β 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 α actins skeletal cardiac smooth one β actin β non muscle and two γ actins γ smooth muscle and γ non muscle ACTB actin β gene codes for β actin It is a cytoskeletal housekeeping protein It is present in the cytoplasm The ACTB gene is located on human chromosome 7p22 1 Monoclonal Anti β Actin mouse IgG2a isotype is derived from the AC 74 hybridoma produced by the fusion of mouse myeloma cells and splenocytes from an immunized mouse
    Catalog Number:
    a5316
    Price:
    None
    Applications:
    Western blot analysis of MDCK cell lysates were performed using monoclonal anti-actin antibody as a primary antibody.
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    Structured Review

    Millipore β 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 α actins skeletal cardiac smooth one β actin β non muscle and two γ actins γ smooth muscle and γ non muscle ACTB actin β gene codes for β actin It is a cytoskeletal housekeeping protein It is present in the cytoplasm The ACTB gene is located on human chromosome 7p22 1 Monoclonal Anti β Actin mouse IgG2a isotype is derived from the AC 74 hybridoma produced by the fusion of mouse myeloma cells and splenocytes from an immunized mouse
    https://www.bioz.com/result/β actin/product/Millipore
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    β actin - by Bioz Stars, 2020-01
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    Images

    1) Product Images from "Leukocyte RhoA exchange factor Arhgef1 mediates vascular inflammation and atherosclerosis"

    Article Title: Leukocyte RhoA exchange factor Arhgef1 mediates vascular inflammation and atherosclerosis

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI92702

    Deletion of Arhgef1 inhibits leukocyte rolling and adhesion. ( A ) Time-dependent in vivo effect of Ang II (30 pmol) on leukocyte rolling and adhesion in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( B ) Effect of losartan on leukocyte rolling and adhesion induced by Ang II (30 pmol, 4 hours) in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( C ) Representative immunoblot of VCAM1, ICAM1, and β-actin in lysates of aortas from Arhgef1 lox/lox and Arhgef1 –/– mice before (0) and 4 and 8 hours after Ang II treatment ( n = 3) and corresponding quantification. All lanes were run on the same gel, but lanes 3 and 4 were noncontiguous as indicated by the black dividing line. ( D ) In vitro static adhesion of Arhgef1 lox/lox and Arhgef1 –/– leukocytes on ICAM before (0) and 1 and 4 hours after Ang II treatment ( n = 6 experiments). ( E ) In vitro analysis of Arhgef1 lox/lox and Arhgef1 –/– leukocyte rolling and adhesion on HUVECs under shear flow, before (–) and 4 hours after (+) Ang II treatment ( n = 5). * P
    Figure Legend Snippet: Deletion of Arhgef1 inhibits leukocyte rolling and adhesion. ( A ) Time-dependent in vivo effect of Ang II (30 pmol) on leukocyte rolling and adhesion in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( B ) Effect of losartan on leukocyte rolling and adhesion induced by Ang II (30 pmol, 4 hours) in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( C ) Representative immunoblot of VCAM1, ICAM1, and β-actin in lysates of aortas from Arhgef1 lox/lox and Arhgef1 –/– mice before (0) and 4 and 8 hours after Ang II treatment ( n = 3) and corresponding quantification. All lanes were run on the same gel, but lanes 3 and 4 were noncontiguous as indicated by the black dividing line. ( D ) In vitro static adhesion of Arhgef1 lox/lox and Arhgef1 –/– leukocytes on ICAM before (0) and 1 and 4 hours after Ang II treatment ( n = 6 experiments). ( E ) In vitro analysis of Arhgef1 lox/lox and Arhgef1 –/– leukocyte rolling and adhesion on HUVECs under shear flow, before (–) and 4 hours after (+) Ang II treatment ( n = 5). * P

    Techniques Used: In Vivo, Mouse Assay, In Vitro, Flow Cytometry

    2) Product Images from "An Extract of Artemisia dracunculus L. stimulates insulin secretion from β cells, activates AMPK and suppresses inflammation"

    Article Title: An Extract of Artemisia dracunculus L. stimulates insulin secretion from β cells, activates AMPK and suppresses inflammation

    Journal: Journal of ethnopharmacology

    doi: 10.1016/j.jep.2015.05.003

    Effect of PMI-5011 on AMP-activated protein Kinase and protein kinase B (PKB) phosphorylation in NIT-1 cells A) NIT-1 cells were incubated in the absence or presence of 10 μg/mL PMI-5011 for the indicated times. B) NIT-1 cells were incubated in the absence or presence of 10 μg/mL PMI-5011, or metformin. Then, Total cell lysates were subjected to Western blot analysis by using anti-pThr172 AMPKα, anti-pSer79 ACC, anti-pSer473 AKT, anti-AMPKα, anti-AKT and anti-GAPDH antibodies. The images are representative image from three independent experiments. C) Anti-inflammatory effect of PMI-5011 on macrophage. Bone marrow derived macrophages were treated with different concentration of PMI-5011 (10–30 μg/ml) followed by stimulation with LPS/IFNγ (0.1 μg/50 U/ml). Post 20 h of treatment, cell supernatant was used for NO estimation using Griess reagent and D) cell lysate was processed for immunoblot analysis for iNOS (BD Bioscience). Beta-actin was used for equal protein loading control. E. Levels of IL6 were determined by ELISA (BD Bioscience) in cell supernatant. Metformin (10 mM) was used as control in this experiment. Values are means + SD of three experiments.
    Figure Legend Snippet: Effect of PMI-5011 on AMP-activated protein Kinase and protein kinase B (PKB) phosphorylation in NIT-1 cells A) NIT-1 cells were incubated in the absence or presence of 10 μg/mL PMI-5011 for the indicated times. B) NIT-1 cells were incubated in the absence or presence of 10 μg/mL PMI-5011, or metformin. Then, Total cell lysates were subjected to Western blot analysis by using anti-pThr172 AMPKα, anti-pSer79 ACC, anti-pSer473 AKT, anti-AMPKα, anti-AKT and anti-GAPDH antibodies. The images are representative image from three independent experiments. C) Anti-inflammatory effect of PMI-5011 on macrophage. Bone marrow derived macrophages were treated with different concentration of PMI-5011 (10–30 μg/ml) followed by stimulation with LPS/IFNγ (0.1 μg/50 U/ml). Post 20 h of treatment, cell supernatant was used for NO estimation using Griess reagent and D) cell lysate was processed for immunoblot analysis for iNOS (BD Bioscience). Beta-actin was used for equal protein loading control. E. Levels of IL6 were determined by ELISA (BD Bioscience) in cell supernatant. Metformin (10 mM) was used as control in this experiment. Values are means + SD of three experiments.

    Techniques Used: Incubation, Western Blot, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Proposed mechanistic action of PMI-5011 in beta cells On one side PMI-5011 activates cellular target of energy balance (AMPK), its substrate, ACC and cell survival target AKT in β cells. On another side PMI-5011 decreases the secretion of pro-inflammatory cytokines, NO/iNOS in macrophages. Collectively, PMI-5011 leading to insulin secretion from β cells and protection of β cells and contributes to preserve metabolic homeostasis of insulin and β cells.
    Figure Legend Snippet: Proposed mechanistic action of PMI-5011 in beta cells On one side PMI-5011 activates cellular target of energy balance (AMPK), its substrate, ACC and cell survival target AKT in β cells. On another side PMI-5011 decreases the secretion of pro-inflammatory cytokines, NO/iNOS in macrophages. Collectively, PMI-5011 leading to insulin secretion from β cells and protection of β cells and contributes to preserve metabolic homeostasis of insulin and β cells.

    Techniques Used:

    3) Product Images from "Extract of Ganoderma formosanum Mycelium as a Highly Potent Tyrosinase Inhibitor"

    Article Title: Extract of Ganoderma formosanum Mycelium as a Highly Potent Tyrosinase Inhibitor

    Journal: Scientific Reports

    doi: 10.1038/srep32854

    GFE-EA inhibits tyrosinase activity and attenuates the protein level of tyrosinase in B16-F10 melanoma cells. ( a ) For assays of tyrosinase activity, 50 μg of total protein from B16-F10 melanoma cells was incubated with 2 mM L -DOPA, and the level of dopachrome was determined by a photometric method as described in Material and Methods and expressed as percentage of control. ( b ) Whole cell lysates, treated with 100–200 ppm of GFE-EA for 48 hours, were analyzed by western blotting with antibody against tyrosinase. Equal protein loading was confirmed by antibody against β-actin (full-length gels and blots are presented in Supplementary Figure S2 ).
    Figure Legend Snippet: GFE-EA inhibits tyrosinase activity and attenuates the protein level of tyrosinase in B16-F10 melanoma cells. ( a ) For assays of tyrosinase activity, 50 μg of total protein from B16-F10 melanoma cells was incubated with 2 mM L -DOPA, and the level of dopachrome was determined by a photometric method as described in Material and Methods and expressed as percentage of control. ( b ) Whole cell lysates, treated with 100–200 ppm of GFE-EA for 48 hours, were analyzed by western blotting with antibody against tyrosinase. Equal protein loading was confirmed by antibody against β-actin (full-length gels and blots are presented in Supplementary Figure S2 ).

    Techniques Used: Activity Assay, Incubation, Western Blot

    4) Product Images from "Altered Protease–Activated Receptor-1 Expression and Signaling in a Malignant Pleural Mesothelioma Cell Line, NCI-H28, with Homozygous Deletion of the β-Catenin Gene"

    Article Title: Altered Protease–Activated Receptor-1 Expression and Signaling in a Malignant Pleural Mesothelioma Cell Line, NCI-H28, with Homozygous Deletion of the β-Catenin Gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111550

    Neither β-catenin rescue nor deletion modify cell surface PAR 1 expression. NCI-H28 cells were transiently transfected with plasmide vector containing CTNNB1 or empty vector (Ctrl) while Met-5A cells were transfected with nonspecific (Ctrls) or specific β-catenin siRNA as described in Materials and Methods. A, relative expression levels of β-catenin. Transfected cells were lysed and total cell proteins were analysed by immunoblot using an anti-β-catenin antibody. Then membranes were reprobed with an anti-β-actin antibody. Data are expressed as arbitrary unit (fold variation over Ctrl) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences of β-catenin relative levels between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (*P≤0.05) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). B, a representative immunoblot. C, cell surface PAR 1 expression measured by ELISA assay. Antibody binding to fixed transfected cells was detected by horseradish peroxidise-conjugated secondary antibody. Data represent the mean ± SEM of three independent experiments performed in triplicate. The differences in cell surface PAR 1 expression between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).
    Figure Legend Snippet: Neither β-catenin rescue nor deletion modify cell surface PAR 1 expression. NCI-H28 cells were transiently transfected with plasmide vector containing CTNNB1 or empty vector (Ctrl) while Met-5A cells were transfected with nonspecific (Ctrls) or specific β-catenin siRNA as described in Materials and Methods. A, relative expression levels of β-catenin. Transfected cells were lysed and total cell proteins were analysed by immunoblot using an anti-β-catenin antibody. Then membranes were reprobed with an anti-β-actin antibody. Data are expressed as arbitrary unit (fold variation over Ctrl) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences of β-catenin relative levels between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (*P≤0.05) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). B, a representative immunoblot. C, cell surface PAR 1 expression measured by ELISA assay. Antibody binding to fixed transfected cells was detected by horseradish peroxidise-conjugated secondary antibody. Data represent the mean ± SEM of three independent experiments performed in triplicate. The differences in cell surface PAR 1 expression between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay

    NCI-H28 cells over-express PAR 1 . A, relative expression levels of PAR 1 mRNA in Met-5A and NCI-H28 cells as determined by real time RT-PCR. B, relative expression levels of PAR 1 protein in primary mesothelial cells, Met-5A, NCI-H28, REN, Ist-Mes2, and Mero-14 cell lines as determined by immunoblot analysis followed by densitometric quantitation. Data are expressed as arbitrary unit (fold increase over Ctrl, Met-5A cells) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences in PAR 1 expression levels between Ctrl (Met-5A or primary mesothelial cells) and MPM cells were significant (*P≤0.05, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). C, a representative immunoblot.
    Figure Legend Snippet: NCI-H28 cells over-express PAR 1 . A, relative expression levels of PAR 1 mRNA in Met-5A and NCI-H28 cells as determined by real time RT-PCR. B, relative expression levels of PAR 1 protein in primary mesothelial cells, Met-5A, NCI-H28, REN, Ist-Mes2, and Mero-14 cell lines as determined by immunoblot analysis followed by densitometric quantitation. Data are expressed as arbitrary unit (fold increase over Ctrl, Met-5A cells) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences in PAR 1 expression levels between Ctrl (Met-5A or primary mesothelial cells) and MPM cells were significant (*P≤0.05, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). C, a representative immunoblot.

    Techniques Used: Expressing, Quantitative RT-PCR, Quantitation Assay

    5) Product Images from "Genistein Disrupts Glucocorticoid Receptor Signaling in Human Uterine Endometrial Ishikawa Cells"

    Article Title: Genistein Disrupts Glucocorticoid Receptor Signaling in Human Uterine Endometrial Ishikawa Cells

    Journal: Environmental Health Perspectives

    doi: 10.1289/ehp.1408437

    The GR and ER were required for transcriptional antagonism of three commonly regulated genes. ( A ) Cells treated 7 hr with the ER antagonist ICI or the GR antagonist RU486 assayed for ERα and GR protein levels by Western blot (left) and quantitated ER protein level (right). ( B ) mRNA expression of CA12 (carbonic anhydrase 12; top), LEFTY1 (left-right determination factor 1; center), and GILZ (glucocorticoid-induced leucine zipper; bottom) in cells pretreated 1 hr with ICI (left) or RU486 (RU; right) and then treated with vehicle (Veh), Dex, Gen, or Dex + Gen for 6 hr. ( C ) GR and ERα protein levels in cells transfected with non-targeting control (NTC) siRNA, GR siRNA, or ER α siRNA as assessed by Western blotting (left), and the extent of knockdown compared with NTC (right). ( D ) mRNA expression of CA12 (top), LEFTY1 (center), and GILZ (right) in cells transfected with NTC siRNA, GR siRNA, or ER α siRNA and treated for 6 hr with vehicle, Dex, Gen, or Dex + Gen. All protein values were normalized to β-actin and compared with vehicle-treated cells; all mRNA values were normalized to the housekeeping gene PPIB . Values are presented as mean ± SE of four biological replicates. * p
    Figure Legend Snippet: The GR and ER were required for transcriptional antagonism of three commonly regulated genes. ( A ) Cells treated 7 hr with the ER antagonist ICI or the GR antagonist RU486 assayed for ERα and GR protein levels by Western blot (left) and quantitated ER protein level (right). ( B ) mRNA expression of CA12 (carbonic anhydrase 12; top), LEFTY1 (left-right determination factor 1; center), and GILZ (glucocorticoid-induced leucine zipper; bottom) in cells pretreated 1 hr with ICI (left) or RU486 (RU; right) and then treated with vehicle (Veh), Dex, Gen, or Dex + Gen for 6 hr. ( C ) GR and ERα protein levels in cells transfected with non-targeting control (NTC) siRNA, GR siRNA, or ER α siRNA as assessed by Western blotting (left), and the extent of knockdown compared with NTC (right). ( D ) mRNA expression of CA12 (top), LEFTY1 (center), and GILZ (right) in cells transfected with NTC siRNA, GR siRNA, or ER α siRNA and treated for 6 hr with vehicle, Dex, Gen, or Dex + Gen. All protein values were normalized to β-actin and compared with vehicle-treated cells; all mRNA values were normalized to the housekeeping gene PPIB . Values are presented as mean ± SE of four biological replicates. * p

    Techniques Used: Western Blot, Expressing, Transfection

    6) Product Images from "Chk1 promotes replication fork progression by controlling replication initiation"

    Article Title: Chk1 promotes replication fork progression by controlling replication initiation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1005031107

    Cdc7 codepletion rescues fork slowing in Chk1-depleted cells. ( A ) Protein levels of Cdc7, Chk1, and β-Actin (loading control) in U2OS cells after 48 hours depletion with Cdc7, Chk1, Cdc7, and Chk1 or control siRNA. ( B ) Quantification of origin firing in Cdc7-, Chk1- or control-depleted cells. First label origins (green-red-green) are shown as percentage of all red (CldU) labeled tracks. ( C ) Distribution of replication fork speeds in Cdc7- or control-depleted cells. ( D ) Distribution of replication fork speeds in Chk1- or control-depleted cells. ( E ) Distribution of replication fork speeds in Cdc7- or Cdc7 and Chk1-depleted cells. ( F ) Average replication fork speeds in Cdc7-, Chk1-, or control-depleted cells. Means and standard deviation (S.D.) (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (student’s t -test, * p
    Figure Legend Snippet: Cdc7 codepletion rescues fork slowing in Chk1-depleted cells. ( A ) Protein levels of Cdc7, Chk1, and β-Actin (loading control) in U2OS cells after 48 hours depletion with Cdc7, Chk1, Cdc7, and Chk1 or control siRNA. ( B ) Quantification of origin firing in Cdc7-, Chk1- or control-depleted cells. First label origins (green-red-green) are shown as percentage of all red (CldU) labeled tracks. ( C ) Distribution of replication fork speeds in Cdc7- or control-depleted cells. ( D ) Distribution of replication fork speeds in Chk1- or control-depleted cells. ( E ) Distribution of replication fork speeds in Cdc7- or Cdc7 and Chk1-depleted cells. ( F ) Average replication fork speeds in Cdc7-, Chk1-, or control-depleted cells. Means and standard deviation (S.D.) (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (student’s t -test, * p

    Techniques Used: Labeling, Standard Deviation

    7) Product Images from "Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus"

    Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

    Journal: Environmental Health Perspectives

    doi: 10.1289/EHP1575

    Effect of neonatal genistein exposure on ligand and GR expression. ( A ) Serum levels of corticosterone were measured in adult mice with intact ovaries and adrenal glands from serum collected between 0930 and 1130 hours in the morning. Data represent the mean of 11–12 animals ± SEM. ( B ) Relative mRNA expression of GR ( Nr3c1 ) was measured by qRT-PCR in adult ADX/OVX control and genistein-exposed mice. Expression was normalized to the reference gene Ppib and set relative to expression in control mice. GR protein levels, quantified by western blot analysis, were normalized to levels of the reference protein β-actin, which was not altered by treatment group (see Figure S5). GR protein levels were set relative to control mice. The results and image represent the mean of 4–5 animals ± SEM. ( C ) Representative images of GR expression and localization (red) in luminal epithelial and endometrial stromal cells. Hoescht 33342 was used to visualize nuclei (blue). Images were taken at 630 × . ADX/OVX, adrenalectomized/ovariectomized; Dex, dexamethasone; GR, glucocorticoid receptor; qRT-PCR, quantitative real-time polymerase chain reaction; Veh, vehicle.
    Figure Legend Snippet: Effect of neonatal genistein exposure on ligand and GR expression. ( A ) Serum levels of corticosterone were measured in adult mice with intact ovaries and adrenal glands from serum collected between 0930 and 1130 hours in the morning. Data represent the mean of 11–12 animals ± SEM. ( B ) Relative mRNA expression of GR ( Nr3c1 ) was measured by qRT-PCR in adult ADX/OVX control and genistein-exposed mice. Expression was normalized to the reference gene Ppib and set relative to expression in control mice. GR protein levels, quantified by western blot analysis, were normalized to levels of the reference protein β-actin, which was not altered by treatment group (see Figure S5). GR protein levels were set relative to control mice. The results and image represent the mean of 4–5 animals ± SEM. ( C ) Representative images of GR expression and localization (red) in luminal epithelial and endometrial stromal cells. Hoescht 33342 was used to visualize nuclei (blue). Images were taken at 630 × . ADX/OVX, adrenalectomized/ovariectomized; Dex, dexamethasone; GR, glucocorticoid receptor; qRT-PCR, quantitative real-time polymerase chain reaction; Veh, vehicle.

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

    8) Product Images from "The beneficial effect of Zinc(II) on low-dose chemotherapeutic sensitivity involves p53 activation in wild-type p53-carrying colorectal cancer cells"

    Article Title: The beneficial effect of Zinc(II) on low-dose chemotherapeutic sensitivity involves p53 activation in wild-type p53-carrying colorectal cancer cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-015-0206-x

    ZnCl 2 induces p53 nuclear accumulation and activation in the presence of low-dose ADR. ( a ) HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), for 4-8-16-24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53 and p21. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. One representative set of blot from three independent experiments, all generating similar results, is shown here. ( b ) RKO were treated with ZnCl 2 (100 μM), for 4-8-16-24 h and the expression levels of p53 was detected by western blotting. A positive control for p53 expression in the same cells, is included (ADR 2 μg/ml for 16 h). Anti-β-actin was used as protein loading control. ( c ) RKO and HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), 8 h. Equal amounts of nuclear extracts were separated by SDS-PAGE and p53 levels detected by western blotting. Anti-Lamin A was used as protein loading control. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here
    Figure Legend Snippet: ZnCl 2 induces p53 nuclear accumulation and activation in the presence of low-dose ADR. ( a ) HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), for 4-8-16-24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53 and p21. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. One representative set of blot from three independent experiments, all generating similar results, is shown here. ( b ) RKO were treated with ZnCl 2 (100 μM), for 4-8-16-24 h and the expression levels of p53 was detected by western blotting. A positive control for p53 expression in the same cells, is included (ADR 2 μg/ml for 16 h). Anti-β-actin was used as protein loading control. ( c ) RKO and HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), 8 h. Equal amounts of nuclear extracts were separated by SDS-PAGE and p53 levels detected by western blotting. Anti-Lamin A was used as protein loading control. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here

    Techniques Used: Activation Assay, Expressing, Western Blot, Positive Control, SDS Page

    ZnCl 2 increases the low-dose ADR-induced p53 stabilization in colon cancer cells. ( a ) RKO and ( b ) HCT116, plated under the same confluence condition, were treated with increasing doses (0.1 to 2 μg/ml) of ADR in the presence or absence of ZnCl 2 (100 μM), for 24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53, γH2AX, and PARP (cleaved form). The samples derive from the same experiment and gels/blots were processed in parallel. ( c ) The phosphorylation of p53 at Ser15 and Ser46 was detected in HCT116 treated with ADR (0.1-1-2 μg/ml) in the presence or absence of ZnCl 2 (100 μM) for 24 h, by western blotting. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here. ( d ) HCT116 cells were treated with ADR (0.2 μg/ml) and ZnCl 2 (100 μM) for 24 h. After treatments, total cell extracts were immunoprecipitated with anti-p53 antibody. Western blot analysis was performed with anti-p53 and anti-MDM2 antibodies. IP: immunoprecipitation. IB: immunoblotting
    Figure Legend Snippet: ZnCl 2 increases the low-dose ADR-induced p53 stabilization in colon cancer cells. ( a ) RKO and ( b ) HCT116, plated under the same confluence condition, were treated with increasing doses (0.1 to 2 μg/ml) of ADR in the presence or absence of ZnCl 2 (100 μM), for 24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53, γH2AX, and PARP (cleaved form). The samples derive from the same experiment and gels/blots were processed in parallel. ( c ) The phosphorylation of p53 at Ser15 and Ser46 was detected in HCT116 treated with ADR (0.1-1-2 μg/ml) in the presence or absence of ZnCl 2 (100 μM) for 24 h, by western blotting. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here. ( d ) HCT116 cells were treated with ADR (0.2 μg/ml) and ZnCl 2 (100 μM) for 24 h. After treatments, total cell extracts were immunoprecipitated with anti-p53 antibody. Western blot analysis was performed with anti-p53 and anti-MDM2 antibodies. IP: immunoprecipitation. IB: immunoblotting

    Techniques Used: Expressing, Western Blot, Immunoprecipitation

    9) Product Images from "Arsenic-Specific Stem Cell Selection During Malignant Transformation"

    Article Title: Arsenic-Specific Stem Cell Selection During Malignant Transformation

    Journal: JNCI Journal of the National Cancer Institute

    doi: 10.1093/jnci/djq093

    Levels of arsenite-induced apoptosis and innate expression levels of apoptosis- and arsenite stress–related factors. A ) Apoptotic rates in RWPE-1 and WPE-stem cells treated with 30 μM arsenite for 24 hours. Data are presented as the mean percent increase in apoptosis over the respective untreated control cells (n = 3). B ) Mean transcript expression levels of various general apoptosis-related factors (n = 3). NS = not statistically significant. C ) Mean BCL2/BAX and BCL2L1/BAX transcript ratios (n = 3). D ) Mean MT1A and MT2A transcript levels in WPE-stem and RWPE-1 cells (n = 3). E ) Western blot analyses comparing various apoptosis-related factors in RWPE-1 ( left three lanes ) and WPE-stem cells ( right three lanes ). Each lane (25 μg protein per lane) corresponds to an independently prepared protein sample (n = 3). β-Actin was used as the control for equal protein loading. F ) Mean transcript expression levels of arsenic efflux–related factors in WPE-stem and RWPE-1 cells (n = 3). G ) Mean innate glutathione levels in RWPE-1 and WPE-stem cells (n = 3). H ) Mean transcript expression of various arsenic-specific and metabolism-related stress factors in RWPE-1 and WPE-stem cells (n = 3). For comparisons of transcript expression levels, data are presented as the percent expression vs RWPE-1 (control) cells. Error bars represent 95% confidence intervals. All P values are two-sided (Student t test).
    Figure Legend Snippet: Levels of arsenite-induced apoptosis and innate expression levels of apoptosis- and arsenite stress–related factors. A ) Apoptotic rates in RWPE-1 and WPE-stem cells treated with 30 μM arsenite for 24 hours. Data are presented as the mean percent increase in apoptosis over the respective untreated control cells (n = 3). B ) Mean transcript expression levels of various general apoptosis-related factors (n = 3). NS = not statistically significant. C ) Mean BCL2/BAX and BCL2L1/BAX transcript ratios (n = 3). D ) Mean MT1A and MT2A transcript levels in WPE-stem and RWPE-1 cells (n = 3). E ) Western blot analyses comparing various apoptosis-related factors in RWPE-1 ( left three lanes ) and WPE-stem cells ( right three lanes ). Each lane (25 μg protein per lane) corresponds to an independently prepared protein sample (n = 3). β-Actin was used as the control for equal protein loading. F ) Mean transcript expression levels of arsenic efflux–related factors in WPE-stem and RWPE-1 cells (n = 3). G ) Mean innate glutathione levels in RWPE-1 and WPE-stem cells (n = 3). H ) Mean transcript expression of various arsenic-specific and metabolism-related stress factors in RWPE-1 and WPE-stem cells (n = 3). For comparisons of transcript expression levels, data are presented as the percent expression vs RWPE-1 (control) cells. Error bars represent 95% confidence intervals. All P values are two-sided (Student t test).

    Techniques Used: Expressing, Western Blot

    10) Product Images from "A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells"

    Article Title: A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-32-72

    Zn-curc restores wild-type p53-DNA binding and transactivating activities. (A) SKBR3 and U373 cells (6x10 6 ) were plated in 150 mm dish and the day after treated with Zn-curc (100 μM) for 16 h before assayed for chromatin immunoprecipitation analysis (ChIP) with anti-p53 or anti-p73 antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoters (p21, Puma, p53AIP1, MDM2) or for mtp53 target promoters (MDR1, cyclin B2). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (No Ab). (B) Cells (3x10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24/48 h. p53 target genes were detected by RT-PCR analysis. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (C) SKBR3 and U373 cells were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc (100 μM) for 24 h, with or without p53 inhibitor pifithrin-α (PFT-α) (30 μM). p53 target genes were dtected by RT-PCR analysis. β-actin was used as control. (D) Gene expression as in (C) , was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for the indicated hours and total cell extracts were subjected to immunoblot analysis. (F) U373 cells were plated at subconfluence in 60 mm dish and the day after treated with curcumin (Curc) (50, 100 μM) for 24 h. Zn-curc (100 μM for 24 h) was used as control of p53 activation. p53 target genes were detected by RT-PCR. β-actin was used as control.
    Figure Legend Snippet: Zn-curc restores wild-type p53-DNA binding and transactivating activities. (A) SKBR3 and U373 cells (6x10 6 ) were plated in 150 mm dish and the day after treated with Zn-curc (100 μM) for 16 h before assayed for chromatin immunoprecipitation analysis (ChIP) with anti-p53 or anti-p73 antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoters (p21, Puma, p53AIP1, MDM2) or for mtp53 target promoters (MDR1, cyclin B2). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (No Ab). (B) Cells (3x10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24/48 h. p53 target genes were detected by RT-PCR analysis. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (C) SKBR3 and U373 cells were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc (100 μM) for 24 h, with or without p53 inhibitor pifithrin-α (PFT-α) (30 μM). p53 target genes were dtected by RT-PCR analysis. β-actin was used as control. (D) Gene expression as in (C) , was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for the indicated hours and total cell extracts were subjected to immunoblot analysis. (F) U373 cells were plated at subconfluence in 60 mm dish and the day after treated with curcumin (Curc) (50, 100 μM) for 24 h. Zn-curc (100 μM for 24 h) was used as control of p53 activation. p53 target genes were detected by RT-PCR. β-actin was used as control.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Activation Assay

    Zn-curc impairs survival of mutant p53-carrying cells. (A) Tumor cells (4 x 10 4 ) were plated in 60 mm dish and 24 h later treated with increased amount of Zn-curc (20, 50, 100 μM). Twenty-four hours later, plates were washed with PBS and fresh medium was added. Death-resistant colonies were stained with crystal violet 14 days later. (B) Death-resistant colonies as in (A) were counted and plotted as percentage ± SD of two independent experiments performed in duplicate. (C) Cells (3 x 10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24 and 48 h. Cell viability was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. (D) Cytofluorimetric analysis of the SubG1 peak evaluated by Propidium Iodide (PI) staining (upper panel) and microscopical analysis of SKBR3 cells, mock-treated or treated with Zn-curc (100 μM) for 24 h (lower panel). Percentage of apoptotic cells is shown ± SD of two independent experiments. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-PARP (cleaved form, 87 Kd) or anti-β-actin antibodies. (F) RKO cells were treated with Zn-curc (100 μM), ZnCl 2 (100 μM) or adryamicin (ADR, 2 μg/ml) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-γH2AX (phopho-Ser139) or anti-β-actin antibodies.
    Figure Legend Snippet: Zn-curc impairs survival of mutant p53-carrying cells. (A) Tumor cells (4 x 10 4 ) were plated in 60 mm dish and 24 h later treated with increased amount of Zn-curc (20, 50, 100 μM). Twenty-four hours later, plates were washed with PBS and fresh medium was added. Death-resistant colonies were stained with crystal violet 14 days later. (B) Death-resistant colonies as in (A) were counted and plotted as percentage ± SD of two independent experiments performed in duplicate. (C) Cells (3 x 10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24 and 48 h. Cell viability was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. (D) Cytofluorimetric analysis of the SubG1 peak evaluated by Propidium Iodide (PI) staining (upper panel) and microscopical analysis of SKBR3 cells, mock-treated or treated with Zn-curc (100 μM) for 24 h (lower panel). Percentage of apoptotic cells is shown ± SD of two independent experiments. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-PARP (cleaved form, 87 Kd) or anti-β-actin antibodies. (F) RKO cells were treated with Zn-curc (100 μM), ZnCl 2 (100 μM) or adryamicin (ADR, 2 μg/ml) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-γH2AX (phopho-Ser139) or anti-β-actin antibodies.

    Techniques Used: Mutagenesis, Staining, Trypan Blue Exclusion Assay

    Zn-curc reactivates mtp53 in an orthotopic U373 glioblastoma model. (A) U373MG-LUC cells (2.5x10 5 ) were injected into the brain of athymic mice and left to growth for 6 days before treating animals with Zn-curc every day for 7 days. Mock- or Zn-curc-treated U373M-derived tumors were then harvested and analysed with a fluorescent microscope that showed as diffuse fluorescence only in Zn-curc - treated tumors. (B) Quantification of tumor cell fluorescence positivity in U373-derived tumors, untreated or Zn-curc-treated, ±SD. (C) Total mRNA was extracted from harvested U373-derived tumors, untreated or Zn-curc-treated, and p53 target gene expression as well as VEGF, MDR1 and Bcl2 expression were assayed by PCR of reverse-transcribed cDNA. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD.
    Figure Legend Snippet: Zn-curc reactivates mtp53 in an orthotopic U373 glioblastoma model. (A) U373MG-LUC cells (2.5x10 5 ) were injected into the brain of athymic mice and left to growth for 6 days before treating animals with Zn-curc every day for 7 days. Mock- or Zn-curc-treated U373M-derived tumors were then harvested and analysed with a fluorescent microscope that showed as diffuse fluorescence only in Zn-curc - treated tumors. (B) Quantification of tumor cell fluorescence positivity in U373-derived tumors, untreated or Zn-curc-treated, ±SD. (C) Total mRNA was extracted from harvested U373-derived tumors, untreated or Zn-curc-treated, and p53 target gene expression as well as VEGF, MDR1 and Bcl2 expression were assayed by PCR of reverse-transcribed cDNA. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD.

    Techniques Used: Injection, Mouse Assay, Derivative Assay, Microscopy, Fluorescence, Expressing, Polymerase Chain Reaction

    11) Product Images from "Gentian violet induces wtp53 transactivation in cancer cells"

    Article Title: Gentian violet induces wtp53 transactivation in cancer cells

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2014.2304

    Gentian violet (GV)-induced cell death is impaired by p53 inhibition. (A) ADF cells (2×10 5 ) were plated at subconfluence in 60-mm Petri dish and the day after treated with GV (0.2–1–2 μ M) for 24 h. Cell death was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. In the lower panel is shown western immunoblotting performed on equal amount of total cell extracts to detect PARP cleavage level. Anti-β-actin was used as protein loading control. (B) Semi-quantitative RT-PCR analyses of p53 expression in HCT116-p53 +/+ and -p53 −/− cells and in RKO-Ctr and -sip53 cells. β-actin was used as internal control. (C) Cell death analyses of the indicated cells were (2×10 5 ) at subconfluence in 60-mm Petri dish and the day after treated with GV (1 μ M) for 24 h. Cell death was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. * P
    Figure Legend Snippet: Gentian violet (GV)-induced cell death is impaired by p53 inhibition. (A) ADF cells (2×10 5 ) were plated at subconfluence in 60-mm Petri dish and the day after treated with GV (0.2–1–2 μ M) for 24 h. Cell death was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. In the lower panel is shown western immunoblotting performed on equal amount of total cell extracts to detect PARP cleavage level. Anti-β-actin was used as protein loading control. (B) Semi-quantitative RT-PCR analyses of p53 expression in HCT116-p53 +/+ and -p53 −/− cells and in RKO-Ctr and -sip53 cells. β-actin was used as internal control. (C) Cell death analyses of the indicated cells were (2×10 5 ) at subconfluence in 60-mm Petri dish and the day after treated with GV (1 μ M) for 24 h. Cell death was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. * P

    Techniques Used: Inhibition, Trypan Blue Exclusion Assay, Western Blot, Quantitative RT-PCR, Expressing

    Gentian violet (GV) induces DNA damage with p53 stabilization. (A) RKO and HCT116 cells were treated with GV (1 μ M) for 16 and 24 h. Western immunoblotting was performed on equal amount of total cell extracts to detect phospho-Histone H2A.X (γH2AX) levels. Adriamycin (ADR) (2 μ g/ml) was used as control of DNA damage. Anti-β-actin was shown as protein loading control. (B) RKO and HCT116 cells were treated with GV (1 μ M) 24 h. Western immunoblotting was performed on equal amount of total cell extracts to detect p53 levels. Anti-β-actin was used as protein loading control.
    Figure Legend Snippet: Gentian violet (GV) induces DNA damage with p53 stabilization. (A) RKO and HCT116 cells were treated with GV (1 μ M) for 16 and 24 h. Western immunoblotting was performed on equal amount of total cell extracts to detect phospho-Histone H2A.X (γH2AX) levels. Adriamycin (ADR) (2 μ g/ml) was used as control of DNA damage. Anti-β-actin was shown as protein loading control. (B) RKO and HCT116 cells were treated with GV (1 μ M) 24 h. Western immunoblotting was performed on equal amount of total cell extracts to detect p53 levels. Anti-β-actin was used as protein loading control.

    Techniques Used: Western Blot

    Gentian violet (GV) induces p53/DNA binding and transcriptional activity. (A) ADF, HCT116 and RKO cells (4×10 6 ) were plated in 150-mm dish and the day after treated with GV (1 μ M) for 16 h before assayed for chromatin immunoprecipitation (ChIP) analysis with anti-p53 (FL393) antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoters (p21, Puma, p53AIP1). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (IgG). (B) H1299 cells were co-transfected with Noxa-luc (1 μ g) reporter and wtp53 (0.5 μ g/sample) expression vector. Twenty-four hours after transfection GV (1 μ M) was added for 24 h before luciferase activity was assayed. The shown data represent the mean ± SD from three independent experiments performed in duplicate. Relative luciferase unit. (C, left panel) Semi-quantitative RT-PCR analyses of p53 target genes in H1299 cells transfected and treated with GV as in (B). β-actin was used as internal control. (C, right panel) Gene expression was measured by densitometric analyses and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ± SD. (D) HCT116 and ADF cells were plated at subconfluence in 60-mm Petri dish and the day after treated with GV (1 μ M) in the presence or absence of pifithryin-α (PFT-α) (30 μ M), for 24 h. P53 target gene expression was detected by RT-PCR analyses. β-actin was used as internal control. (E) RKO and ADF cells were treated with GV (1 and 2 μ M) for 24 h. Western immuno blotting was performed on equal amount of total cell extracts to detect Bax levels. Anti-β-actin was used as protein loading control.
    Figure Legend Snippet: Gentian violet (GV) induces p53/DNA binding and transcriptional activity. (A) ADF, HCT116 and RKO cells (4×10 6 ) were plated in 150-mm dish and the day after treated with GV (1 μ M) for 16 h before assayed for chromatin immunoprecipitation (ChIP) analysis with anti-p53 (FL393) antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoters (p21, Puma, p53AIP1). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (IgG). (B) H1299 cells were co-transfected with Noxa-luc (1 μ g) reporter and wtp53 (0.5 μ g/sample) expression vector. Twenty-four hours after transfection GV (1 μ M) was added for 24 h before luciferase activity was assayed. The shown data represent the mean ± SD from three independent experiments performed in duplicate. Relative luciferase unit. (C, left panel) Semi-quantitative RT-PCR analyses of p53 target genes in H1299 cells transfected and treated with GV as in (B). β-actin was used as internal control. (C, right panel) Gene expression was measured by densitometric analyses and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ± SD. (D) HCT116 and ADF cells were plated at subconfluence in 60-mm Petri dish and the day after treated with GV (1 μ M) in the presence or absence of pifithryin-α (PFT-α) (30 μ M), for 24 h. P53 target gene expression was detected by RT-PCR analyses. β-actin was used as internal control. (E) RKO and ADF cells were treated with GV (1 and 2 μ M) for 24 h. Western immuno blotting was performed on equal amount of total cell extracts to detect Bax levels. Anti-β-actin was used as protein loading control.

    Techniques Used: Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Amplification, Transfection, Expressing, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot

    12) Product Images from "Neuroprotective Effects of Citicoline in in Vitro Models of Retinal Neurodegeneration"

    Article Title: Neuroprotective Effects of Citicoline in in Vitro Models of Retinal Neurodegeneration

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms15046286

    Citicoline treatment does not induce cytotoxic effect in primary retinal cultures. Primary retinal cultures, treated or not with 100 μM citicoline for 96 h, were immunolabeled with antibodies against rhodopsin ( A , D rhod), GABA ( B , E ) and CRALBP ( C , F ), in order to evidence photoreceptors, GABAergic neurons and Müller glia, respectively. Cell nuclei were counterstained with Hoechst 33258. Antibody immunoreactivity was unaffected in citicoline-treated samples, showing that the agent does not influence cell composition and differentiation in primary retinal cultures ( D – F ); Apoptosis was evaluated by TUNEL assay. Apoptotic nuclei were quantified as percentage of TUNEL-positive nuclei over total nuclei. Bars represent the mean ± SEM of at least five independent experiments ( G ); Absence of proapoptotic effects after citicoline treatment is also shown by unchanged levels of activated cleaved caspase 3 ( H ). Whole cell lysates were prepared from primary retinal cultures. Equal amounts of total protein from each lysate were resolved on 15% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with anti-cleaved caspase 3. β-actin levels were used as control of protein loading. Blots are representative of three independent experiments. Bars = 20 μm.
    Figure Legend Snippet: Citicoline treatment does not induce cytotoxic effect in primary retinal cultures. Primary retinal cultures, treated or not with 100 μM citicoline for 96 h, were immunolabeled with antibodies against rhodopsin ( A , D rhod), GABA ( B , E ) and CRALBP ( C , F ), in order to evidence photoreceptors, GABAergic neurons and Müller glia, respectively. Cell nuclei were counterstained with Hoechst 33258. Antibody immunoreactivity was unaffected in citicoline-treated samples, showing that the agent does not influence cell composition and differentiation in primary retinal cultures ( D – F ); Apoptosis was evaluated by TUNEL assay. Apoptotic nuclei were quantified as percentage of TUNEL-positive nuclei over total nuclei. Bars represent the mean ± SEM of at least five independent experiments ( G ); Absence of proapoptotic effects after citicoline treatment is also shown by unchanged levels of activated cleaved caspase 3 ( H ). Whole cell lysates were prepared from primary retinal cultures. Equal amounts of total protein from each lysate were resolved on 15% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with anti-cleaved caspase 3. β-actin levels were used as control of protein loading. Blots are representative of three independent experiments. Bars = 20 μm.

    Techniques Used: Immunolabeling, TUNEL Assay, SDS Page

    13) Product Images from "Impaired Insulin Signaling Affects Renal Organic Anion Transporter 3 (Oat3) Function in Streptozotocin-Induced Diabetic Rats"

    Article Title: Impaired Insulin Signaling Affects Renal Organic Anion Transporter 3 (Oat3) Function in Streptozotocin-Induced Diabetic Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096236

    Effect of diabetes on Oat3 expression in the renal cortex. (A) and (C); Western blot analysis for Oat3 in the membrane and whole cell lysate fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of Oat3 in membrane and whole cell lysate fractions normalized to β-actin, respectively. Na + -K + -ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. ** p
    Figure Legend Snippet: Effect of diabetes on Oat3 expression in the renal cortex. (A) and (C); Western blot analysis for Oat3 in the membrane and whole cell lysate fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of Oat3 in membrane and whole cell lysate fractions normalized to β-actin, respectively. Na + -K + -ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. ** p

    Techniques Used: Expressing, Western Blot, Marker

    Effects of diabetes on Akt and phospho-Akt expressions in the renal cortex. (A) and (C); Western blot analysis for Akt and phospho-Akt in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of Akt and phospho-Akt in the membrane and cytosolic fractions normalized to β - actin, respectively. Na + -K + -ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. ** p
    Figure Legend Snippet: Effects of diabetes on Akt and phospho-Akt expressions in the renal cortex. (A) and (C); Western blot analysis for Akt and phospho-Akt in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of Akt and phospho-Akt in the membrane and cytosolic fractions normalized to β - actin, respectively. Na + -K + -ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. ** p

    Techniques Used: Western Blot, Marker

    Effect of diabetes on PI3K expression in the renal cortex. (A) and (C); Western blot analysis for PI3K in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of PI3K in membrane and cytosolic fractions normalized to β - actin, respectively. Na + -K + -ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. ** p
    Figure Legend Snippet: Effect of diabetes on PI3K expression in the renal cortex. (A) and (C); Western blot analysis for PI3K in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of PI3K in membrane and cytosolic fractions normalized to β - actin, respectively. Na + -K + -ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. ** p

    Techniques Used: Expressing, Western Blot, Marker

    Effects of diabetes on PKCζ and phospho-PKCζ expressions in the renal cortex. (A) and (C); Western blot analysis for PKCζ and phospho-PKCζ in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of PKCζ and phospho-PKCζ in the membrane and cytosolic fractions normalized to β - actin, respectively. Na + -K + -ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. β - actin for the membrane fractions of PKCζ (A) and phospho-PKCζ (C) were obtained from the same membrane/loading protein. Bar graphs indicate means ± SEM from five independent experiments. ** p
    Figure Legend Snippet: Effects of diabetes on PKCζ and phospho-PKCζ expressions in the renal cortex. (A) and (C); Western blot analysis for PKCζ and phospho-PKCζ in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of PKCζ and phospho-PKCζ in the membrane and cytosolic fractions normalized to β - actin, respectively. Na + -K + -ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. β - actin for the membrane fractions of PKCζ (A) and phospho-PKCζ (C) were obtained from the same membrane/loading protein. Bar graphs indicate means ± SEM from five independent experiments. ** p

    Techniques Used: Western Blot, Marker

    14) Product Images from "Differentiation of Neurons Restricts Arbovirus Replication and Increases Expression of the Alpha Isoform of IRF-7"

    Article Title: Differentiation of Neurons Restricts Arbovirus Replication and Increases Expression of the Alpha Isoform of IRF-7

    Journal: Journal of Virology

    doi: 10.1128/JVI.02394-14

    SINV intracellular replication is delayed in differentiated AP-7 neurons. AP-7 neurons were infected with the TE strain of SINV (MOI, 5). (A) cAP-7 and dAP-7 neurons were fixed at 24 h after infection, and ultrastructure analysis was performed by transmission electron microscopy of thin sections. Bar, 100 nM. (B) Total cellular RNA was collected at various times after infection. cDNA was produced using SINV-specific primers and quantified by qPCR compared to standard SINV genomic DNA. SINV RNA levels in cAP-7 (dashed lines) or dAP-7 (solid lines) neurons are expressed as the mean SINV copy number (log 10 ) ± standard deviation of triplicate samples from a representative of two experiments. (C) Immunoblot analysis of total cell lysates prepared at the indicated times from mock-infected (M) or SINV-infected cAP-7 (left) or dAP-7 (right) neurons with anti-nsP3 (top), anti-E2 (middle), or anti-β-actin (bottom) antibodies. A representative of 2 experiments is shown. Significant differences between cAP-7 and dAP-7 at each time point were determined by two-way ANOVA with Bonferroni's posttest. ***, P
    Figure Legend Snippet: SINV intracellular replication is delayed in differentiated AP-7 neurons. AP-7 neurons were infected with the TE strain of SINV (MOI, 5). (A) cAP-7 and dAP-7 neurons were fixed at 24 h after infection, and ultrastructure analysis was performed by transmission electron microscopy of thin sections. Bar, 100 nM. (B) Total cellular RNA was collected at various times after infection. cDNA was produced using SINV-specific primers and quantified by qPCR compared to standard SINV genomic DNA. SINV RNA levels in cAP-7 (dashed lines) or dAP-7 (solid lines) neurons are expressed as the mean SINV copy number (log 10 ) ± standard deviation of triplicate samples from a representative of two experiments. (C) Immunoblot analysis of total cell lysates prepared at the indicated times from mock-infected (M) or SINV-infected cAP-7 (left) or dAP-7 (right) neurons with anti-nsP3 (top), anti-E2 (middle), or anti-β-actin (bottom) antibodies. A representative of 2 experiments is shown. Significant differences between cAP-7 and dAP-7 at each time point were determined by two-way ANOVA with Bonferroni's posttest. ***, P

    Techniques Used: Infection, Transmission Assay, Electron Microscopy, Produced, Real-time Polymerase Chain Reaction, Standard Deviation

    Irf-3 and Irf-7 mRNAs and IRF-7 protein increase during differentiation. (A) Total cellular RNA was collected at various times after plating of AP-7 neurons. Neurons were incubated at the permissive temperature in DMEM–10% FBS, 1 day prior to day 0 collection, corresponding to cAP-7 neurons. After day 0, neurons were maintained in differentiation medium at the nonpermissive temperature as described for the differentiation procedures. cDNA was produced using random primers, and Irf-3 and Irf-7 mRNAs were measured by qPCR and normalized to glyceraldehyde 3-phosphate dehydrogenase levels. mRNA levels are expressed as the mean value compared to levels in uninfected day 0 cAP-7 neurons ± standard deviations of six samples. (B) Total RNA was collected from day 0 (9 replicates) and day 7 (12 replicates) AP-7 neuron cultures or from triplicate cultures of day 0 or day 28 CSM14.1 neurons. mRNA levels, measured as described for panel A, are expressed as the mean value of differentiated cultures compared to levels in day 0 AP-7 or CSM14.1 neurons ± standard deviations. (C) Total cell lysates prepared from AP-7 neurons during differentiation were analyzed by immunoblot analysis with anti-IRF-7 (top row), anti-IRF-3 (middle row), or anti-β-actin (bottom row) antibodies. Multiple isoforms of IRF-7 are detected by the IRF-7 antibody. (D) Total cell lysates prepared from CSM neurons at the indicated days during differentiation were analyzed by immunoblot analysis with anti-IRF-7 (top row) or anti-β-actin (bottom row) antibodies. (E) Brain homogenates (10%) from 3-day-old or 6-week-old C57BL/6J mice were analyzed by immunoblot analysis with anti-IRF-7 (top row), anti-IRF-3 (middle row), or anti-β-actin (bottom row) antibodies. Results of a representative experiment of at least 2 experiments are shown. Significant differences between cAP-7 and dAP-7 at each time point were determined by one-way ANOVA with Dunnett's posttest. ***, P
    Figure Legend Snippet: Irf-3 and Irf-7 mRNAs and IRF-7 protein increase during differentiation. (A) Total cellular RNA was collected at various times after plating of AP-7 neurons. Neurons were incubated at the permissive temperature in DMEM–10% FBS, 1 day prior to day 0 collection, corresponding to cAP-7 neurons. After day 0, neurons were maintained in differentiation medium at the nonpermissive temperature as described for the differentiation procedures. cDNA was produced using random primers, and Irf-3 and Irf-7 mRNAs were measured by qPCR and normalized to glyceraldehyde 3-phosphate dehydrogenase levels. mRNA levels are expressed as the mean value compared to levels in uninfected day 0 cAP-7 neurons ± standard deviations of six samples. (B) Total RNA was collected from day 0 (9 replicates) and day 7 (12 replicates) AP-7 neuron cultures or from triplicate cultures of day 0 or day 28 CSM14.1 neurons. mRNA levels, measured as described for panel A, are expressed as the mean value of differentiated cultures compared to levels in day 0 AP-7 or CSM14.1 neurons ± standard deviations. (C) Total cell lysates prepared from AP-7 neurons during differentiation were analyzed by immunoblot analysis with anti-IRF-7 (top row), anti-IRF-3 (middle row), or anti-β-actin (bottom row) antibodies. Multiple isoforms of IRF-7 are detected by the IRF-7 antibody. (D) Total cell lysates prepared from CSM neurons at the indicated days during differentiation were analyzed by immunoblot analysis with anti-IRF-7 (top row) or anti-β-actin (bottom row) antibodies. (E) Brain homogenates (10%) from 3-day-old or 6-week-old C57BL/6J mice were analyzed by immunoblot analysis with anti-IRF-7 (top row), anti-IRF-3 (middle row), or anti-β-actin (bottom row) antibodies. Results of a representative experiment of at least 2 experiments are shown. Significant differences between cAP-7 and dAP-7 at each time point were determined by one-way ANOVA with Dunnett's posttest. ***, P

    Techniques Used: Incubation, Produced, Real-time Polymerase Chain Reaction, Mouse Assay

    Stat1 is not phosphorylated during SINV infection of AP-7 neurons. (A) cAP-7 or dAP-7 neurons were mock infected (M) or infected with SINV (MOI, 5). Total cell lysates prepared at the indicated times after infection were analyzed with anti-pSTAT1 (P-Y701) (top row), anti-STAT1 (middle row), or anti-β-actin (bottom row) antibodies. Results of a representative experiment of 2 experiments are shown. (B) cAP-7 or dAP-7 neurons were treated with 500 IU/ml of recombinant rIFN-α, -β, or -γ. Total cell lysates prepared 4 h after treatment were analyzed by immunoblotting using anti-STAT1 (P-Y701) (top row) or anti-β-actin (bottom row) antibodies. Results of a representative experiment of 2 experiments are shown.
    Figure Legend Snippet: Stat1 is not phosphorylated during SINV infection of AP-7 neurons. (A) cAP-7 or dAP-7 neurons were mock infected (M) or infected with SINV (MOI, 5). Total cell lysates prepared at the indicated times after infection were analyzed with anti-pSTAT1 (P-Y701) (top row), anti-STAT1 (middle row), or anti-β-actin (bottom row) antibodies. Results of a representative experiment of 2 experiments are shown. (B) cAP-7 or dAP-7 neurons were treated with 500 IU/ml of recombinant rIFN-α, -β, or -γ. Total cell lysates prepared 4 h after treatment were analyzed by immunoblotting using anti-STAT1 (P-Y701) (top row) or anti-β-actin (bottom row) antibodies. Results of a representative experiment of 2 experiments are shown.

    Techniques Used: Infection, Recombinant

    15) Product Images from "Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival"

    Article Title: Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118311

    Deletion of SNAP-23 leads to acute death of MEFs. MEF lines were generated from SNAP-23 fl/+ mice (clones 75.1 and 88.2) or SNAP-23 fl/- mice (clones 75.2 and 88.3). (A) Infection of a typical SNAP-23 fl/+ MEF culture infected with empty retrovirus (left panel) or GFP-Cre retrovirus (right panel) shows that two days after infection the vast majority of MEFs were viable (Annexin V - ) and expressed GFP-Cre. (B) The indicated MEF lines were infected with GFP-Cre-expressing retrovirus and the number of live cells present in each culture (based on staining with PI) was determined at different times. The absolute cell recovery in each condition was expressed relative to the amount of cells present two days after infection (control experiments showed that there was no Cre-dependent cell death in any line after only two days of infection). The data shown are representative of two independent experiments analyzed at day 2, 4, 6, 8 and one experiment analyzed at day 1, 3, 5, 7. (C) Adherent SNAP-23 fl/- MEFs were isolated 4 days after retroviral transduction with Cre (Cre+) or after mock-transduction (Cre-). Equal numbers of cells from each culture were analyzed by SDS-PAGE and immunoblotting using a SNAP-23 antibody. The blot was re-probed for anti-β actin mAb as a loading control. The amount of SNAP-23 present in each cell lysate was normalized to the amount of actin present and the data shown are mean +/- SD of three independent experiments (*p
    Figure Legend Snippet: Deletion of SNAP-23 leads to acute death of MEFs. MEF lines were generated from SNAP-23 fl/+ mice (clones 75.1 and 88.2) or SNAP-23 fl/- mice (clones 75.2 and 88.3). (A) Infection of a typical SNAP-23 fl/+ MEF culture infected with empty retrovirus (left panel) or GFP-Cre retrovirus (right panel) shows that two days after infection the vast majority of MEFs were viable (Annexin V - ) and expressed GFP-Cre. (B) The indicated MEF lines were infected with GFP-Cre-expressing retrovirus and the number of live cells present in each culture (based on staining with PI) was determined at different times. The absolute cell recovery in each condition was expressed relative to the amount of cells present two days after infection (control experiments showed that there was no Cre-dependent cell death in any line after only two days of infection). The data shown are representative of two independent experiments analyzed at day 2, 4, 6, 8 and one experiment analyzed at day 1, 3, 5, 7. (C) Adherent SNAP-23 fl/- MEFs were isolated 4 days after retroviral transduction with Cre (Cre+) or after mock-transduction (Cre-). Equal numbers of cells from each culture were analyzed by SDS-PAGE and immunoblotting using a SNAP-23 antibody. The blot was re-probed for anti-β actin mAb as a loading control. The amount of SNAP-23 present in each cell lysate was normalized to the amount of actin present and the data shown are mean +/- SD of three independent experiments (*p

    Techniques Used: Generated, Mouse Assay, Infection, Expressing, Staining, Isolation, Transduction, SDS Page

    16) Product Images from "Activation of the P2X7 receptor in midbrain periaqueductal gray participates in the analgesic effect of tramadol in bone cancer pain rats"

    Article Title: Activation of the P2X7 receptor in midbrain periaqueductal gray participates in the analgesic effect of tramadol in bone cancer pain rats

    Journal: Molecular Pain

    doi: 10.1177/1744806918803039

    Alterations of P2X 7 receptor protein level in the vlPAG induced by cancer cell inoculation or tramadol injections. (a) By using Western blot analysis, we detected a protein band of ≈68 kDa, coinciding with the known molecular weight of the P2X 7 receptor. (b) β-actin was used as loading control. The P2X 7 receptor protein levels in different groups were expressed as ROD. No distinct change in P2X 7 receptor protein level was observed in vlPAG between normal and sham-BCP groups. The P2X 7 receptor protein level in vlPAG in BCP group was increased compared with normal and sham-BCP groups. Intraperitoneal tramadol injection at doses of 10, 20, and 40 mg/kg showed significantly and dose dependently upregulated the P2X 7 receptor protein level in vlPAG. n = 6; ⋆⋆ p
    Figure Legend Snippet: Alterations of P2X 7 receptor protein level in the vlPAG induced by cancer cell inoculation or tramadol injections. (a) By using Western blot analysis, we detected a protein band of ≈68 kDa, coinciding with the known molecular weight of the P2X 7 receptor. (b) β-actin was used as loading control. The P2X 7 receptor protein levels in different groups were expressed as ROD. No distinct change in P2X 7 receptor protein level was observed in vlPAG between normal and sham-BCP groups. The P2X 7 receptor protein level in vlPAG in BCP group was increased compared with normal and sham-BCP groups. Intraperitoneal tramadol injection at doses of 10, 20, and 40 mg/kg showed significantly and dose dependently upregulated the P2X 7 receptor protein level in vlPAG. n = 6; ⋆⋆ p

    Techniques Used: Western Blot, Molecular Weight, Injection

    17) Product Images from "Activation of the P2X7 receptor in midbrain periaqueductal gray participates in the analgesic effect of tramadol in bone cancer pain rats"

    Article Title: Activation of the P2X7 receptor in midbrain periaqueductal gray participates in the analgesic effect of tramadol in bone cancer pain rats

    Journal: Molecular Pain

    doi: 10.1177/1744806918803039

    Alterations of P2X 7 receptor protein level in the vlPAG induced by cancer cell inoculation or tramadol injections. (a) By using Western blot analysis, we detected a protein band of ≈68 kDa, coinciding with the known molecular weight of the P2X 7 receptor. (b) β-actin was used as loading control. The P2X 7 receptor protein levels in different groups were expressed as ROD. No distinct change in P2X 7 receptor protein level was observed in vlPAG between normal and sham-BCP groups. The P2X 7 receptor protein level in vlPAG in BCP group was increased compared with normal and sham-BCP groups. Intraperitoneal tramadol injection at doses of 10, 20, and 40 mg/kg showed significantly and dose dependently upregulated the P2X 7 receptor protein level in vlPAG. n = 6; ⋆⋆ p
    Figure Legend Snippet: Alterations of P2X 7 receptor protein level in the vlPAG induced by cancer cell inoculation or tramadol injections. (a) By using Western blot analysis, we detected a protein band of ≈68 kDa, coinciding with the known molecular weight of the P2X 7 receptor. (b) β-actin was used as loading control. The P2X 7 receptor protein levels in different groups were expressed as ROD. No distinct change in P2X 7 receptor protein level was observed in vlPAG between normal and sham-BCP groups. The P2X 7 receptor protein level in vlPAG in BCP group was increased compared with normal and sham-BCP groups. Intraperitoneal tramadol injection at doses of 10, 20, and 40 mg/kg showed significantly and dose dependently upregulated the P2X 7 receptor protein level in vlPAG. n = 6; ⋆⋆ p

    Techniques Used: Western Blot, Molecular Weight, Injection

    18) Product Images from "Cortactin Is a Substrate of Activated Cdc42-Associated Kinase 1 (ACK1) during Ligand-induced Epidermal Growth Factor Receptor Downregulation"

    Article Title: Cortactin Is a Substrate of Activated Cdc42-Associated Kinase 1 (ACK1) during Ligand-induced Epidermal Growth Factor Receptor Downregulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044363

    ACK1 directly phosphorylates cortactin at Y421/466/482. (A) Characterization of purified ACK1. Recombinant 6X-histidine tagged recombinant ACK1 was purified from SF9 cells and 5 micrograms stained with either Coomassie blue ( left ) or analyzed by Western blotting with anti-ACK antibodies ( middle ). Kinase activity was assayed by analyzing autophosphorylation (Autophos) using 0.5 micrograms of 6X-His ACK1 incubated with gamma- 32 P-ATP for 10 min at 30°C. The reaction was evaluated by SDS-PAGE and autoradiography ( right ). The position of molecular weight standards is shown on the left and 6X-His ACK1 on the right. (B) Identification of cortactin tyrosine residues phosphorylated by ACK1. Purified GST-cortactin murine wild type (WT) or Y421/466/482F (triple tyrosine mutant; TYM) (0.5 micrograms) were incubated in kinase assays without or with 3U of purified Src ( left ) or with the indicated amounts of purified 6X-His ACK1 ( middle and right ) in the presence of [gamma- 32 P]ATP. Reactions were separated by SDS-PAGE and analyzed by audioradiography. Arrows indicate the positions of phosphorylated GST-cortactin and 6X-His ACK1 ( left ). (C) Determination of the K M of ACK1 for cortactin. Graphical representation showing ACK1 phosphorylation kinetics for cortactin calculated from the kinase assay in B ( right panel). Percent of maximum phosphorylation signal measured by densitometry is represented on the ordinate versus concentration of ACK1 (in micrograms) on the abscissa. The calculated K M for cortactin phosphorylation is shown. Data in B and C are representative from three independent experiments. (D) ACK1 knockdown reduces cortactin phosphorylation on tyrosine 421. 1483 cells transfected with scrambled control (Ctl) siRNA or ACK1-targeting siRNA were lysed and analyzed by Western blotting for ACK1 knockdown (ACK1) and cortactin pY421 phosphorylation (Cort pY421). Beta actin was blotted to verify equivalent protein loading. Blots are representative of two independent experiments.
    Figure Legend Snippet: ACK1 directly phosphorylates cortactin at Y421/466/482. (A) Characterization of purified ACK1. Recombinant 6X-histidine tagged recombinant ACK1 was purified from SF9 cells and 5 micrograms stained with either Coomassie blue ( left ) or analyzed by Western blotting with anti-ACK antibodies ( middle ). Kinase activity was assayed by analyzing autophosphorylation (Autophos) using 0.5 micrograms of 6X-His ACK1 incubated with gamma- 32 P-ATP for 10 min at 30°C. The reaction was evaluated by SDS-PAGE and autoradiography ( right ). The position of molecular weight standards is shown on the left and 6X-His ACK1 on the right. (B) Identification of cortactin tyrosine residues phosphorylated by ACK1. Purified GST-cortactin murine wild type (WT) or Y421/466/482F (triple tyrosine mutant; TYM) (0.5 micrograms) were incubated in kinase assays without or with 3U of purified Src ( left ) or with the indicated amounts of purified 6X-His ACK1 ( middle and right ) in the presence of [gamma- 32 P]ATP. Reactions were separated by SDS-PAGE and analyzed by audioradiography. Arrows indicate the positions of phosphorylated GST-cortactin and 6X-His ACK1 ( left ). (C) Determination of the K M of ACK1 for cortactin. Graphical representation showing ACK1 phosphorylation kinetics for cortactin calculated from the kinase assay in B ( right panel). Percent of maximum phosphorylation signal measured by densitometry is represented on the ordinate versus concentration of ACK1 (in micrograms) on the abscissa. The calculated K M for cortactin phosphorylation is shown. Data in B and C are representative from three independent experiments. (D) ACK1 knockdown reduces cortactin phosphorylation on tyrosine 421. 1483 cells transfected with scrambled control (Ctl) siRNA or ACK1-targeting siRNA were lysed and analyzed by Western blotting for ACK1 knockdown (ACK1) and cortactin pY421 phosphorylation (Cort pY421). Beta actin was blotted to verify equivalent protein loading. Blots are representative of two independent experiments.

    Techniques Used: Purification, Recombinant, Staining, Western Blot, Activity Assay, Incubation, SDS Page, Autoradiography, Molecular Weight, Mutagenesis, Kinase Assay, Concentration Assay, Transfection, CTL Assay

    19) Product Images from "Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus"

    Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

    Journal: Environmental Health Perspectives

    doi: 10.1289/EHP1575

    Effect of neonatal genistein exposure on ligand and GR expression. ( A ) Serum levels of corticosterone were measured in adult mice with intact ovaries and adrenal glands from serum collected between 0930 and 1130 hours in the morning. Data represent the mean of 11 – 12 animals ± SEM . ( B ) Relative mRNA expression of GR ( Nr3c1 ) was measured by qRT-PCR in adult ADX/OVX control and genistein-exposed mice. Expression was normalized to the reference gene Ppib and set relative to expression in control mice. GR protein levels, quantified by western blot analysis, were normalized to levels of the reference protein β -actin , which was not altered by treatment group (see Figure S5). GR protein levels were set relative to control mice. The results and image represent the mean of 4 – 5 animals ± SEM . ( C ) Representative images of GR expression and localization (red) in luminal epithelial and endometrial stromal cells. Hoescht 33342 was used to visualize nuclei (blue). Images were taken at 630 × . ADX/OVX, adrenalectomized/ovariectomized; Dex, dexamethasone; GR, glucocorticoid receptor; qRT-PCR, quantitative real-time polymerase chain reaction; Veh, vehicle.
    Figure Legend Snippet: Effect of neonatal genistein exposure on ligand and GR expression. ( A ) Serum levels of corticosterone were measured in adult mice with intact ovaries and adrenal glands from serum collected between 0930 and 1130 hours in the morning. Data represent the mean of 11 – 12 animals ± SEM . ( B ) Relative mRNA expression of GR ( Nr3c1 ) was measured by qRT-PCR in adult ADX/OVX control and genistein-exposed mice. Expression was normalized to the reference gene Ppib and set relative to expression in control mice. GR protein levels, quantified by western blot analysis, were normalized to levels of the reference protein β -actin , which was not altered by treatment group (see Figure S5). GR protein levels were set relative to control mice. The results and image represent the mean of 4 – 5 animals ± SEM . ( C ) Representative images of GR expression and localization (red) in luminal epithelial and endometrial stromal cells. Hoescht 33342 was used to visualize nuclei (blue). Images were taken at 630 × . ADX/OVX, adrenalectomized/ovariectomized; Dex, dexamethasone; GR, glucocorticoid receptor; qRT-PCR, quantitative real-time polymerase chain reaction; Veh, vehicle.

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

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    Electrophoresis:

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    Article Snippet: Paragraph title: Western blot analysis ... Following protein transfer into 0.45 μM nitrocellulose (NC) membrane (BD, Cat.#1620115), membranes were blocked with 5% skim-milk in TBS-T buffer (0.05%) for 2 h. Then, membranes that washed and were probed using the following antibodies: Nuclear factor kappa B (NF-κB) p65 (Cell signaling Technology, Inc. Cat.#8242; 1:500 dilutions), phospho-NF-κB p65 (Ser468) (Cell signaling Technology, Inc. Cat.#3039; 1:500 dilutions) and anti–β-actin mouse mAb (Sigma-Aldrich, Inc. Cat.#A5316; 1:5,000 dilutions), and the secondary antibodies.

    Article Title: Beneficial Effects of Antecedent Exercise Training on Limb Motor Function and Calpain Expression in a Rat Model of Stroke
    Article Snippet: The hippocampus was homogenized in lysis buffer B (137 mM NaCl, 20 mM Tris, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 0.5 mM sodium vanadate, pH 8.0) for western blot analysis. .. After incubation with the rabbit polyclonal anti-m-calpain antibody (1:1,000 Triple Point Biologics; Forest Grove, OR, USA) and the monoclonal anti-β actin (A-5316, 1:5,000, Sigma, USA) antibody, the membranes were washed with TBST and incubated with appropriate horse radish peroxidase-conjugated secondary antibody (1:4,000 dilution).

    Article Title: Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study
    Article Snippet: Paragraph title: 4.10. Western Blotting ... The membrane was blocked with 4% dry non-fat milk in 0.1% Tween/Tris Buffer Saline (T-TBS) and incubated overnight at 4 °C with anti β-actin mouse monoclonal antibody (A-5316, Sigma-Aldrich) or anti-HPRT rabbit monoclonal antibody (ab109021, Abcam, Cambridge, UK) diluted 1:2000 or 1:10,000 in T-TBS, respectively.

    Article Title: The Effects of Antecedent Exercise on Motor Function Recovery and Brain-derived Neurotrophic Factor Expression after Focal Cerebral Ischemia in Rats
    Article Snippet: The hippocampus was homogenized in lysis buffer B (137 mM NaCl, 20 mM Tris, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg/Ml aprotinin, 1 µg/Ml leupeptin, 0.5 mM sodium vanadate, pH8.0) for western blot analysis. .. After incubation with the anti-BDNF primary antibody (sc-546, Santa Cruz, USA), monoclonal anti-β actin (A-5316, 1:5,000, Sigma, USA) antibodies membranes were washed with TBST and incubated with the appropriate horse radish peroxidase-conjugated secondary antibody (1:4,000 dilution).

    Article Title: Escherichia coli Subtilase Cytotoxin Induces Apoptosis Regulated by Host Bcl-2 Family Proteins Bax/Bak ▿
    Article Snippet: Paragraph title: Western immunoblotting. ... Anti-Puma (catalog no. P-4618) and anti-β-actin (AC-74) (catalog no. A-5316) were from Sigma.

    Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients
    Article Snippet: Paragraph title: Western blot analysis ... Equal loading and transfer were assessed by reprobing the blots with anti-β -actin antibody (Sigma A-5316) and Ponceau red (Sigma 19 976-1), respectively. β -Actin was used as an internal standard to account for variations in the amount of protein (usually 20 μ g) loaded in each lane.

    Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation
    Article Snippet: Paragraph title: Western blot analysis ... A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or β-actin (1:20,000; Sigma, Cat#A5316 RRID:AB_476743) were used as loading controls.

    Electrotransfer:

    Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation
    Article Snippet: After the electrotransfer of the proteins, CRMP2 protein was detected using a rabbit antibody directed against CRMP2 (1:500; Abcam, Cat#ab36201, RRID:AB_731750) and the different synaptic markers were detected using mouse monoclonal antibodies directed against PSD-95 (1:20,000; Millipore, Merck, Cat#P246, RRID:AB_260911), synaptophysin (1:2000–1:20,000; Sigma, Cat#S5768, RRID:AB_477523), and syntaxin 1 A (1:2000; Sigma, Cat#S0664, RRID:AB_477483), guinea-pig polyclonal anti-vGluT1 (1:10,000; Synaptic Systems, Goettingen, Germany, Cat#135304 RRID:AB_887878), anti-vGAT (1:1000; Synaptic Systems; Cat#131004, RRID:AB_887873), and rabbit polyclonal anti-gephyrin (1:10,000; Abcam, Cat#ab25784 RRID:AB_1209349), all diluted in TBS-T (Tris-buffered saline, 0.1% Tween 20) containing 5% dried milk. .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or β-actin (1:20,000; Sigma, Cat#A5316 RRID:AB_476743) were used as loading controls.

    TCA Precipitation:

    Article Title: Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells
    Article Snippet: Western blot analysis Proteins from the same volume of each fraction (200 to 250 μl) were concentrated by TCA precipitation, dissolved in SDS/β-mercaptoethanol loading buffer, and resolved on a 4% to 15% gradient SDS/PAGE gel (Bio-Rad, Hercules, CA). .. Antibodies against H3 (ab1791), DNMT3A (ab2850), and SUV39h1 (ab12405) were purchased from Abcam Inc. (Cambridge, UK); DNMT1 (sc-20701) and DNMT3B (sc-10235) from Santa Cruz Biotech (Santa Cruz, CA); Myc epitope tag (05-724) from Upstate (now Millipore, Billerica, MA) and G9a (G 6919); β-Actin (A 5316) from Sigma (Saint Louis, MO).

    Cell Culture:

    Article Title: LXCXE-independent chromatin remodeling by Rb/E2f mediates neuronal quiescence
    Article Snippet: .. Protein was isolated from cultured cortical neurons and western blot analyses performed as previously described, with antibodies directed toward cleaved caspase-3 (Cell Signaling, Cat No. 9664S), Rb (PharMingen, Cat No. 554136), Cyclin A2 (Abcam, Cat No. ab7965), PCNA (Millpore, Cat No. MAB424) and β-actin (Sigma, Cat No. A-5316). ..

    Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation
    Article Snippet: Protein extracts from cultured hippocampal neurons or hippocampal homogenates were diluted in 6× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer ((0.5 M Tris, 0.4% SDS, pH 6.8), 30% glycerol, 10% SDS, 0.6 M dithiothreitol, and 0.012% of bromophenol blue), boiled at 95 °C for 5 min and then separated by 10% SDS-PAGE electrophoresis. .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or β-actin (1:20,000; Sigma, Cat#A5316 RRID:AB_476743) were used as loading controls.

    other:

    Article Title: Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death
    Article Snippet: Monoclonal anti-β-actin (Catalogue No. A-5316) was from Sigma Aldrich.

    Imaging:

    Article Title: Escherichia coli Subtilase Cytotoxin Induces Apoptosis Regulated by Host Bcl-2 Family Proteins Bax/Bak ▿
    Article Snippet: Anti-Puma (catalog no. P-4618) and anti-β-actin (AC-74) (catalog no. A-5316) were from Sigma. .. Images were captured using a Kodak 4000MM image station (Carestream Molecular Imaging), and net band intensities (mean pixel intensity by number of pixels) were determined using MI imaging software (Carestream Molecular Imaging).

    Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients
    Article Snippet: Equal loading and transfer were assessed by reprobing the blots with anti-β -actin antibody (Sigma A-5316) and Ponceau red (Sigma 19 976-1), respectively. β -Actin was used as an internal standard to account for variations in the amount of protein (usually 20 μ g) loaded in each lane. .. The levels of protein expression were quantified using the Kodak 1D Image analysing software (Scientific Imaging Systems, Eastman Kodak Company, Rochester, NY, USA) and normalised to the β -actin levels.

    Protein Concentration:

    Article Title: Effects of Balance and Gait Training on the Recovery of the Motor Function in an Animal Model of Parkinson's Disease
    Article Snippet: The samples were centrifuged for 20 min at 15,000 rpm to obtain supernatant, and the protein concentration in the solution was quantified using Bradford method (Bio-Rad protein assay). .. To confirm if the equivalent protein, monoclonal anti-β actin (A-5316, 1:5,000, Sigma, USA), had been loaded, goat anti-mouse IgG (BD Bioscience, Flanklin Lakes, NJ, USA) was used for comparison.

    Article Title: Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study
    Article Snippet: Sample lysates were centrifuged at 15,000 rpm for 20 min at 4 °C and protein concentration in the supernatants was determined by the bicinchoninic acid (BCA; Pierce, Rockford, IL, USA) microplate method. .. The membrane was blocked with 4% dry non-fat milk in 0.1% Tween/Tris Buffer Saline (T-TBS) and incubated overnight at 4 °C with anti β-actin mouse monoclonal antibody (A-5316, Sigma-Aldrich) or anti-HPRT rabbit monoclonal antibody (ab109021, Abcam, Cambridge, UK) diluted 1:2000 or 1:10,000 in T-TBS, respectively.

    Binding Assay:

    Article Title: Effects of Balance and Gait Training on the Recovery of the Motor Function in an Animal Model of Parkinson's Disease
    Article Snippet: Thereafter, the gel was transferred to a nitrocellulose membrane (Whatman GmbH, Dassel, Germany) and underwent reacted with TBX and 5% non fat dried milk for 1 hr at 4°C to prevent non-specific binding with the antibody. .. To confirm if the equivalent protein, monoclonal anti-β actin (A-5316, 1:5,000, Sigma, USA), had been loaded, goat anti-mouse IgG (BD Bioscience, Flanklin Lakes, NJ, USA) was used for comparison.

    Article Title: Beneficial Effects of Antecedent Exercise Training on Limb Motor Function and Calpain Expression in a Rat Model of Stroke
    Article Snippet: After electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes and nonspecific binding was blocked with 5% nonfat dry milk in tris-buffered saline and Tween 20. .. After incubation with the rabbit polyclonal anti-m-calpain antibody (1:1,000 Triple Point Biologics; Forest Grove, OR, USA) and the monoclonal anti-β actin (A-5316, 1:5,000, Sigma, USA) antibody, the membranes were washed with TBST and incubated with appropriate horse radish peroxidase-conjugated secondary antibody (1:4,000 dilution).

    Nucleic Acid Electrophoresis:

    Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation
    Article Snippet: Protein extracts from cultured hippocampal neurons or hippocampal homogenates were diluted in 6× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer ((0.5 M Tris, 0.4% SDS, pH 6.8), 30% glycerol, 10% SDS, 0.6 M dithiothreitol, and 0.012% of bromophenol blue), boiled at 95 °C for 5 min and then separated by 10% SDS-PAGE electrophoresis. .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or β-actin (1:20,000; Sigma, Cat#A5316 RRID:AB_476743) were used as loading controls.

    Isolation:

    Article Title: LXCXE-independent chromatin remodeling by Rb/E2f mediates neuronal quiescence
    Article Snippet: .. Protein was isolated from cultured cortical neurons and western blot analyses performed as previously described, with antibodies directed toward cleaved caspase-3 (Cell Signaling, Cat No. 9664S), Rb (PharMingen, Cat No. 554136), Cyclin A2 (Abcam, Cat No. ab7965), PCNA (Millpore, Cat No. MAB424) and β-actin (Sigma, Cat No. A-5316). ..

    Protein Extraction:

    Article Title: Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function
    Article Snippet: Paragraph title: Protein extraction and quantification ... After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies overnight at 4°C (BAP1, Santa Cruz sc-4 sc-28383, 1/500; CDH2, Cell signaling #14215, 1/200; β-actin, Sigma-Aldrich A-5316, 1/10000, PGC1-α, Santa Cruz sc-13067, 1/500, BAF47, BD Pharmingen #612111, 1/1000, OXR1, Novus Biologicals NBP1-86393, 1/1000).

    Blocking Assay:

    Article Title: Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function
    Article Snippet: .. After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies overnight at 4°C (BAP1, Santa Cruz sc-4 sc-28383, 1/500; CDH2, Cell signaling #14215, 1/200; β-actin, Sigma-Aldrich A-5316, 1/10000, PGC1-α, Santa Cruz sc-13067, 1/500, BAF47, BD Pharmingen #612111, 1/1000, OXR1, Novus Biologicals NBP1-86393, 1/1000). .. Following incubation with appropriate fluorescent secondary antibody (1/10 000 mixed with 0.1% Tween-20), the immunoreactive bands were visualized using LI-COR-Odyssey infra-red scanner (LI-COR).

    Article Title: Transcriptomic analysis reveals sex-specific differences in the expression of Dcl1 and Fis1 genes in the radio-adaptive response of thymocytes to TRP53-mediated apoptosis
    Article Snippet: After blocking, membranes were incubated with each primary antibody in either 5 % w/v BSA or nonfat dry milk, 1× TBS, 0.1 % Tween 20 overnight at 4 °C. .. For β-actin (A 5316; Sigma) was used a dilution of 1:5000.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: High fat diet increases melanoma cell growth in the bone marrow by inducing osteopontin and interleukin 6
    Article Snippet: .. Following protein transfer into 0.45 μM nitrocellulose (NC) membrane (BD, Cat.#1620115), membranes were blocked with 5% skim-milk in TBS-T buffer (0.05%) for 2 h. Then, membranes that washed and were probed using the following antibodies: Nuclear factor kappa B (NF-κB) p65 (Cell signaling Technology, Inc. Cat.#8242; 1:500 dilutions), phospho-NF-κB p65 (Ser468) (Cell signaling Technology, Inc. Cat.#3039; 1:500 dilutions) and anti–β-actin mouse mAb (Sigma-Aldrich, Inc. Cat.#A5316; 1:5,000 dilutions), and the secondary antibodies. .. Chemiluminescent reagent (GE Healthcare, Cat.#RPN2106) and detected on Amersham Hyperfilm ECL (GE Healthcare, Cat.#28906839) were used for the revelation of the western blot.

    Article Title: Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma
    Article Snippet: .. Protein detection was performed using anti-NELFE (Abcam, cat# ab170104), anti-β–actin (Sigma-Aldrich, catA5316), anti-Cyclin B1 (Cell Signaling Technology, cat #4138), anti-HLA-A (Abcam, cat#52922), anti-HRAS (Santa Cruz, cat#sc-520) and anti-SV40 T Ag (Santa Cruz, cat#sc-147). ..

    Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation
    Article Snippet: .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Catab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or β-actin (1:20,000; Sigma, Cat#A5316 RRID:AB_476743) were used as loading controls. .. The membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse or goat anti-guinea-pig secondary antibodies (1:10,000; Pierce) and subsequently with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Alfagene) or with Luminata Forte Western HRP Substrate (Millipore).

    SDS Page:

    Article Title: Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells
    Article Snippet: Western blot analysis Proteins from the same volume of each fraction (200 to 250 μl) were concentrated by TCA precipitation, dissolved in SDS/β-mercaptoethanol loading buffer, and resolved on a 4% to 15% gradient SDS/PAGE gel (Bio-Rad, Hercules, CA). .. Antibodies against H3 (ab1791), DNMT3A (ab2850), and SUV39h1 (ab12405) were purchased from Abcam Inc. (Cambridge, UK); DNMT1 (sc-20701) and DNMT3B (sc-10235) from Santa Cruz Biotech (Santa Cruz, CA); Myc epitope tag (05-724) from Upstate (now Millipore, Billerica, MA) and G9a (G 6919); β-Actin (A 5316) from Sigma (Saint Louis, MO).

    Article Title: Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function
    Article Snippet: 50μg of protein extracts were resolved by 4-15% SDS-PAGE (SDS-PAGE Mini- PROTEAN® TGX™ #456-1086 Biorad) and transferred to 0.2 μm nitrocellulose membranes (Biorad). .. After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies overnight at 4°C (BAP1, Santa Cruz sc-4 sc-28383, 1/500; CDH2, Cell signaling #14215, 1/200; β-actin, Sigma-Aldrich A-5316, 1/10000, PGC1-α, Santa Cruz sc-13067, 1/500, BAF47, BD Pharmingen #612111, 1/1000, OXR1, Novus Biologicals NBP1-86393, 1/1000).

    Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation
    Article Snippet: Protein extracts from cultured hippocampal neurons or hippocampal homogenates were diluted in 6× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer ((0.5 M Tris, 0.4% SDS, pH 6.8), 30% glycerol, 10% SDS, 0.6 M dithiothreitol, and 0.012% of bromophenol blue), boiled at 95 °C for 5 min and then separated by 10% SDS-PAGE electrophoresis. .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or β-actin (1:20,000; Sigma, Cat#A5316 RRID:AB_476743) were used as loading controls.

    Plasmid Preparation:

    Article Title: A polymorphism in the lysyl oxidase propeptide domain accelerates carcinogen-induced cancer
    Article Snippet: .. Antibodies used were Ki-67 (cloneSP6; Thermo Scientific), LOX-PP (rabbit polyclonal IgG) used as described previously , β-actin (#A-5316; Sigma–Aldrich) and biotinylated goat anti-rabbit IgG (Vector). ..

    Software:

    Article Title: Beneficial Effects of Antecedent Exercise Training on Limb Motor Function and Calpain Expression in a Rat Model of Stroke
    Article Snippet: After incubation with the rabbit polyclonal anti-m-calpain antibody (1:1,000 Triple Point Biologics; Forest Grove, OR, USA) and the monoclonal anti-β actin (A-5316, 1:5,000, Sigma, USA) antibody, the membranes were washed with TBST and incubated with appropriate horse radish peroxidase-conjugated secondary antibody (1:4,000 dilution). .. The film signals were digitally scanned and then quantified with NIH image J software.

    Article Title: The Effects of Antecedent Exercise on Motor Function Recovery and Brain-derived Neurotrophic Factor Expression after Focal Cerebral Ischemia in Rats
    Article Snippet: After incubation with the anti-BDNF primary antibody (sc-546, Santa Cruz, USA), monoclonal anti-β actin (A-5316, 1:5,000, Sigma, USA) antibodies membranes were washed with TBST and incubated with the appropriate horse radish peroxidase-conjugated secondary antibody (1:4,000 dilution). .. The film signals were digitally scanned and then quantified using NIH image J software.

    Article Title: Escherichia coli Subtilase Cytotoxin Induces Apoptosis Regulated by Host Bcl-2 Family Proteins Bax/Bak ▿
    Article Snippet: Anti-Puma (catalog no. P-4618) and anti-β-actin (AC-74) (catalog no. A-5316) were from Sigma. .. Images were captured using a Kodak 4000MM image station (Carestream Molecular Imaging), and net band intensities (mean pixel intensity by number of pixels) were determined using MI imaging software (Carestream Molecular Imaging).

    Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients
    Article Snippet: Equal loading and transfer were assessed by reprobing the blots with anti-β -actin antibody (Sigma A-5316) and Ponceau red (Sigma 19 976-1), respectively. β -Actin was used as an internal standard to account for variations in the amount of protein (usually 20 μ g) loaded in each lane. .. The levels of protein expression were quantified using the Kodak 1D Image analysing software (Scientific Imaging Systems, Eastman Kodak Company, Rochester, NY, USA) and normalised to the β -actin levels.

    Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation
    Article Snippet: A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or β-actin (1:20,000; Sigma, Cat#A5316 RRID:AB_476743) were used as loading controls. .. The immunoreactivity was visualized using a VersaDoc3000 apparatus and analyzed using the ImageLab software (BioRad, Amadora, Portugal).

    Irradiation:

    Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients
    Article Snippet: Western blot analysis Nearly confluent nonirradiated and irradiated with 8 Gy fibroblasts were detached by trypsinisation, lyzed in RIPA buffer (10 mM tris-HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100) containing protease inhibitors (2 μ g ml−1 aprotinin, 2 μ g ml−1 leupeptin, 5 μ g ml−1 pepstatin, 1 mM phenylmethane sulphonyl fluoride) for 30 min on ice and subsequently centrifuged 10 min at 500 g . .. Equal loading and transfer were assessed by reprobing the blots with anti-β -actin antibody (Sigma A-5316) and Ponceau red (Sigma 19 976-1), respectively. β -Actin was used as an internal standard to account for variations in the amount of protein (usually 20 μ g) loaded in each lane.

    Negative Control:

    Article Title: Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study
    Article Snippet: Western Blotting At the endpoint of the culture, both cell/scaffolds constructs (n = 3) and controls (scaffolds without cells as a negative control, and cells grown in monolayer up to 80% confluence—4 days—as a positive control) were washed in D-PBS and resuspended in a cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and antiproteases 1×, all from Sigma Aldrich). .. The membrane was blocked with 4% dry non-fat milk in 0.1% Tween/Tris Buffer Saline (T-TBS) and incubated overnight at 4 °C with anti β-actin mouse monoclonal antibody (A-5316, Sigma-Aldrich) or anti-HPRT rabbit monoclonal antibody (ab109021, Abcam, Cambridge, UK) diluted 1:2000 or 1:10,000 in T-TBS, respectively.

    Radio Immunoprecipitation:

    Article Title: Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function
    Article Snippet: Cell pellets were lysed in radioimmunoprecipitation assay buffer (RIPA) [100mM Tris-HCl/pH7.5, 0.1M NaCl, 1mM EDTA, 1% Triton, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)], supplemented with NaF, Na3 VO4 , phenylmethanesulfonylfluoride (PMSF) and a cocktail of protease inhibitors (cOmplete©, Roche). .. After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies overnight at 4°C (BAP1, Santa Cruz sc-4 sc-28383, 1/500; CDH2, Cell signaling #14215, 1/200; β-actin, Sigma-Aldrich A-5316, 1/10000, PGC1-α, Santa Cruz sc-13067, 1/500, BAF47, BD Pharmingen #612111, 1/1000, OXR1, Novus Biologicals NBP1-86393, 1/1000).

    Lysis:

    Article Title: Beneficial Effects of Antecedent Exercise Training on Limb Motor Function and Calpain Expression in a Rat Model of Stroke
    Article Snippet: The tissue was homogenized in freshly prepared lysis buffer (1:10 w/v) and centrifuged at 12,000 × g for 30 min. .. After incubation with the rabbit polyclonal anti-m-calpain antibody (1:1,000 Triple Point Biologics; Forest Grove, OR, USA) and the monoclonal anti-β actin (A-5316, 1:5,000, Sigma, USA) antibody, the membranes were washed with TBST and incubated with appropriate horse radish peroxidase-conjugated secondary antibody (1:4,000 dilution).

    Article Title: Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study
    Article Snippet: Western Blotting At the endpoint of the culture, both cell/scaffolds constructs (n = 3) and controls (scaffolds without cells as a negative control, and cells grown in monolayer up to 80% confluence—4 days—as a positive control) were washed in D-PBS and resuspended in a cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and antiproteases 1×, all from Sigma Aldrich). .. The membrane was blocked with 4% dry non-fat milk in 0.1% Tween/Tris Buffer Saline (T-TBS) and incubated overnight at 4 °C with anti β-actin mouse monoclonal antibody (A-5316, Sigma-Aldrich) or anti-HPRT rabbit monoclonal antibody (ab109021, Abcam, Cambridge, UK) diluted 1:2000 or 1:10,000 in T-TBS, respectively.

    Article Title: The Effects of Antecedent Exercise on Motor Function Recovery and Brain-derived Neurotrophic Factor Expression after Focal Cerebral Ischemia in Rats
    Article Snippet: Tissue was homogenized in freshly prepared lysis buffer (1:10 w/v) and centrifuged at 12,000 ×g for 30 min. .. After incubation with the anti-BDNF primary antibody (sc-546, Santa Cruz, USA), monoclonal anti-β actin (A-5316, 1:5,000, Sigma, USA) antibodies membranes were washed with TBST and incubated with the appropriate horse radish peroxidase-conjugated secondary antibody (1:4,000 dilution).

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    Millipore β actin
    Deletion of Arhgef1 inhibits leukocyte rolling and adhesion. ( A ) Time-dependent in vivo effect of Ang II (30 pmol) on leukocyte rolling and adhesion in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( B ) Effect of losartan on leukocyte rolling and adhesion induced by Ang II (30 pmol, 4 hours) in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( C ) Representative immunoblot of VCAM1, ICAM1, and <t>β-actin</t> in lysates of aortas from Arhgef1 lox/lox and Arhgef1 –/– mice before (0) and 4 and 8 hours after Ang II treatment ( n = 3) and corresponding quantification. All lanes were run on the same gel, but lanes 3 and 4 were noncontiguous as indicated by the black dividing line. ( D ) In vitro static adhesion of Arhgef1 lox/lox and Arhgef1 –/– leukocytes on ICAM before (0) and 1 and 4 hours after Ang II treatment ( n = 6 experiments). ( E ) In vitro analysis of Arhgef1 lox/lox and Arhgef1 –/– leukocyte rolling and adhesion on HUVECs under shear flow, before (–) and 4 hours after (+) Ang II treatment ( n = 5). * P
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin/product/Millipore
    Average 90 stars, based on 1329 article reviews
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    β actin - by Bioz Stars, 2020-01
    90/100 stars
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    iPS cell line derived with OSKM retrovirus Lines have been evaluated for exogenous gene silencing and concomitant expression of corresponding endogenous genes
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    Deletion of Arhgef1 inhibits leukocyte rolling and adhesion. ( A ) Time-dependent in vivo effect of Ang II (30 pmol) on leukocyte rolling and adhesion in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( B ) Effect of losartan on leukocyte rolling and adhesion induced by Ang II (30 pmol, 4 hours) in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( C ) Representative immunoblot of VCAM1, ICAM1, and β-actin in lysates of aortas from Arhgef1 lox/lox and Arhgef1 –/– mice before (0) and 4 and 8 hours after Ang II treatment ( n = 3) and corresponding quantification. All lanes were run on the same gel, but lanes 3 and 4 were noncontiguous as indicated by the black dividing line. ( D ) In vitro static adhesion of Arhgef1 lox/lox and Arhgef1 –/– leukocytes on ICAM before (0) and 1 and 4 hours after Ang II treatment ( n = 6 experiments). ( E ) In vitro analysis of Arhgef1 lox/lox and Arhgef1 –/– leukocyte rolling and adhesion on HUVECs under shear flow, before (–) and 4 hours after (+) Ang II treatment ( n = 5). * P

    Journal: The Journal of Clinical Investigation

    Article Title: Leukocyte RhoA exchange factor Arhgef1 mediates vascular inflammation and atherosclerosis

    doi: 10.1172/JCI92702

    Figure Lengend Snippet: Deletion of Arhgef1 inhibits leukocyte rolling and adhesion. ( A ) Time-dependent in vivo effect of Ang II (30 pmol) on leukocyte rolling and adhesion in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( B ) Effect of losartan on leukocyte rolling and adhesion induced by Ang II (30 pmol, 4 hours) in mesenteric vessels of Arhgef1 lox/lox and Arhgef1 –/– mice ( n = 5 mice). ( C ) Representative immunoblot of VCAM1, ICAM1, and β-actin in lysates of aortas from Arhgef1 lox/lox and Arhgef1 –/– mice before (0) and 4 and 8 hours after Ang II treatment ( n = 3) and corresponding quantification. All lanes were run on the same gel, but lanes 3 and 4 were noncontiguous as indicated by the black dividing line. ( D ) In vitro static adhesion of Arhgef1 lox/lox and Arhgef1 –/– leukocytes on ICAM before (0) and 1 and 4 hours after Ang II treatment ( n = 6 experiments). ( E ) In vitro analysis of Arhgef1 lox/lox and Arhgef1 –/– leukocyte rolling and adhesion on HUVECs under shear flow, before (–) and 4 hours after (+) Ang II treatment ( n = 5). * P

    Article Snippet: Equal loading was checked by reprobing of the membrane with mAb to β-actin (A-5316, MilliporeSigma).

    Techniques: In Vivo, Mouse Assay, In Vitro, Flow Cytometry