were collected at the indicated points, and whole cell lysates were harvested for Western blot assays. p17 (1–118)-transfected and mock-infected cells were used as negative controls. β-actin was included as a loading control. ( B ) The level of CDK2 was examined in Vero cells without treatment or pretreated with MG132 followed by mock infection, ARV infection, and p17 transfection, respectively. Levels of CDK 2 mRNA in ARV-infected and pcDNA3.1-flag-p17-transfected Vero cells were analyzed by semi-quantitative RT-PCR. Mock infection (cells alone) was used as a negative control. The graph represents the mean ± SD calculated from three independent experiments. ( C ) The amount of CDK2 and cyclin A2 association were examined in either ARV-infected or p17-transfected Vero cells. ( D ) An in vitro GST pull-down assay was carried out. Elution fractions were boiled and examined by Western blot analysis. 30% total input of TrxA-His-17 or TrxA-His-17(1–118) mutant represented the internal loading control. ( E ) To confirm whether CDK2 phosphorylates Akt, knockdown of CDK2 with an shRNA and overexpression of CDK2 in p17-transfected cells were carried out, followed by Western blot analysis with indicated antibodies. For negative controls, cells were transfected as indicated. ( F ) To test whether insulin and CDK2 overexpression counteract the inhibitory effect of p17 on mTORC2 complex association, Vero cells were pretreated with insulin (0.2 μm) or transfected with pCI-neo-CDK2 plasmid for 3 hours, respectively, followed by transfection with pcDNA3.1-Flag-p17 for 18 hours. Vero cells were collected and washed twice in phosphate-buffered saline (PBS) and scraped in 200 μl of CHAPS lysis buffer. ( G ) To determine the effects of Akt and CDK2 on ARV replication, individual 24-well plates of Vero cells were infected with ARV at an MOI of 5 for 6 hours, followed by transfection with Akt and CDK2 shRNAs or the pCI-neo-CDK2 plasmid for 24 hours, respectively. The ARV-infected cell supernatant was collected at 24 hpi for determining virus titer. All the data shown represent the mean ± SD calculated from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The uncropped blots with molecular weights are shown in Figs S7 and S8 .

    Article Snippet: Mouse anti-β-actin antibody was from Millipore (Billerica, USA).

    Techniques: Infection, Transfection, Western Blot, Quantitative RT-PCR, Negative Control, In Vitro, Pull Down Assay, Mutagenesis, shRNA, Over Expression, Plasmid Preparation, Lysis, Activation Assay

    ARV σA protein enhances the proteasome activity and expression level of PSMB6. ( A ) Vero cell lysates from ARV-infected, σA-transfected, and PSMB6- depleted cells with ARV at an MOI of 10 for 24 hours were used to quantify relative proteasome activity. ( B ) Examination of the PSMB6 levels. Vero cells were infected with ARV at an MOI of 10 at indicated time points, followed by Western blot assay with an anti-PSMB6 antibody. ( C ) Analysis of mRNA levels of PSMB6 and other subunits by real-time RT-PCR.The data reveal that PSMB6 is transcriptionally upregulated by σA. ( D ) To confirm whether PSMB6 mediates ribosomal protein ubiquitin-proteasome-mediated degradation and inhibits Akt phosphorylation at S473, Vero cells were transfected with plasmids overexpressing PSMB6 followed by Western blot assay with the indicated antibodies ( E ) In the presence of MG132, the decrease in the levels of p-Akt (S473) in ARV-infected vero cells could be reversed in ARV-infected cells. ( F ) The levels of PSMB6, Rpl26, Rpl27, and p-Akt (S473) were examined in ARV-infected cells co-transfected with an shRNA against σA. ( G ) To confirm whether both PSMB6 and MDM2 mediate ribosomal proteins degradation, knockdown of either PSMB6 or MDM2 with shRNAs was performed followed by Western blot analysis with the indicated antibodies. ( H ) Vero cells were transfected with pcDNA3.1-p17 or co-transfected with pcDNA3.1-p17 and pcDNA3.1-σA plasmids for 24 hours, respectively, followed by Western blot assays with the indicated antibodies. ( I ) Individual 24-well plates of Vero cells were transfected with a PSMB6 shRNA for 24 hours, followed by ARV infection at an MOI of 5 for 24 hours. The ARV-infected cell supernatant was collected at 24 hpi for determining the virus titer. All data shown represent the mean ± SD calculated from three independent experiments. ( J ) A model depicting the cooperation between p17 and σA proteins of ARV to trigger ribosomal protein degradation is shown. The protein levels were normalized to those for β-actin. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The activation and inactivation folds indicated below each lane were normalized against against values for the mock control or at 0 h. The uncropped blots with molecular weights are shown in Figs S5 and S6 .

    Journal: Scientific Reports

    Article Title: Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6

    doi: 10.1038/s41598-017-05510-x

    Figure Lengend Snippet: ARV σA protein enhances the proteasome activity and expression level of PSMB6. ( A ) Vero cell lysates from ARV-infected, σA-transfected, and PSMB6- depleted cells with ARV at an MOI of 10 for 24 hours were used to quantify relative proteasome activity. ( B ) Examination of the PSMB6 levels. Vero cells were infected with ARV at an MOI of 10 at indicated time points, followed by Western blot assay with an anti-PSMB6 antibody. ( C ) Analysis of mRNA levels of PSMB6 and other subunits by real-time RT-PCR.The data reveal that PSMB6 is transcriptionally upregulated by σA. ( D ) To confirm whether PSMB6 mediates ribosomal protein ubiquitin-proteasome-mediated degradation and inhibits Akt phosphorylation at S473, Vero cells were transfected with plasmids overexpressing PSMB6 followed by Western blot assay with the indicated antibodies ( E ) In the presence of MG132, the decrease in the levels of p-Akt (S473) in ARV-infected vero cells could be reversed in ARV-infected cells. ( F ) The levels of PSMB6, Rpl26, Rpl27, and p-Akt (S473) were examined in ARV-infected cells co-transfected with an shRNA against σA. ( G ) To confirm whether both PSMB6 and MDM2 mediate ribosomal proteins degradation, knockdown of either PSMB6 or MDM2 with shRNAs was performed followed by Western blot analysis with the indicated antibodies. ( H ) Vero cells were transfected with pcDNA3.1-p17 or co-transfected with pcDNA3.1-p17 and pcDNA3.1-σA plasmids for 24 hours, respectively, followed by Western blot assays with the indicated antibodies. ( I ) Individual 24-well plates of Vero cells were transfected with a PSMB6 shRNA for 24 hours, followed by ARV infection at an MOI of 5 for 24 hours. The ARV-infected cell supernatant was collected at 24 hpi for determining the virus titer. All data shown represent the mean ± SD calculated from three independent experiments. ( J ) A model depicting the cooperation between p17 and σA proteins of ARV to trigger ribosomal protein degradation is shown. The protein levels were normalized to those for β-actin. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The activation and inactivation folds indicated below each lane were normalized against against values for the mock control or at 0 h. The uncropped blots with molecular weights are shown in Figs S5 and S6 .

    Article Snippet: Mouse anti-β-actin antibody was from Millipore (Billerica, USA).

    Techniques: Activity Assay, Expressing, Infection, Transfection, Western Blot, Quantitative RT-PCR, shRNA, Activation Assay

    p17 deregulates mTORC2 assembly. ( A ) To study the effect of insulin on ribosomal proteins and Akt phosphorylation at S473, cells were pretreated with insulin (0.2 μm) for 1 hour, followed by transfection with pcDNA3.1-Flag-p17 for 24 hours at 37 °C. Whole cell lysates were harvested for Western blot assays with the indicated antibodies. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against values for the mock control. those at mock. The levels of indicated proteins in the mock control were considered 1-fold. ( B ) Upper panel: in co-immunoprecipitation experiments, the binding of rictor, mSN1, and MlST8 to mTOR was examined in ARV-infected or p17-transfected Vero cells. Cells were mock-infected or infected with ARV at an MOI of 10 and transfected with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag (vector only) plasmid for 24 hours. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot analysis, and proteins were detected with the indicated antibodies. Lower panel: Data were obtained in three independent experiments, error bars indicate the means ± SD. ( C ) Upper panel: for analysis of the effect of insulin on mTORC2, cells were treated with insulin (0.2 uM) for 1 hour, followed by transfection of cells with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag plasmid for 24 hours at 37 °C. The interaction of mTOR and rictor with Rpl7 and Rpl26 was examined by co-immunoprecipitation experiments described in panel A. Lower panel: Data shown represent the mean ± SD calculated from three independent experiments. ( D ) To study whether MDM2 and PSMB6 affect the association of mTORC2 and ribosomal proteins, depletion of MDM2 and PSMB6 with shRNAs was performed. The uncropped blots with molecular weights are shown in Figs S6 and S7 .

    Journal: Scientific Reports

    Article Title: Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6

    doi: 10.1038/s41598-017-05510-x

    Figure Lengend Snippet: p17 deregulates mTORC2 assembly. ( A ) To study the effect of insulin on ribosomal proteins and Akt phosphorylation at S473, cells were pretreated with insulin (0.2 μm) for 1 hour, followed by transfection with pcDNA3.1-Flag-p17 for 24 hours at 37 °C. Whole cell lysates were harvested for Western blot assays with the indicated antibodies. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against values for the mock control. those at mock. The levels of indicated proteins in the mock control were considered 1-fold. ( B ) Upper panel: in co-immunoprecipitation experiments, the binding of rictor, mSN1, and MlST8 to mTOR was examined in ARV-infected or p17-transfected Vero cells. Cells were mock-infected or infected with ARV at an MOI of 10 and transfected with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag (vector only) plasmid for 24 hours. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot analysis, and proteins were detected with the indicated antibodies. Lower panel: Data were obtained in three independent experiments, error bars indicate the means ± SD. ( C ) Upper panel: for analysis of the effect of insulin on mTORC2, cells were treated with insulin (0.2 uM) for 1 hour, followed by transfection of cells with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag plasmid for 24 hours at 37 °C. The interaction of mTOR and rictor with Rpl7 and Rpl26 was examined by co-immunoprecipitation experiments described in panel A. Lower panel: Data shown represent the mean ± SD calculated from three independent experiments. ( D ) To study whether MDM2 and PSMB6 affect the association of mTORC2 and ribosomal proteins, depletion of MDM2 and PSMB6 with shRNAs was performed. The uncropped blots with molecular weights are shown in Figs S6 and S7 .

    Article Snippet: Mouse anti-β-actin antibody was from Millipore (Billerica, USA).

    Techniques: Transfection, Western Blot, Activation Assay, Immunoprecipitation, Binding Assay, Infection, Plasmid Preparation, SDS Page

    p17 promotes E3 ligase MDM2 targeting to ribosomal proteins. ( A ) Vero cells were infected with ARV at an MOI of 10 or transfected with pcDNA3.1-Flag-p17 or pcDNA3.1-Flag-p17(1–118) plasmids for 24 hours, followed by Western blot assays with the indicated antibodies. ( B ) The expression levels of Rpl26 and Rpl27 in ARV-infected and p17-transfected cells were examined in the presence or absence of MG132 (25 uM), respectively. Whole cell lysates were harvested for Western blot assays with the indicated antibodies. The experiments were repeated three times, and representative blots are shown. ( C ) In co-immunoprecipitation experiments, the binding of E3 ligase MDM2 to ribosomal proteins was examinedinp17-transfected Vero cells. Vero cells were transfected with pcDNA3.1-Flag-p17 plasmid and pcDNA3.1-Flag, respectively. Cell lysates were immunoprecipitated with MDM2 or Rpl26 and interacting proteins were detected with the indicated antibodies. ( D ) Vero cells were transfected with pcDNA3.1-Flag-p17 with or without co-transfection with MDM2 shRNA.The interaction of MDM2 with Rpl26 and Rpl27 was examined. ( E ) Vero cells without treatment or pretreated with MG132 followed by mock infection, ARV infection, and p17 transfection, respectively. The interaction of ubiquitin with Rpl26 and Rpl27was examined. Similar results were obtained in three independent experiments. The protein levels were normalized to those for β-actin. The levels of indicated protein in the mock control or at 0 h were considered 1-fold.The activation and inactivation folds indicated below each lane were normalized against values for the mock control or at 0 h. The uncropped blots with molecular weights are shown in Fig. S5 .

    Journal: Scientific Reports

    Article Title: Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6

    doi: 10.1038/s41598-017-05510-x

    Figure Lengend Snippet: p17 promotes E3 ligase MDM2 targeting to ribosomal proteins. ( A ) Vero cells were infected with ARV at an MOI of 10 or transfected with pcDNA3.1-Flag-p17 or pcDNA3.1-Flag-p17(1–118) plasmids for 24 hours, followed by Western blot assays with the indicated antibodies. ( B ) The expression levels of Rpl26 and Rpl27 in ARV-infected and p17-transfected cells were examined in the presence or absence of MG132 (25 uM), respectively. Whole cell lysates were harvested for Western blot assays with the indicated antibodies. The experiments were repeated three times, and representative blots are shown. ( C ) In co-immunoprecipitation experiments, the binding of E3 ligase MDM2 to ribosomal proteins was examinedinp17-transfected Vero cells. Vero cells were transfected with pcDNA3.1-Flag-p17 plasmid and pcDNA3.1-Flag, respectively. Cell lysates were immunoprecipitated with MDM2 or Rpl26 and interacting proteins were detected with the indicated antibodies. ( D ) Vero cells were transfected with pcDNA3.1-Flag-p17 with or without co-transfection with MDM2 shRNA.The interaction of MDM2 with Rpl26 and Rpl27 was examined. ( E ) Vero cells without treatment or pretreated with MG132 followed by mock infection, ARV infection, and p17 transfection, respectively. The interaction of ubiquitin with Rpl26 and Rpl27was examined. Similar results were obtained in three independent experiments. The protein levels were normalized to those for β-actin. The levels of indicated protein in the mock control or at 0 h were considered 1-fold.The activation and inactivation folds indicated below each lane were normalized against values for the mock control or at 0 h. The uncropped blots with molecular weights are shown in Fig. S5 .

    Article Snippet: Mouse anti-β-actin antibody was from Millipore (Billerica, USA).

    Techniques: Infection, Transfection, Western Blot, Expressing, Immunoprecipitation, Binding Assay, Plasmid Preparation, Cotransfection, shRNA, Activation Assay

    Nuclear import of p17 is important for induction of autophagy. ( A ) A GFP-LC3 plasmid was used to observe LC3 punta in Vero cells under a fluorescence microscope. All conditions for the treated- and untreated-cells are described in the Material and Method section. Scale Bar: 20 μm. ( B ) GFP-LC3 plasmid was applied to observe LC3 punta under a fluorescence microscope. Quantitation results from Fig. 6A represents mean GFP-LC3 puncta per cell. n = 15. ( C ) Levels of LC3-II in different treatments were examined. Whole cell lysates were harvested for Western blot assays. pcDNA3.1-Flag-p17 (1–118)-transfected and mock-infected cells were used as negative controls. β-actin was included as a loading control. The LC3-II level of mock control (cells alone) was considered 1-fold. The activation folds indicated below each lane were normalized against values for the mock control. The uncropped blots with molecular weights are shown in Fig. S9 .

    Journal: Scientific Reports

    Article Title: Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6

    doi: 10.1038/s41598-017-05510-x

    Figure Lengend Snippet: Nuclear import of p17 is important for induction of autophagy. ( A ) A GFP-LC3 plasmid was used to observe LC3 punta in Vero cells under a fluorescence microscope. All conditions for the treated- and untreated-cells are described in the Material and Method section. Scale Bar: 20 μm. ( B ) GFP-LC3 plasmid was applied to observe LC3 punta under a fluorescence microscope. Quantitation results from Fig. 6A represents mean GFP-LC3 puncta per cell. n = 15. ( C ) Levels of LC3-II in different treatments were examined. Whole cell lysates were harvested for Western blot assays. pcDNA3.1-Flag-p17 (1–118)-transfected and mock-infected cells were used as negative controls. β-actin was included as a loading control. The LC3-II level of mock control (cells alone) was considered 1-fold. The activation folds indicated below each lane were normalized against values for the mock control. The uncropped blots with molecular weights are shown in Fig. S9 .

    Article Snippet: Mouse anti-β-actin antibody was from Millipore (Billerica, USA).

    Techniques: Plasmid Preparation, Fluorescence, Microscopy, Quantitation Assay, Western Blot, Transfection, Infection, Activation Assay

    Formation of nitrosative damage maker 3-nitrotyrosine (3NT) in brain microvessels of low intensity blast range. ( A ) A representative of immunofluorescent staining of 3NT in microvessel of whole brain tissue cross section in control and blast exposed animals. ( B ) Corresponding Western Blot of 3NT and housekeeping protein, β-actin. ( C ) Bar graphs of quantification of the 3NT immunoreactive fluorescence. Values are mean ± SEM, (n = 4), and asterisk indicates statistical significant (p-value

    Journal: Scientific Reports

    Article Title: Primary blast causes mild, moderate, severe and lethal TBI with increasing blast overpressures: Experimental rat injury model

    doi: 10.1038/srep26992

    Figure Lengend Snippet: Formation of nitrosative damage maker 3-nitrotyrosine (3NT) in brain microvessels of low intensity blast range. ( A ) A representative of immunofluorescent staining of 3NT in microvessel of whole brain tissue cross section in control and blast exposed animals. ( B ) Corresponding Western Blot of 3NT and housekeeping protein, β-actin. ( C ) Bar graphs of quantification of the 3NT immunoreactive fluorescence. Values are mean ± SEM, (n = 4), and asterisk indicates statistical significant (p-value

    Article Snippet: Rabbit anti-zonula occluden-1 (ZO-1) was from US Biological (Massachusetts, MA) and mouse anti-β-actin was purchased from Millipore (Billerica, MA).

    Techniques: Staining, Western Blot, Fluorescence, Significance Assay

    Mild TBI range of blast-wave exposure induces NADPH oxidase expression in rat brain microvessels. ( A ) A representative of immunofluorescent staining of NOX1 in intact microvessels of brain cross sections from rats subjected to a single exposure to 60, 100, or 130 kPa peak overpressure, and control. ( B ) Corresponding Western blot of NOX1 and housekeeping protein, β-actin. ( C ) Bar graphs show the quantitative results of the NOX1 immunoreactive fluorescence intensities. Values are mean ± SEM (n = 4) with p-value ≤0.01 compared with control.

    Journal: Scientific Reports

    Article Title: Primary blast causes mild, moderate, severe and lethal TBI with increasing blast overpressures: Experimental rat injury model

    doi: 10.1038/srep26992

    Figure Lengend Snippet: Mild TBI range of blast-wave exposure induces NADPH oxidase expression in rat brain microvessels. ( A ) A representative of immunofluorescent staining of NOX1 in intact microvessels of brain cross sections from rats subjected to a single exposure to 60, 100, or 130 kPa peak overpressure, and control. ( B ) Corresponding Western blot of NOX1 and housekeeping protein, β-actin. ( C ) Bar graphs show the quantitative results of the NOX1 immunoreactive fluorescence intensities. Values are mean ± SEM (n = 4) with p-value ≤0.01 compared with control.

    Article Snippet: Rabbit anti-zonula occluden-1 (ZO-1) was from US Biological (Massachusetts, MA) and mouse anti-β-actin was purchased from Millipore (Billerica, MA).

    Techniques: Expressing, Staining, Western Blot, Fluorescence

    Mild TBI range of blast-wave exposure dose-dependently increased the levels of inducible nitric oxide synthase (iNOS) in rat brain microvessels. ( A ) A representative of immunofluorescent staining of iNOS in microvessel of whole brain tissue cross section in control and blast exposed animals. ( B ) Corresponding Western Blot of iNOS and housekeeping protein, β-actin. ( C ) Bar graphs show the quantitative results of the iNOS immunoreactive fluorescence intensities. Values are mean ± SEM, (n = 4), and asterisks indicate statistical significance (p-value

    Journal: Scientific Reports

    Article Title: Primary blast causes mild, moderate, severe and lethal TBI with increasing blast overpressures: Experimental rat injury model

    doi: 10.1038/srep26992

    Figure Lengend Snippet: Mild TBI range of blast-wave exposure dose-dependently increased the levels of inducible nitric oxide synthase (iNOS) in rat brain microvessels. ( A ) A representative of immunofluorescent staining of iNOS in microvessel of whole brain tissue cross section in control and blast exposed animals. ( B ) Corresponding Western Blot of iNOS and housekeeping protein, β-actin. ( C ) Bar graphs show the quantitative results of the iNOS immunoreactive fluorescence intensities. Values are mean ± SEM, (n = 4), and asterisks indicate statistical significance (p-value

    Article Snippet: Rabbit anti-zonula occluden-1 (ZO-1) was from US Biological (Massachusetts, MA) and mouse anti-β-actin was purchased from Millipore (Billerica, MA).

    Techniques: Staining, Western Blot, Fluorescence, Significance Assay

    Formation of oxidative damage maker 4-hydroxynenonal (4HNE) in rat brain microvessels. ( A ) A representative of immunofluorescent staining of 4HNE in microvessel of whole brain tissue cross section in control and blast exposed animals. ( B ) Corresponding Western Blot of 4HNE and housekeeping protein, β-actin. ( C ) Bar graphs show quantification results of the 4HNE immunoreactive fluorescence intensities. Values are mean ± SEM, (n = 4), and asterisk indicates statistical significant (p-value

    Journal: Scientific Reports

    Article Title: Primary blast causes mild, moderate, severe and lethal TBI with increasing blast overpressures: Experimental rat injury model

    doi: 10.1038/srep26992

    Figure Lengend Snippet: Formation of oxidative damage maker 4-hydroxynenonal (4HNE) in rat brain microvessels. ( A ) A representative of immunofluorescent staining of 4HNE in microvessel of whole brain tissue cross section in control and blast exposed animals. ( B ) Corresponding Western Blot of 4HNE and housekeeping protein, β-actin. ( C ) Bar graphs show quantification results of the 4HNE immunoreactive fluorescence intensities. Values are mean ± SEM, (n = 4), and asterisk indicates statistical significant (p-value

    Article Snippet: Rabbit anti-zonula occluden-1 (ZO-1) was from US Biological (Massachusetts, MA) and mouse anti-β-actin was purchased from Millipore (Billerica, MA).

    Techniques: Staining, Western Blot, Fluorescence, Significance Assay

    Proliferation potential of Tcf21 lineage–traced fibroblasts residing in stable scar. ( A ) Experimental scheme whereby Tcf21 MCM/+ ; R26 EGFP mice previously treated with tamoxifen were subjected to MI and then treated with Ang II/PE through osmotic pump 4 weeks after MI. Mice were treated with EdU through daily i.p. injections for 6 days starting at day 2 after pump implantation, and hearts were harvested 4 hours after the last EdU injection for IHC analysis. ( B and C ) Quantification ( B ) and representative IHC images from 3 hearts analyzed ( C ) of EdU + (white) and Ki67 + (red) Tcf21 lineage–traced (EGFP + ) fibroblasts in the infarct region and septum of hearts from Tcf21 MCM/+ ; R26 EGFP mice that received treatment as shown in A . Nuclei are shown with DAPI (blue). Scale bars: 20 μm. ( D and E ) Quantification ( D ) and representative immunocytochemistry from 3 separate experiments ( E ) of EdU + (white) Tcf21 lineage–traced (EGFP + ) fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks later. EdU was given for 6 hours with and without TGF-β stimulation. Scale bars: 200 μm. ( F ) Representative immunocytochemistry images from 3 separate experiments showing αSMA stress fibers (red) in Tcf21 lineage–traced fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks after MI. Cells were also treated with TGF-β for 3 days. Scale bars: 10 μm. ( B and D ) Data are shown as mean ± SD ( n = 3). ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

    doi: 10.1172/JCI98215

    Figure Lengend Snippet: Proliferation potential of Tcf21 lineage–traced fibroblasts residing in stable scar. ( A ) Experimental scheme whereby Tcf21 MCM/+ ; R26 EGFP mice previously treated with tamoxifen were subjected to MI and then treated with Ang II/PE through osmotic pump 4 weeks after MI. Mice were treated with EdU through daily i.p. injections for 6 days starting at day 2 after pump implantation, and hearts were harvested 4 hours after the last EdU injection for IHC analysis. ( B and C ) Quantification ( B ) and representative IHC images from 3 hearts analyzed ( C ) of EdU + (white) and Ki67 + (red) Tcf21 lineage–traced (EGFP + ) fibroblasts in the infarct region and septum of hearts from Tcf21 MCM/+ ; R26 EGFP mice that received treatment as shown in A . Nuclei are shown with DAPI (blue). Scale bars: 20 μm. ( D and E ) Quantification ( D ) and representative immunocytochemistry from 3 separate experiments ( E ) of EdU + (white) Tcf21 lineage–traced (EGFP + ) fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks later. EdU was given for 6 hours with and without TGF-β stimulation. Scale bars: 200 μm. ( F ) Representative immunocytochemistry images from 3 separate experiments showing αSMA stress fibers (red) in Tcf21 lineage–traced fibroblasts isolated from uninjured hearts and the infarct region of hearts 4 weeks after MI. Cells were also treated with TGF-β for 3 days. Scale bars: 10 μm. ( B and D ) Data are shown as mean ± SD ( n = 3). ** P

    Article Snippet: Primary antibodies and dilutions used in immunocytochemical staining (ICC) included anti-EGFP antibody (Abcam ab13970, 1:200) and anti-αSMA antibody (MilliporeSigma A2547, 1:200).

    Techniques: Mouse Assay, Injection, Immunohistochemistry, Immunocytochemistry, Isolation

    Effect of ECM maturation on αSMA expression in myofibroblasts. ( A and B ) Representative IHC images ( A ) and quantitation ( B ) of αSMA + (red) Tcf21 lineage–traced (EGFP + ) fibroblasts from the infarct region of hearts from Tcf21 MCM/+ ; R26 EGFP mice treated with BAPN or PBS as a control (Cont.). Nuclei are shown with DAPI (blue). Scale bars: 20 μm. Data are shown as mean ± SD ( n = 3). *** P

    Journal: The Journal of Clinical Investigation

    Article Title: Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

    doi: 10.1172/JCI98215

    Figure Lengend Snippet: Effect of ECM maturation on αSMA expression in myofibroblasts. ( A and B ) Representative IHC images ( A ) and quantitation ( B ) of αSMA + (red) Tcf21 lineage–traced (EGFP + ) fibroblasts from the infarct region of hearts from Tcf21 MCM/+ ; R26 EGFP mice treated with BAPN or PBS as a control (Cont.). Nuclei are shown with DAPI (blue). Scale bars: 20 μm. Data are shown as mean ± SD ( n = 3). *** P

    Article Snippet: Primary antibodies and dilutions used in immunocytochemical staining (ICC) included anti-EGFP antibody (Abcam ab13970, 1:200) and anti-αSMA antibody (MilliporeSigma A2547, 1:200).

    Techniques: Expressing, Immunohistochemistry, Quantitation Assay, Mouse Assay

    Representative mouse and human heart IHC of selected proteins underlying the matrifibrocyte. ( A ) Representative IHC heart images showing Comp protein expression (red) along with Tcf21 lineage–traced (EGFP + ) fibroblasts from uninjured mice or the infarct region 7 and 28 days after MI. Nuclei are shown with DAPI (blue). Scale bars: 20 μm. ( B ) Representative IHC mouse heart images showing Chad protein expression (red) along with Tcf21 lineage–traced (EGFP + ) fibroblasts from uninjured heart or the infarct region 7 and 28 days after MI. Nuclei are shown with DAPI (blue). The insets show higher magnification (×4) of double-positive cell for EGFP and Chad. Scale bars: 20 μm. ( C ) Representative IHC images showing protein expression of Chad (red), Comp (red), αSMA (red), Col3 (red), or vimentin (Vim, green) on the same or adjacent serial sections of ischemic LV human heart samples with scar and/or uninjured (control). Nuclei are shown with DAPI (blue). n = 3 (ischemic LVAD). n = 1 (healthy control). Scale bars: 20 μm.

    Journal: The Journal of Clinical Investigation

    Article Title: Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

    doi: 10.1172/JCI98215

    Figure Lengend Snippet: Representative mouse and human heart IHC of selected proteins underlying the matrifibrocyte. ( A ) Representative IHC heart images showing Comp protein expression (red) along with Tcf21 lineage–traced (EGFP + ) fibroblasts from uninjured mice or the infarct region 7 and 28 days after MI. Nuclei are shown with DAPI (blue). Scale bars: 20 μm. ( B ) Representative IHC mouse heart images showing Chad protein expression (red) along with Tcf21 lineage–traced (EGFP + ) fibroblasts from uninjured heart or the infarct region 7 and 28 days after MI. Nuclei are shown with DAPI (blue). The insets show higher magnification (×4) of double-positive cell for EGFP and Chad. Scale bars: 20 μm. ( C ) Representative IHC images showing protein expression of Chad (red), Comp (red), αSMA (red), Col3 (red), or vimentin (Vim, green) on the same or adjacent serial sections of ischemic LV human heart samples with scar and/or uninjured (control). Nuclei are shown with DAPI (blue). n = 3 (ischemic LVAD). n = 1 (healthy control). Scale bars: 20 μm.

    Article Snippet: Primary antibodies and dilutions used in immunocytochemical staining (ICC) included anti-EGFP antibody (Abcam ab13970, 1:200) and anti-αSMA antibody (MilliporeSigma A2547, 1:200).

    Techniques: Immunohistochemistry, Expressing, Mouse Assay

    Model of MI injury–dependent cardiac fibroblast states. Model showing the different states of cardiac fibroblasts at different post-MI stages. In uninjured heart, cardiac fibroblasts reside within the interstitial space, but after MI injury, they become maximally activated by 2 to 4 days, and they elongate and begin to express αSMA. By days 4 to 7, the myofibroblast differentiated state is maximal, with high levels of αSMA protein in elongated processes within these cells. Finally, these fibroblasts stop proliferating and lose αSMA expression as they further differentiate into the matrifibrocyte by day 10 and onwards within the scar region.

    Journal: The Journal of Clinical Investigation

    Article Title: Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

    doi: 10.1172/JCI98215

    Figure Lengend Snippet: Model of MI injury–dependent cardiac fibroblast states. Model showing the different states of cardiac fibroblasts at different post-MI stages. In uninjured heart, cardiac fibroblasts reside within the interstitial space, but after MI injury, they become maximally activated by 2 to 4 days, and they elongate and begin to express αSMA. By days 4 to 7, the myofibroblast differentiated state is maximal, with high levels of αSMA protein in elongated processes within these cells. Finally, these fibroblasts stop proliferating and lose αSMA expression as they further differentiate into the matrifibrocyte by day 10 and onwards within the scar region.

    Article Snippet: Primary antibodies and dilutions used in immunocytochemical staining (ICC) included anti-EGFP antibody (Abcam ab13970, 1:200) and anti-αSMA antibody (MilliporeSigma A2547, 1:200).

    Techniques: Expressing

    Differentiation of Tcf21 lineage–traced fibroblasts into myofibroblasts after MI. ( A and B ) Quantification of αSMA + and EdU + Tcf21 lineage–traced fibroblasts in the MI region ( A ) and border zone ( B ) after a single EdU injection at the indicated time points after MI by IHC. ( C ) Representative IHC images showing αSMA + (red) and EdU + (white) Tcf21 lineage–traced (EGFP + ) fibroblasts quantified in A and B . Nuclei are shown with DAPI (blue). These same images from C are shown in Supplemental Figure 1D in a larger temporal array. ( D – F ) Quantification of αSMA + (red) and EdU + (white) Tcf21 lineage–traced fibroblasts in the MI region ( D ) and border zone ( E ) after 7 daily EdU injections during the indicated time periods after MI by IHC and representative IHC images ( F ). Nuclei are shown with DAPI (blue). ( A , B , D and E ) Data are shown as mean ± SD ( n = 3). C and F show representative images from 3 separate hearts analyzed. Scale bars: 20 μm.

    Journal: The Journal of Clinical Investigation

    Article Title: Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

    doi: 10.1172/JCI98215

    Figure Lengend Snippet: Differentiation of Tcf21 lineage–traced fibroblasts into myofibroblasts after MI. ( A and B ) Quantification of αSMA + and EdU + Tcf21 lineage–traced fibroblasts in the MI region ( A ) and border zone ( B ) after a single EdU injection at the indicated time points after MI by IHC. ( C ) Representative IHC images showing αSMA + (red) and EdU + (white) Tcf21 lineage–traced (EGFP + ) fibroblasts quantified in A and B . Nuclei are shown with DAPI (blue). These same images from C are shown in Supplemental Figure 1D in a larger temporal array. ( D – F ) Quantification of αSMA + (red) and EdU + (white) Tcf21 lineage–traced fibroblasts in the MI region ( D ) and border zone ( E ) after 7 daily EdU injections during the indicated time periods after MI by IHC and representative IHC images ( F ). Nuclei are shown with DAPI (blue). ( A , B , D and E ) Data are shown as mean ± SD ( n = 3). C and F show representative images from 3 separate hearts analyzed. Scale bars: 20 μm.

    Article Snippet: Primary antibodies and dilutions used in immunocytochemical staining (ICC) included anti-EGFP antibody (Abcam ab13970, 1:200) and anti-αSMA antibody (MilliporeSigma A2547, 1:200).

    Techniques: Injection, Immunohistochemistry

    Apoptosis and turnover of Tcf21 lineage–traced fibroblasts after MI. ( A ) Experimental scheme of tamoxifen treatment of Tcf21 MCM/+ ; R26 EGFP mice before MI. Hearts were harvested at the indicated time points after MI for TUNEL staining. ( B ) Representative TUNEL staining (red) images from 3 separate hearts analyzed showing apoptotic Tcf21 lineage–traced (EGFP + ) fibroblasts after MI. Nuclei are shown with DAPI (blue). The inset shows a higher magnification (×8) image of a TUNEL + EGFP + cell. ( C ) Experimental scheme of tamoxifen treatment of Tcf21 MCM/+ ; R26 EGFP mice before MI followed by 7 daily EdU injections during the first week after MI. Hearts were then harvested 4 hours and 28 days after the last EdU injection for IHC analysis. ( D and E ) Quantification ( D ) of EdU + (white) Tcf21 lineage–traced (EGFP + ) fibroblasts in the infarcted area at 4 hours and 28 days after the last injection of 7 daily EdU injections. ( E ) Nuclei are shown with DAPI (blue), and αSMA (red) was stained to show myofibroblast identity. Data are shown as mean ± SD ( n = 3 hearts analyzed). Two-tailed t test showed no significance. For E , representative IHC images are shown from 3 separate hearts analyzed. ( F ) Representative IHC images from 3 separate hearts analyzed showing EdU + (white) CD31 + endothelial cells (red) versus Tcf21 lineage–traced fibroblasts (EGFP + ) in hearts before MI and within infarct region at day 4 and day 10 after MI 4 hours after a single EdU injection. ( G ) Representative IHC images from 3 hearts analyzed showing EdU + (white) CD45 + leukocytes (red) versus Tcf21 lineage–traced fibroblasts (EGFP + ) in hearts before MI and within infarct region at day 4 and day 10 after MI 4 hours after a single EdU injection. Scale bars: 20 μm.

    Journal: The Journal of Clinical Investigation

    Article Title: Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

    doi: 10.1172/JCI98215

    Figure Lengend Snippet: Apoptosis and turnover of Tcf21 lineage–traced fibroblasts after MI. ( A ) Experimental scheme of tamoxifen treatment of Tcf21 MCM/+ ; R26 EGFP mice before MI. Hearts were harvested at the indicated time points after MI for TUNEL staining. ( B ) Representative TUNEL staining (red) images from 3 separate hearts analyzed showing apoptotic Tcf21 lineage–traced (EGFP + ) fibroblasts after MI. Nuclei are shown with DAPI (blue). The inset shows a higher magnification (×8) image of a TUNEL + EGFP + cell. ( C ) Experimental scheme of tamoxifen treatment of Tcf21 MCM/+ ; R26 EGFP mice before MI followed by 7 daily EdU injections during the first week after MI. Hearts were then harvested 4 hours and 28 days after the last EdU injection for IHC analysis. ( D and E ) Quantification ( D ) of EdU + (white) Tcf21 lineage–traced (EGFP + ) fibroblasts in the infarcted area at 4 hours and 28 days after the last injection of 7 daily EdU injections. ( E ) Nuclei are shown with DAPI (blue), and αSMA (red) was stained to show myofibroblast identity. Data are shown as mean ± SD ( n = 3 hearts analyzed). Two-tailed t test showed no significance. For E , representative IHC images are shown from 3 separate hearts analyzed. ( F ) Representative IHC images from 3 separate hearts analyzed showing EdU + (white) CD31 + endothelial cells (red) versus Tcf21 lineage–traced fibroblasts (EGFP + ) in hearts before MI and within infarct region at day 4 and day 10 after MI 4 hours after a single EdU injection. ( G ) Representative IHC images from 3 hearts analyzed showing EdU + (white) CD45 + leukocytes (red) versus Tcf21 lineage–traced fibroblasts (EGFP + ) in hearts before MI and within infarct region at day 4 and day 10 after MI 4 hours after a single EdU injection. Scale bars: 20 μm.

    Article Snippet: Primary antibodies and dilutions used in immunocytochemical staining (ICC) included anti-EGFP antibody (Abcam ab13970, 1:200) and anti-αSMA antibody (MilliporeSigma A2547, 1:200).

    Techniques: Mouse Assay, TUNEL Assay, Staining, Injection, Immunohistochemistry, Two Tailed Test

    Lineage tracing of myofibroblasts in Acta2 CreERT2 ; R26 EGFP mice. ( A ) Schematic of the Acta2 BAC with a tamoxifen-regulated CreERT2 cDNA cassette inserted into exon 1, and mice containing this transgene were crossed with R26 EGFP reporter mice containing a loxP site–flanked stop cassette upstream of EGFP to allow for Cre-dependent lineage tracing. ( B ) Experimental scheme whereby Acta2 CreERT2 ; R26 EGFP mice were given tamoxifen through daily i.p. injections from day –1 to day 4 after MI. Mice were treated with a single EdU injection at the indicated time points after MI, and hearts were harvested 4 hours afterward for IHC analysis. ( C and D ) Representative IHC images from 3 hearts analyzed showing αSMA protein (red) and EdU + (white) in αSMA lineage–traced (EGFP + ) fibroblasts in the infarct region after a single EdU injection at the indicated time points after MI ( C ) and quantification ( D ). Nuclei are shown with DAPI (blue). Data are shown as mean ± SD ( n = 3). Scale bars: 20 μm. ( E ) Representative IHC images from 3 separate hearts analyzed showing αSMA lineage–traced (EGFP + ) fibroblasts and expression of αSMA protein (red) in the infarct region and border zone at 7 days and 8 weeks after MI. Nuclei are shown with DAPI (blue). Scale bars: 200 μm.

    Journal: The Journal of Clinical Investigation

    Article Title: Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

    doi: 10.1172/JCI98215

    Figure Lengend Snippet: Lineage tracing of myofibroblasts in Acta2 CreERT2 ; R26 EGFP mice. ( A ) Schematic of the Acta2 BAC with a tamoxifen-regulated CreERT2 cDNA cassette inserted into exon 1, and mice containing this transgene were crossed with R26 EGFP reporter mice containing a loxP site–flanked stop cassette upstream of EGFP to allow for Cre-dependent lineage tracing. ( B ) Experimental scheme whereby Acta2 CreERT2 ; R26 EGFP mice were given tamoxifen through daily i.p. injections from day –1 to day 4 after MI. Mice were treated with a single EdU injection at the indicated time points after MI, and hearts were harvested 4 hours afterward for IHC analysis. ( C and D ) Representative IHC images from 3 hearts analyzed showing αSMA protein (red) and EdU + (white) in αSMA lineage–traced (EGFP + ) fibroblasts in the infarct region after a single EdU injection at the indicated time points after MI ( C ) and quantification ( D ). Nuclei are shown with DAPI (blue). Data are shown as mean ± SD ( n = 3). Scale bars: 20 μm. ( E ) Representative IHC images from 3 separate hearts analyzed showing αSMA lineage–traced (EGFP + ) fibroblasts and expression of αSMA protein (red) in the infarct region and border zone at 7 days and 8 weeks after MI. Nuclei are shown with DAPI (blue). Scale bars: 200 μm.

    Article Snippet: Primary antibodies and dilutions used in immunocytochemical staining (ICC) included anti-EGFP antibody (Abcam ab13970, 1:200) and anti-αSMA antibody (MilliporeSigma A2547, 1:200).

    Techniques: Mouse Assay, BAC Assay, Injection, Immunohistochemistry, Expressing