anti beta actin  (Cell Signaling Technology Inc)

 
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  • 99
    Name:
    β Actin Antibody
    Description:
    Actin a ubiquitous eukaryotic protein is the major component of the cytoskeleton At least six isoforms are known in mammals Nonmuscle β and γ actin also known as cytoplasmic actin are predominantly expressed in nonmuscle cells controlling cell structure and motility 1 α cardiac and α skeletal actin are expressed in striated cardiac and skeletal muscles respectively two smooth muscle actins α and γ actin are found primarily in vascular smooth muscle and enteric smooth muscle respectively These actin isoforms regulate the contractile potential of muscle cells 1 Actin exists mainly as a fibrous polymer F actin In response to cytoskeletal reorganizing signals during processes such as cytokinesis endocytosis or stress cofilin promotes fragmentation and depolymerization of F actin resulting in an increase in the monomeric globular form G actin 2 The ARP2 3 complex stabilizes F actin fragments and promotes formation of new actin filaments 2 Research studies have shown that actin is hyperphosphorylated in primary breast tumors 3 Cleavage of actin under apoptotic conditions has been observed in vitro and in cardiac and skeletal muscle as shown in research studies 4 6 Actin cleavage by caspase 3 may accelerate ubiquitin proteasome dependent muscle proteolysis 6
    Catalog Number:
    4967
    Price:
    None
    Applications:
    Western Blot
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino-terminal residues of human β-actin. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Hamster Monkey Mink D melanogaster Zebrafish Bovine
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    Structured Review

    Cell Signaling Technology Inc anti beta actin
    Thyroid hormone mediated ERK activation stimulates PIMT phosphorylation and enhances hepatic gluconeogenesis in rats. Rats (n=5) were injected intraperitoneally (0.1mL/animal) with L-thyroxine solution for 2 weeks. Post treatment animals were sacrificed, serum was collected for detecting levels of T 3 ( A ) and T 4 ( B ) and whole body weight ( C ) were measured. ( D ) PIMT immunoprecipitated lysates from liver tissue were analyzed by western blotting with phospho-ERK1/2 and total ERK1/2 antibodies. ( E )PIMT was immunoprecipitated from liver tissue lysates (Set1: 3 control and 3 treated; Set2: 2 control and 2 treated) separated on 10% SDS PAGE and probed with Anti-MPM2 followed by Anti-PIMT as mentioned. ( F ) Chromatin immunoprecipitation assay was performed in liver tissue lysates (one control and one treated in both Set1 and Set2) using Anti-PIMT or mock Anti-goat IgG on rat PEPCK promoter. ( G ) The liver homogenates were analyzed using Anti-PEPCK (top panel) or Anti <t>beta</t> actin (bottom panel). ( H ) Overnight fasting blood sugar was measured using glucose strip.
    Actin a ubiquitous eukaryotic protein is the major component of the cytoskeleton At least six isoforms are known in mammals Nonmuscle β and γ actin also known as cytoplasmic actin are predominantly expressed in nonmuscle cells controlling cell structure and motility 1 α cardiac and α skeletal actin are expressed in striated cardiac and skeletal muscles respectively two smooth muscle actins α and γ actin are found primarily in vascular smooth muscle and enteric smooth muscle respectively These actin isoforms regulate the contractile potential of muscle cells 1 Actin exists mainly as a fibrous polymer F actin In response to cytoskeletal reorganizing signals during processes such as cytokinesis endocytosis or stress cofilin promotes fragmentation and depolymerization of F actin resulting in an increase in the monomeric globular form G actin 2 The ARP2 3 complex stabilizes F actin fragments and promotes formation of new actin filaments 2 Research studies have shown that actin is hyperphosphorylated in primary breast tumors 3 Cleavage of actin under apoptotic conditions has been observed in vitro and in cardiac and skeletal muscle as shown in research studies 4 6 Actin cleavage by caspase 3 may accelerate ubiquitin proteasome dependent muscle proteolysis 6
    https://www.bioz.com/result/anti beta actin/product/Cell Signaling Technology Inc
    Average 99 stars, based on 115 article reviews
    Price from $9.99 to $1999.99
    anti beta actin - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis"

    Article Title: ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083787

    Thyroid hormone mediated ERK activation stimulates PIMT phosphorylation and enhances hepatic gluconeogenesis in rats. Rats (n=5) were injected intraperitoneally (0.1mL/animal) with L-thyroxine solution for 2 weeks. Post treatment animals were sacrificed, serum was collected for detecting levels of T 3 ( A ) and T 4 ( B ) and whole body weight ( C ) were measured. ( D ) PIMT immunoprecipitated lysates from liver tissue were analyzed by western blotting with phospho-ERK1/2 and total ERK1/2 antibodies. ( E )PIMT was immunoprecipitated from liver tissue lysates (Set1: 3 control and 3 treated; Set2: 2 control and 2 treated) separated on 10% SDS PAGE and probed with Anti-MPM2 followed by Anti-PIMT as mentioned. ( F ) Chromatin immunoprecipitation assay was performed in liver tissue lysates (one control and one treated in both Set1 and Set2) using Anti-PIMT or mock Anti-goat IgG on rat PEPCK promoter. ( G ) The liver homogenates were analyzed using Anti-PEPCK (top panel) or Anti beta actin (bottom panel). ( H ) Overnight fasting blood sugar was measured using glucose strip.
    Figure Legend Snippet: Thyroid hormone mediated ERK activation stimulates PIMT phosphorylation and enhances hepatic gluconeogenesis in rats. Rats (n=5) were injected intraperitoneally (0.1mL/animal) with L-thyroxine solution for 2 weeks. Post treatment animals were sacrificed, serum was collected for detecting levels of T 3 ( A ) and T 4 ( B ) and whole body weight ( C ) were measured. ( D ) PIMT immunoprecipitated lysates from liver tissue were analyzed by western blotting with phospho-ERK1/2 and total ERK1/2 antibodies. ( E )PIMT was immunoprecipitated from liver tissue lysates (Set1: 3 control and 3 treated; Set2: 2 control and 2 treated) separated on 10% SDS PAGE and probed with Anti-MPM2 followed by Anti-PIMT as mentioned. ( F ) Chromatin immunoprecipitation assay was performed in liver tissue lysates (one control and one treated in both Set1 and Set2) using Anti-PIMT or mock Anti-goat IgG on rat PEPCK promoter. ( G ) The liver homogenates were analyzed using Anti-PEPCK (top panel) or Anti beta actin (bottom panel). ( H ) Overnight fasting blood sugar was measured using glucose strip.

    Techniques Used: Activation Assay, Injection, Immunoprecipitation, Western Blot, SDS Page, Chromatin Immunoprecipitation, Stripping Membranes

    2) Product Images from "The cell and stress specific dynamics of in vivo and in vitro canonical and non-canonical tRNA cleavage"

    Article Title: The cell and stress specific dynamics of in vivo and in vitro canonical and non-canonical tRNA cleavage

    Journal: bioRxiv

    doi: 10.1101/2020.02.04.934695

    Plasmid hAng or RNH1 overexpression in PC12 and their impact on tRNA cleavage after stress. A: western blotting confirmation of Flag labeled human angiogenin expression in PC12. B: Western blotting confirmation of RNH1 overexpression after plasmid induction. RNH1 expression increased 2.5 folds when corrected to beta-actin expression (graph represent relative quantification using ImageJ). C: SYBR gold staining after hAng overexpression and stress shows that hAng overexpression increased tRNA cleavage after As stress only and had not impact on A.M stress. D: RNH1 overexpression did not suppress tRNA cleavage after As stress. Arrow: tRNA, dotted line: tiRNA.
    Figure Legend Snippet: Plasmid hAng or RNH1 overexpression in PC12 and their impact on tRNA cleavage after stress. A: western blotting confirmation of Flag labeled human angiogenin expression in PC12. B: Western blotting confirmation of RNH1 overexpression after plasmid induction. RNH1 expression increased 2.5 folds when corrected to beta-actin expression (graph represent relative quantification using ImageJ). C: SYBR gold staining after hAng overexpression and stress shows that hAng overexpression increased tRNA cleavage after As stress only and had not impact on A.M stress. D: RNH1 overexpression did not suppress tRNA cleavage after As stress. Arrow: tRNA, dotted line: tiRNA.

    Techniques Used: Plasmid Preparation, Over Expression, Western Blot, Labeling, Expressing, Staining

    3) Product Images from "Global Identification of EVI1 Target Genes in Acute Myeloid Leukemia"

    Article Title: Global Identification of EVI1 Target Genes in Acute Myeloid Leukemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067134

    Increased endogenous STAT1 phosphorylation in human Evi1 overexpressed leukemic cell lines. a) Western blot analysis using anti-total-STAT1 antibody. Lane 1 from left shows total STAT1 protein expression level in Kasumi 1 cells. Lane 2 shows total STAT1 protein level in Kasumi 3 cells. Evi1 overexpressed myeloid leukemic cells demonstrate a higher baseline of STAT1 protein, consistent with our mRNA findings. b) Western blot analysis using anti-phospho-STAT1 antibody. Lane 1 from left shows endogenous phosphorylated STAT1 protein expression level in Kasumi 1 cells (human leukemic cell line without Evi1 expression. Lane 2 shows the STAT1 protein level in Kasumi 3 cells. c) Beta actin loading control.
    Figure Legend Snippet: Increased endogenous STAT1 phosphorylation in human Evi1 overexpressed leukemic cell lines. a) Western blot analysis using anti-total-STAT1 antibody. Lane 1 from left shows total STAT1 protein expression level in Kasumi 1 cells. Lane 2 shows total STAT1 protein level in Kasumi 3 cells. Evi1 overexpressed myeloid leukemic cells demonstrate a higher baseline of STAT1 protein, consistent with our mRNA findings. b) Western blot analysis using anti-phospho-STAT1 antibody. Lane 1 from left shows endogenous phosphorylated STAT1 protein expression level in Kasumi 1 cells (human leukemic cell line without Evi1 expression. Lane 2 shows the STAT1 protein level in Kasumi 3 cells. c) Beta actin loading control.

    Techniques Used: Western Blot, Expressing

    Significant downregulation of Cebpe in human Evi1 overexpressed leukemic cells. a) Lanes 1 and 2 are beta-actin positive controls for U937 wildtype and U937+ Evi1 cells, respectively. Lane 3 sample is U937 wildtype cells (without Evi1 overexpression) and Lane 4 sample is U937 with Evi1 overexpression. Cebpe is downregulated in EVI1 overexpressed U937 human leukemic cells (Lane 4). b) Quantitative RT-PCR shows significant downregulation of Cebpe in Evi1 overexpressed human leukemic cells. The y-axis value denotes the relative levels of RNA expression based on normalized Ct values. U937+ Evi1 cells had 8 point increase in Ct value (or 256 fold decrease) compared to the U937 wildtype cells (p
    Figure Legend Snippet: Significant downregulation of Cebpe in human Evi1 overexpressed leukemic cells. a) Lanes 1 and 2 are beta-actin positive controls for U937 wildtype and U937+ Evi1 cells, respectively. Lane 3 sample is U937 wildtype cells (without Evi1 overexpression) and Lane 4 sample is U937 with Evi1 overexpression. Cebpe is downregulated in EVI1 overexpressed U937 human leukemic cells (Lane 4). b) Quantitative RT-PCR shows significant downregulation of Cebpe in Evi1 overexpressed human leukemic cells. The y-axis value denotes the relative levels of RNA expression based on normalized Ct values. U937+ Evi1 cells had 8 point increase in Ct value (or 256 fold decrease) compared to the U937 wildtype cells (p

    Techniques Used: Over Expression, Quantitative RT-PCR, RNA Expression

    4) Product Images from "EPH/ephrin profile and EPHB2 expression predicts patient survival in breast cancer"

    Article Title: EPH/ephrin profile and EPHB2 expression predicts patient survival in breast cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7246

    EPHB2 Antibody validation by immunoblot and immunocytochemistry The left arrows indicate bands at ≈ 100 kDa (brain lysate) and 75kDa (all other cell lines and the positive control). The EPHB2 positive control consisted in HEK293 expressing the recombinant human EPHB2/Fc extracellular domain with estimated Mw between 50-90 kDa. Other tested lysates were extracted from mouse brain, human colorectal cancer cells HCT116 and SW620 and breast cancer cells MDA-MB-468 and T47D A. The SW620, a colon cell lined derived from a metastatic lymph node, was selected to knockdown EPHB2 expression with siRNA B. or treated with the AllStars control siRNA. Beta actin (1:1000) was used as loading control. Paraffin embedded HCT116 cells treated with control siRNA C. or EPHB2 siRNAs D. were stained with the rabbit anti-EPHB2 (dilution 1:300).
    Figure Legend Snippet: EPHB2 Antibody validation by immunoblot and immunocytochemistry The left arrows indicate bands at ≈ 100 kDa (brain lysate) and 75kDa (all other cell lines and the positive control). The EPHB2 positive control consisted in HEK293 expressing the recombinant human EPHB2/Fc extracellular domain with estimated Mw between 50-90 kDa. Other tested lysates were extracted from mouse brain, human colorectal cancer cells HCT116 and SW620 and breast cancer cells MDA-MB-468 and T47D A. The SW620, a colon cell lined derived from a metastatic lymph node, was selected to knockdown EPHB2 expression with siRNA B. or treated with the AllStars control siRNA. Beta actin (1:1000) was used as loading control. Paraffin embedded HCT116 cells treated with control siRNA C. or EPHB2 siRNAs D. were stained with the rabbit anti-EPHB2 (dilution 1:300).

    Techniques Used: Immunocytochemistry, Positive Control, Expressing, Recombinant, Multiple Displacement Amplification, Derivative Assay, Staining

    5) Product Images from "The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans"

    Article Title: The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007038

    hpk-1 prevents sumoylation of HSF-1. (A) Changes in HSF-1 post-translational modifications between early L4 wild-type and hpk-1(pk1393) animals were examined by western blot to HSF-1; smo-1(RNAi) , which targets C . elegans SUMO, was used to block sumoylation, dePhos is lambda protein phosphatase treatment (other samples were mock treated). Beta-actin serves as a loading control. The ratio of modified to unmodified HSF-1 is 0.35, 0.51, and 0.35 for N2/ ev , hpk-1(pk1393)/ev , and hpk-1(pk1393)/smo-1(RNAi) , respectively (see S3A Fig for additional data). (B) hpk-1 prevents sumoylation of HSF-1. Ratio of HSF-1 unmodified (75kD) to modified (90-95kD, sumoylated and sumoylated plus phosphorylated). The S.E.M. from Image J quantification is shown for seven N2 and hpk-1(pk1393) replicates (* p
    Figure Legend Snippet: hpk-1 prevents sumoylation of HSF-1. (A) Changes in HSF-1 post-translational modifications between early L4 wild-type and hpk-1(pk1393) animals were examined by western blot to HSF-1; smo-1(RNAi) , which targets C . elegans SUMO, was used to block sumoylation, dePhos is lambda protein phosphatase treatment (other samples were mock treated). Beta-actin serves as a loading control. The ratio of modified to unmodified HSF-1 is 0.35, 0.51, and 0.35 for N2/ ev , hpk-1(pk1393)/ev , and hpk-1(pk1393)/smo-1(RNAi) , respectively (see S3A Fig for additional data). (B) hpk-1 prevents sumoylation of HSF-1. Ratio of HSF-1 unmodified (75kD) to modified (90-95kD, sumoylated and sumoylated plus phosphorylated). The S.E.M. from Image J quantification is shown for seven N2 and hpk-1(pk1393) replicates (* p

    Techniques Used: Western Blot, Blocking Assay, Modification

    6) Product Images from "Celecoxib increases EGF signaling in colon tumor associated fibroblasts, modulating EGFR expression and degradation"

    Article Title: Celecoxib increases EGF signaling in colon tumor associated fibroblasts, modulating EGFR expression and degradation

    Journal: Oncotarget

    doi:

    Celecoxib increases colon TAFs responsiveness to EGF a ) TAF cell growth was evaluated on day 7 of culture in the presence of Celecoxib (Cel, 10μM), EGF (50ng/ml) or Cel+EGF. Ctrl: control TAFs in absence of stimuli. The test was run in six replicates and repeated three times. b ) Cell adhesion of TAFs seeded on collagen type IV. TAFs were primed or not with Celecoxib in culture flasks, afterwards they were plated in microwells either without additional treatment or with EGF, for 30min. The test was run in quadruplicates and repeated three times. c ) Western blot for p-Akt and p-Erk1-2 of TAFs primed with Celecoxib and/or incubated with EGF for the indicated period of time. Beta-actin was used as a loading control. d ) Relative quantification of WB replicates run as shown in panel c.
    Figure Legend Snippet: Celecoxib increases colon TAFs responsiveness to EGF a ) TAF cell growth was evaluated on day 7 of culture in the presence of Celecoxib (Cel, 10μM), EGF (50ng/ml) or Cel+EGF. Ctrl: control TAFs in absence of stimuli. The test was run in six replicates and repeated three times. b ) Cell adhesion of TAFs seeded on collagen type IV. TAFs were primed or not with Celecoxib in culture flasks, afterwards they were plated in microwells either without additional treatment or with EGF, for 30min. The test was run in quadruplicates and repeated three times. c ) Western blot for p-Akt and p-Erk1-2 of TAFs primed with Celecoxib and/or incubated with EGF for the indicated period of time. Beta-actin was used as a loading control. d ) Relative quantification of WB replicates run as shown in panel c.

    Techniques Used: Western Blot, Incubation

    Celecoxib affects protein turnover mimicking the inhibitors of endo-lysosome acidification a ) Western blot for EGFR and p62/SQSTM1 of colon TAFs pretreated with Celecoxib and triggered for 3 or 16h with EGF. b ) Western blot analysis of the effects of proteasome-lysosome inhibitors. 48h treatment with MG132 (proteasome inhibitor, 2μM), Bafilomycin-A1 (Baf, V-ATPase inhibitor, 25nM), NH 4 Cl (lysosomal pH -buffering molecule, 10mM) were compared to Celecoxib (10μM) as modulators of EGFR, p62, HSP70 and IkBα. c ) Relative quantification of EGFR and p62 levels from replicate tests as shown in panel b; p values were calculated as compared to untreated controls. d ) Western blot comparison of the effects of Celecoxib and lysosome acidification inhibitors. The effects of Celecoxib (10μM), Bafilomycin-A1 (2.5nM) and NH 4 Cl (2.5mM) pretreatment were tested in the absence/presence of EGF (3h) on several target proteins: EGFR, p62, IkBα, EEA1, LAMP1, Rab7, pro Cathepsin-D and Cathepsin-D. Loading controls: beta-actin (1) normalizes EEA1, p62 and Cathepsin-D; beta-actin (2) normalizes EGFR, IkBα, LAMP1 and Rab7. e ) Relative quantification of EGFR, p62, Rab7, pro and active Cathepsin-D levels from replicate tests as shown in panel d; p values defined in Materials and Methods were calculated as compared to untreated controls.
    Figure Legend Snippet: Celecoxib affects protein turnover mimicking the inhibitors of endo-lysosome acidification a ) Western blot for EGFR and p62/SQSTM1 of colon TAFs pretreated with Celecoxib and triggered for 3 or 16h with EGF. b ) Western blot analysis of the effects of proteasome-lysosome inhibitors. 48h treatment with MG132 (proteasome inhibitor, 2μM), Bafilomycin-A1 (Baf, V-ATPase inhibitor, 25nM), NH 4 Cl (lysosomal pH -buffering molecule, 10mM) were compared to Celecoxib (10μM) as modulators of EGFR, p62, HSP70 and IkBα. c ) Relative quantification of EGFR and p62 levels from replicate tests as shown in panel b; p values were calculated as compared to untreated controls. d ) Western blot comparison of the effects of Celecoxib and lysosome acidification inhibitors. The effects of Celecoxib (10μM), Bafilomycin-A1 (2.5nM) and NH 4 Cl (2.5mM) pretreatment were tested in the absence/presence of EGF (3h) on several target proteins: EGFR, p62, IkBα, EEA1, LAMP1, Rab7, pro Cathepsin-D and Cathepsin-D. Loading controls: beta-actin (1) normalizes EEA1, p62 and Cathepsin-D; beta-actin (2) normalizes EGFR, IkBα, LAMP1 and Rab7. e ) Relative quantification of EGFR, p62, Rab7, pro and active Cathepsin-D levels from replicate tests as shown in panel d; p values defined in Materials and Methods were calculated as compared to untreated controls.

    Techniques Used: Western Blot

    7) Product Images from "Fatty acid binding protein 4 enhances prostate cancer progression by upregulating matrix metalloproteinases and stromal cell cytokine production"

    Article Title: Fatty acid binding protein 4 enhances prostate cancer progression by upregulating matrix metalloproteinases and stromal cell cytokine production

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22908

    PrSC or HFD conditions stimulate PCa metastasis and invasiveness in vivo For in vivo metastatic tumor studies, PC-3M-luc-C6 cells were intraperitoneallyinjected into mice randomly assigned to four groups (5 mice per group): control (Ctrl), PrSC, HFD, and HFD+BMS. For the PrSC group, PrSC cells were injected with the tumor cells. Mice in the HFD and HFD+BMS groups were fed HFD, and mice in the Ctrl and PrSC groups were fed a control diet. Mice in the HFD+BMS group were administrated BMS309403 by dissolution in drinking water. (A and B) Increased luciferase activity by PrSC or HFD. At 14 and 28 days inoculation, bioluminescence was used to detect intraperitoneal tumor growth and metastases by the intraperitoneal injection of luciferin. (C) Increased tumor metastasis in the PrSC and HFD groups. At 29 days after the injection of cells, mice were sacrificed, and the proportion of metastatic tumor burden in peritoneal organs, peritoneum, intestine, stomach, liver, and diaphragm were evaluated. (D, E and F) Increased invasive capacity in the PrSC and HFD groups by the increased adipocyte infiltration, stromal fibroblast activation, and upregulation of FABP4 and MMPs. (D) Slides of mouse tumor tissues were subjected to hematoxylin and eosin staining (H E), and immunohistochemistry to detect the expression of FABP4 and αSMA. (E and F) mRNA expressions of FABP4 and MMP2 and 9 in tumors from each group were analyzed by quantitative RT-PCR. The mRNA expression levels of FABP4 and MMP2 and 9 was normalized to the levels of beta-actin , and the relative values were compared with the level of the Ctrl group. * P
    Figure Legend Snippet: PrSC or HFD conditions stimulate PCa metastasis and invasiveness in vivo For in vivo metastatic tumor studies, PC-3M-luc-C6 cells were intraperitoneallyinjected into mice randomly assigned to four groups (5 mice per group): control (Ctrl), PrSC, HFD, and HFD+BMS. For the PrSC group, PrSC cells were injected with the tumor cells. Mice in the HFD and HFD+BMS groups were fed HFD, and mice in the Ctrl and PrSC groups were fed a control diet. Mice in the HFD+BMS group were administrated BMS309403 by dissolution in drinking water. (A and B) Increased luciferase activity by PrSC or HFD. At 14 and 28 days inoculation, bioluminescence was used to detect intraperitoneal tumor growth and metastases by the intraperitoneal injection of luciferin. (C) Increased tumor metastasis in the PrSC and HFD groups. At 29 days after the injection of cells, mice were sacrificed, and the proportion of metastatic tumor burden in peritoneal organs, peritoneum, intestine, stomach, liver, and diaphragm were evaluated. (D, E and F) Increased invasive capacity in the PrSC and HFD groups by the increased adipocyte infiltration, stromal fibroblast activation, and upregulation of FABP4 and MMPs. (D) Slides of mouse tumor tissues were subjected to hematoxylin and eosin staining (H E), and immunohistochemistry to detect the expression of FABP4 and αSMA. (E and F) mRNA expressions of FABP4 and MMP2 and 9 in tumors from each group were analyzed by quantitative RT-PCR. The mRNA expression levels of FABP4 and MMP2 and 9 was normalized to the levels of beta-actin , and the relative values were compared with the level of the Ctrl group. * P

    Techniques Used: In Vivo, Mouse Assay, Injection, Luciferase, Activity Assay, Activation Assay, Staining, Immunohistochemistry, Expressing, Quantitative RT-PCR

    Expression and secretion of FABP4 promotes PCa invasiveness by activating PrSC to produce proinflammatory cytokines (A and B) Expression and secretion of FABP4 in PCa cells. Overall, 1 × 10 5 PC-3 cells were cultured in a 35-mm dish, and treated with or without 50 nM FABP4 siRNA-1 for 24 hours. Equal amounts of proteins (10 μg) from the cultured cells were subjected to anti-FABP4 and anti-beta-actin staining (A). The mean FABP4 level in the conditioned medium (CM) was significantly lower in PC-3 cells treated with FABP4 siRNA-1 (235.4 ± 11.6 pg per 10 5 cells) compared with control PC-3 cells (938.8 ± 29.0 pg per 10 5 cells) ( P = 0.007, B). ** P
    Figure Legend Snippet: Expression and secretion of FABP4 promotes PCa invasiveness by activating PrSC to produce proinflammatory cytokines (A and B) Expression and secretion of FABP4 in PCa cells. Overall, 1 × 10 5 PC-3 cells were cultured in a 35-mm dish, and treated with or without 50 nM FABP4 siRNA-1 for 24 hours. Equal amounts of proteins (10 μg) from the cultured cells were subjected to anti-FABP4 and anti-beta-actin staining (A). The mean FABP4 level in the conditioned medium (CM) was significantly lower in PC-3 cells treated with FABP4 siRNA-1 (235.4 ± 11.6 pg per 10 5 cells) compared with control PC-3 cells (938.8 ± 29.0 pg per 10 5 cells) ( P = 0.007, B). ** P

    Techniques Used: Expressing, Cell Culture, Staining

    8) Product Images from "Polymer Nanoassemblies with Hydrophobic Pendant Groups in the Core Induce False Positive siRNA Transfection in Luciferase Reporter Assays"

    Article Title: Polymer Nanoassemblies with Hydrophobic Pendant Groups in the Core Induce False Positive siRNA Transfection in Luciferase Reporter Assays

    Journal: International journal of pharmaceutics

    doi: 10.1016/j.ijpharm.2017.06.056

    PNA induced ubiquitination of luciferase Western blots detailed the expression of (A) luciferase, and (B) high molecular weight luciferase after 72h dosage of PNAs in HT-29-luc cells. Additionally, cells were dosed as indicated with MG132 proteasome inhibitor for 6h before cell lysis. Cellular expression of luciferase was compared with beta tubulin. * denotes that the column is significantly different from the no particle, no MG132 condition (p
    Figure Legend Snippet: PNA induced ubiquitination of luciferase Western blots detailed the expression of (A) luciferase, and (B) high molecular weight luciferase after 72h dosage of PNAs in HT-29-luc cells. Additionally, cells were dosed as indicated with MG132 proteasome inhibitor for 6h before cell lysis. Cellular expression of luciferase was compared with beta tubulin. * denotes that the column is significantly different from the no particle, no MG132 condition (p

    Techniques Used: Luciferase, Western Blot, Expressing, Molecular Weight, Lysis

    9) Product Images from "Gender- and region-specific changes in estrogen signaling in aging rat brain mitochondria"

    Article Title: Gender- and region-specific changes in estrogen signaling in aging rat brain mitochondria

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101538

    Signaling downstream of estrogen in the hippocampus across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), PKC comparing age ( C ), PKC comparing gender ( D ), cx43 comparing age ( E ), cx43 comparing gender ( F ), p-cx43 ( G ), p-ERK ( H ), ERK ( I ), beta-actin ( J ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the hippocampus across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), PKC comparing age ( C ), PKC comparing gender ( D ), cx43 comparing age ( E ), cx43 comparing gender ( F ), p-cx43 ( G ), p-ERK ( H ), ERK ( I ), beta-actin ( J ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the mitochondria of the corpus callosum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for PKC ( B ), p-cx43 ( C ), estrogen receptor beta ( D ), cx43 ( E ), ATP synthase ( F ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the mitochondria of the corpus callosum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for PKC ( B ), p-cx43 ( C ), estrogen receptor beta ( D ), cx43 ( E ), ATP synthase ( F ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the cerebellum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for for cx43 with age ( B ), p-PKC ( C ), PKC ( D ), ERK ( E ),ERK ( F ), p-cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the cerebellum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for for cx43 with age ( B ), p-PKC ( C ), PKC ( D ), ERK ( E ),ERK ( F ), p-cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the mitochondria of the cerebral cortex across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-cx43 comparing age ( B ), p-cx43 comparing gender ( C ), cx43 ( D ), estrogen receptor beta ( E ), PKC ( F ), ATP synthase ( G ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the mitochondria of the cerebral cortex across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-cx43 comparing age ( B ), p-cx43 comparing gender ( C ), cx43 ( D ), estrogen receptor beta ( E ), PKC ( F ), ATP synthase ( G ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the amygdala across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), PKC ( C ), p-ERK ( D ), ERK ( E ), p-cx43 ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the amygdala across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), PKC ( C ), p-ERK ( D ), ERK ( E ), p-cx43 ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the mitochondria of the hippocampus across age and gender .Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-cx43 comparing age ( B ), p-cx43 comparing gender ( C ), cx43 comparing age ( D ), cx43 comparing gender ( E ), estrogen receptor beta ( F ), PKC ( G ), ATP synthase ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the mitochondria of the hippocampus across age and gender .Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-cx43 comparing age ( B ), p-cx43 comparing gender ( C ), cx43 comparing age ( D ), cx43 comparing gender ( E ), estrogen receptor beta ( F ), PKC ( G ), ATP synthase ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the mitochondria of the amygdala across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for PKC comparing age ( B ), PKC comparing gender ( C ), p-cx43 ( D ), cx43 ( E ), estrogen receptor beta ( F ), ATP synthase ( G ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the mitochondria of the amygdala across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for PKC comparing age ( B ), PKC comparing gender ( C ), p-cx43 ( D ), cx43 ( E ), estrogen receptor beta ( F ), ATP synthase ( G ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the cerebral cortex across age and gender. Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-ERK ( B ), p-PKC ( C ), ERK ( D ), ERK ( E ), p-cx43 ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the cerebral cortex across age and gender. Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-ERK ( B ), p-PKC ( C ), ERK ( D ), ERK ( E ), p-cx43 ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the corpus callosum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), p-ERK ( C ), p-cx43 comparing gender ( D ), PKC ( E ), ERK ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the corpus callosum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), p-ERK ( C ), p-cx43 comparing gender ( D ), PKC ( E ), ERK ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    10) Product Images from "Potential depression and antidepressant-response biomarkers in human lymphoblast cell lines from treatment-responsive and treatment-resistant subjects: roles of SSRIs and omega-3 polyunsaturated fatty acids"

    Article Title: Potential depression and antidepressant-response biomarkers in human lymphoblast cell lines from treatment-responsive and treatment-resistant subjects: roles of SSRIs and omega-3 polyunsaturated fatty acids

    Journal: bioRxiv

    doi: 10.1101/2020.01.22.914358

    Basal expression levels of genes in LCLs from healthy controls and depressed subjects who responded and did not respond to antidepressant treatment. Total RNA was collected and used for reverse transcription. qRT-PCR was used to measure the target gene expression including a FLOT1 , b CAV1 , c GNAS , d GNAL , e ADCY1 , f ADCY3 , g ADCY6 , h ADCY7 , i ADCY9 , j VEGFA , k IL6 and l BDNF . Each experiment was done in triplicate and expression levels were normalized with beta actin (2 -ΔCt ). All genes and gene products investigated were selected a priori as previous studies have focused on lipid rafts (hence Caveolin and flotillin), cAMP generation (hence Gs, Golf and isoforms of adenylyl cyclase), cAMP/regulated growth factors (hence VEGFA and BDNF), and inflammatory mediators linked to n-3 PUFA (hence IL6). Values from each cell line are represented as dots in the scatter plot-bar of mean ± SEM. Adjusted p value from 3 post-hoc pair-wise comparisons among groups of cell lines, * p
    Figure Legend Snippet: Basal expression levels of genes in LCLs from healthy controls and depressed subjects who responded and did not respond to antidepressant treatment. Total RNA was collected and used for reverse transcription. qRT-PCR was used to measure the target gene expression including a FLOT1 , b CAV1 , c GNAS , d GNAL , e ADCY1 , f ADCY3 , g ADCY6 , h ADCY7 , i ADCY9 , j VEGFA , k IL6 and l BDNF . Each experiment was done in triplicate and expression levels were normalized with beta actin (2 -ΔCt ). All genes and gene products investigated were selected a priori as previous studies have focused on lipid rafts (hence Caveolin and flotillin), cAMP generation (hence Gs, Golf and isoforms of adenylyl cyclase), cAMP/regulated growth factors (hence VEGFA and BDNF), and inflammatory mediators linked to n-3 PUFA (hence IL6). Values from each cell line are represented as dots in the scatter plot-bar of mean ± SEM. Adjusted p value from 3 post-hoc pair-wise comparisons among groups of cell lines, * p

    Techniques Used: Expressing, Quantitative RT-PCR

    11) Product Images from "Targeting class I histone deacetylases by the novel small molecule inhibitor 4 SC‐202 blocks oncogenic hedgehog‐ GLI signaling and overcomes smoothened inhibitor resistance"

    Article Title: Targeting class I histone deacetylases by the novel small molecule inhibitor 4 SC‐202 blocks oncogenic hedgehog‐ GLI signaling and overcomes smoothened inhibitor resistance

    Journal: International Journal of Cancer

    doi: 10.1002/ijc.31117

    4SC‐202 inhibits HH/GLI signaling in SMOi‐resistant cancer cells. ( a ) Western blot analysis of GLI1 expression in cells with lentiviral shRNA‐mediated knockdown of SUFU (shSUFU) or control knockdown (shControl). Beta Actin served as loading control. ( b ) qPCR analysis of GLI1 mRNA expression in SUFU knockdown (shSUFU) and control cells (shControl) transduced with lentiviral nontargeting scrambled shRNA. ( c and d ) qPCR analysis of GLI1 ( c ) and HHIP mRNA expression ( d ) as readout for HH/GLI signaling activity in SUFU‐depleted SMOi‐resistant cells showing resistance to vismodegib but sensitivity to 4SC‐202 treatment. ( e ) Western blot analysis of SUFU‐depleted Daoy cells treated with vismodegib or 4SC‐202 at the concentrations indicated. β‐Tubulin served as loading control. Vismodegib was unable to reduce GLI1 protein levels, while 4SC‐202 treatment effectively abolished GLI1 protein expression. ( f ) Relative densitometric quantification of GLI1/β‐Tubulin protein levels shown in ( e ). ( g ) ChIP analysis of MYC‐tagged GLI1 binding to the GLI target promoter PTCH in response to control or 4SC‐202 treatment. Enrichment of GLI1 bound promoter DNA was measured by qPCR. IgG isotype antibody was used as control. ( h ) Murine BCC cells were subcutaneously injected into dorsal flanks of 12 NSG mice. 4SC‐202 was administered by oral gavage at 80 mg/kg/day. The tumor volume at day 0 (i.e., start of drug treatment (arrow)) was set to 100%. ( i ) Western blot analysis of solvent (allografts #1–#4) and 4SC‐202 treated (allografts #5–#8) BCC lysates probed for proliferation‐cell‐nuclear‐antigen (Pcna) and Ccnd1 expression. Erk1/2 expression served as loading control. ( j ) Analysis of in vitro cell proliferation of murine BCC cells in response to 4SC‐202 and entinostat treatment. ChIP: chromatin immunoprecipitation. ** p
    Figure Legend Snippet: 4SC‐202 inhibits HH/GLI signaling in SMOi‐resistant cancer cells. ( a ) Western blot analysis of GLI1 expression in cells with lentiviral shRNA‐mediated knockdown of SUFU (shSUFU) or control knockdown (shControl). Beta Actin served as loading control. ( b ) qPCR analysis of GLI1 mRNA expression in SUFU knockdown (shSUFU) and control cells (shControl) transduced with lentiviral nontargeting scrambled shRNA. ( c and d ) qPCR analysis of GLI1 ( c ) and HHIP mRNA expression ( d ) as readout for HH/GLI signaling activity in SUFU‐depleted SMOi‐resistant cells showing resistance to vismodegib but sensitivity to 4SC‐202 treatment. ( e ) Western blot analysis of SUFU‐depleted Daoy cells treated with vismodegib or 4SC‐202 at the concentrations indicated. β‐Tubulin served as loading control. Vismodegib was unable to reduce GLI1 protein levels, while 4SC‐202 treatment effectively abolished GLI1 protein expression. ( f ) Relative densitometric quantification of GLI1/β‐Tubulin protein levels shown in ( e ). ( g ) ChIP analysis of MYC‐tagged GLI1 binding to the GLI target promoter PTCH in response to control or 4SC‐202 treatment. Enrichment of GLI1 bound promoter DNA was measured by qPCR. IgG isotype antibody was used as control. ( h ) Murine BCC cells were subcutaneously injected into dorsal flanks of 12 NSG mice. 4SC‐202 was administered by oral gavage at 80 mg/kg/day. The tumor volume at day 0 (i.e., start of drug treatment (arrow)) was set to 100%. ( i ) Western blot analysis of solvent (allografts #1–#4) and 4SC‐202 treated (allografts #5–#8) BCC lysates probed for proliferation‐cell‐nuclear‐antigen (Pcna) and Ccnd1 expression. Erk1/2 expression served as loading control. ( j ) Analysis of in vitro cell proliferation of murine BCC cells in response to 4SC‐202 and entinostat treatment. ChIP: chromatin immunoprecipitation. ** p

    Techniques Used: Western Blot, Expressing, shRNA, Real-time Polymerase Chain Reaction, Transduction, Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Injection, Mouse Assay, In Vitro

    12) Product Images from "Gender- and region-specific changes in estrogen signaling in aging rat brain mitochondria"

    Article Title: Gender- and region-specific changes in estrogen signaling in aging rat brain mitochondria

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101538

    Signaling downstream of estrogen in the hippocampus across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), PKC comparing age ( C ), PKC comparing gender ( D ), cx43 comparing age ( E ), cx43 comparing gender ( F ), p-cx43 ( G ), p-ERK ( H ), ERK ( I ), beta-actin ( J ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the hippocampus across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), PKC comparing age ( C ), PKC comparing gender ( D ), cx43 comparing age ( E ), cx43 comparing gender ( F ), p-cx43 ( G ), p-ERK ( H ), ERK ( I ), beta-actin ( J ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the mitochondria of the corpus callosum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for PKC ( B ), p-cx43 ( C ), estrogen receptor beta ( D ), cx43 ( E ), ATP synthase ( F ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the mitochondria of the corpus callosum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for PKC ( B ), p-cx43 ( C ), estrogen receptor beta ( D ), cx43 ( E ), ATP synthase ( F ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the cerebellum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for for cx43 with age ( B ), p-PKC ( C ), PKC ( D ), ERK ( E ),ERK ( F ), p-cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the cerebellum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for for cx43 with age ( B ), p-PKC ( C ), PKC ( D ), ERK ( E ),ERK ( F ), p-cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the mitochondria of the cerebral cortex across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-cx43 comparing age ( B ), p-cx43 comparing gender ( C ), cx43 ( D ), estrogen receptor beta ( E ), PKC ( F ), ATP synthase ( G ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the mitochondria of the cerebral cortex across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-cx43 comparing age ( B ), p-cx43 comparing gender ( C ), cx43 ( D ), estrogen receptor beta ( E ), PKC ( F ), ATP synthase ( G ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the amygdala across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), PKC ( C ), p-ERK ( D ), ERK ( E ), p-cx43 ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the amygdala across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), PKC ( C ), p-ERK ( D ), ERK ( E ), p-cx43 ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the mitochondria of the hippocampus across age and gender .Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-cx43 comparing age ( B ), p-cx43 comparing gender ( C ), cx43 comparing age ( D ), cx43 comparing gender ( E ), estrogen receptor beta ( F ), PKC ( G ), ATP synthase ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the mitochondria of the hippocampus across age and gender .Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-cx43 comparing age ( B ), p-cx43 comparing gender ( C ), cx43 comparing age ( D ), cx43 comparing gender ( E ), estrogen receptor beta ( F ), PKC ( G ), ATP synthase ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the mitochondria of the amygdala across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for PKC comparing age ( B ), PKC comparing gender ( C ), p-cx43 ( D ), cx43 ( E ), estrogen receptor beta ( F ), ATP synthase ( G ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the mitochondria of the amygdala across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for PKC comparing age ( B ), PKC comparing gender ( C ), p-cx43 ( D ), cx43 ( E ), estrogen receptor beta ( F ), ATP synthase ( G ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the cerebral cortex across age and gender. Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-ERK ( B ), p-PKC ( C ), ERK ( D ), ERK ( E ), p-cx43 ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the cerebral cortex across age and gender. Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-ERK ( B ), p-PKC ( C ), ERK ( D ), ERK ( E ), p-cx43 ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the mitochondria of the cerebellum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for estrogen receptor beta ( B ), PKC ( C ), p-cx43 ( D ), cx43 ( E ), ATP synthase ( F ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the mitochondria of the cerebellum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for estrogen receptor beta ( B ), PKC ( C ), p-cx43 ( D ), cx43 ( E ), ATP synthase ( F ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    Signaling downstream of estrogen in the corpus callosum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), p-ERK ( C ), p-cx43 comparing gender ( D ), PKC ( E ), ERK ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P
    Figure Legend Snippet: Signaling downstream of estrogen in the corpus callosum across age and gender . Representative western blots for each protein of interest and a representative Ponceau stain as a load control ( A ). Graphical depiction of the fold change for p-PKC ( B ), p-ERK ( C ), p-cx43 comparing gender ( D ), PKC ( E ), ERK ( F ), cx43 ( G ), beta-actin ( H ). Error bars = SEM. 4W = 4 weeks of age; 3M = 3 months of age; 9M = 9 months of age; 12M = 12 months of age. Pink = female; blue = male. ANOVA with Tukey posthoc, * = P

    Techniques Used: Western Blot, Staining

    13) Product Images from "Genetic dissection of the miR-200–Zeb1 axis reveals its importance in tumor differentiation and invasion"

    Article Title: Genetic dissection of the miR-200–Zeb1 axis reveals its importance in tumor differentiation and invasion

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07130-z

    Mutation of miR-200 sites in Zeb1 is sufficient to phenocopy miR-200 ablation. a Murine Zeb1 3′UTR, miR-200 site conservation, and mutated miR-200 site sequences. b Percent survival of RT2 mice with Zeb1 mutation ( RT2 , RT2_Zeb1 200H , RT2_Zeb1 200M , n = 42, 45, 26, respectively). c Random-fed blood glucose ( RT2 , RT2_Zeb1 200H , RT2_Zeb1 200M , n = 11, 11, 10, respectively). Dotted line represents onset of severe hypoglycemia. d Tumor burden in different age classes (box, 25th and 75th percentiles; central line, median; from left to right: n = 9, 4, 4, 4, 5, 11, 12 mice). e Representative insulin/glucagon and SV40 immunofluorescence staining of whole pancreas (left; insulin-negative tumors outlined; scale bar = 2 mm) and zoomed-in regions (right; all tumors outlined; scale bar = 100 μm). LN lymph node. f Percent tumors per grade ( n = 5 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 55, 17, 16 tumors, respectively). g Percent insulin-positive and -negative tumors ( n = 5 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 67, 12, 15 tumors, respectively). h Mean log 2 FC (with 95% confidence intervals) of beta-cell-identity genes in RT2_Zeb1 200M vs. RT2 islets of 6-week-old mice. i Percent TUNEL-positive nuclei ( n = 2 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 17, 21, 13 lesions, respectively). j Representative H E and SV40 immunofluorescence staining of late-stage RT2_Zeb1 200M liver (scale bar = 2 mm). k Representative images of late-stage RT2_Zeb1 200H and RT2_Zeb1 200M mice. l Percent of mice with macrometastasis in difference age classes. c , i Data are plotted as mean ± SD. Significance was evaluated by b Mantel Cox test, c two-tailed t test with Holm−Sidak correction (vs. RT2 ), d , i one-way ANOVA with Dunnett’s multiple comparisons (vs. RT2 ) and h empirical Bayes method. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001
    Figure Legend Snippet: Mutation of miR-200 sites in Zeb1 is sufficient to phenocopy miR-200 ablation. a Murine Zeb1 3′UTR, miR-200 site conservation, and mutated miR-200 site sequences. b Percent survival of RT2 mice with Zeb1 mutation ( RT2 , RT2_Zeb1 200H , RT2_Zeb1 200M , n = 42, 45, 26, respectively). c Random-fed blood glucose ( RT2 , RT2_Zeb1 200H , RT2_Zeb1 200M , n = 11, 11, 10, respectively). Dotted line represents onset of severe hypoglycemia. d Tumor burden in different age classes (box, 25th and 75th percentiles; central line, median; from left to right: n = 9, 4, 4, 4, 5, 11, 12 mice). e Representative insulin/glucagon and SV40 immunofluorescence staining of whole pancreas (left; insulin-negative tumors outlined; scale bar = 2 mm) and zoomed-in regions (right; all tumors outlined; scale bar = 100 μm). LN lymph node. f Percent tumors per grade ( n = 5 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 55, 17, 16 tumors, respectively). g Percent insulin-positive and -negative tumors ( n = 5 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 67, 12, 15 tumors, respectively). h Mean log 2 FC (with 95% confidence intervals) of beta-cell-identity genes in RT2_Zeb1 200M vs. RT2 islets of 6-week-old mice. i Percent TUNEL-positive nuclei ( n = 2 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 17, 21, 13 lesions, respectively). j Representative H E and SV40 immunofluorescence staining of late-stage RT2_Zeb1 200M liver (scale bar = 2 mm). k Representative images of late-stage RT2_Zeb1 200H and RT2_Zeb1 200M mice. l Percent of mice with macrometastasis in difference age classes. c , i Data are plotted as mean ± SD. Significance was evaluated by b Mantel Cox test, c two-tailed t test with Holm−Sidak correction (vs. RT2 ), d , i one-way ANOVA with Dunnett’s multiple comparisons (vs. RT2 ) and h empirical Bayes method. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001

    Techniques Used: Mutagenesis, Mouse Assay, Immunofluorescence, Staining, TUNEL Assay, Two Tailed Test

    miR-200 ablation promotes tumor growth, malignancy, and invasion. a Diagram of murine miR-200 superfamily (seed regions boxed). b Percent survival of RT2 mice with one or both miR-200 genomic clusters ablated ( RT2 , RT2_Rip-Cre_200a fl/fl , RT2_141~200cKO , RT2_DKO , n = 42, 8, 43, 36, respectively). c Random-fed blood glucose ( RT2 , RT2_Rip-Cre_200a fl/fl , RT2_141~200cKO , RT2_DKO , n = 11, 7, 11, 13, respectively). Dotted line represents onset of severe hypoglycemia. d Tumor burden in different age classes (box, 25th and 75th percentiles; central line, median; left to right: n = 9, 5, 4, 6, 9, 7, 11, 14 mice). e Representative insulin/glucagon and SV40 immunofluorescence staining of whole pancreas (left; insulin-negative tumors outlined; scale bar = 2 mm) and zoomed-in regions (right; all tumors outlined; scale bar = 100 μm). LN lymph node. f Percent tumors per grade ( n = 5 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO , n = 55, 9, 31 tumors respectively). g Percent insulin-positive and -negative tumors ( n = 5 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO , n = 67, 6, 31 tumors, respectively). h Mean log 2 FC (with 95% confidence intervals) of beta-cell-identity genes in RT2_DKO vs. RT2 islets of 6-week-old mice. i Percent of TUNEL-positive nuclei ( n = 2 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO, n = 17, 15, 17 lesions per group). j Representative H E staining and SV40 immunofluorescence staining of late-stage RT2_DKO liver (scale bar = 2 mm). k Representative images of late-stage RT2 and RT2_DKO mice. l Percent mice with macrometastasis in difference age classes. Data in c , i are plotted as mean ± SD. Significance was evaluated by b Mantel Cox test, c two-tailed t test with Holm−Sidak correction (vs. RT2 ), d , i one-way ANOVA with Dunnett’s multiple comparisons test (vs. RT2 ) and h empirical Bayes method. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001
    Figure Legend Snippet: miR-200 ablation promotes tumor growth, malignancy, and invasion. a Diagram of murine miR-200 superfamily (seed regions boxed). b Percent survival of RT2 mice with one or both miR-200 genomic clusters ablated ( RT2 , RT2_Rip-Cre_200a fl/fl , RT2_141~200cKO , RT2_DKO , n = 42, 8, 43, 36, respectively). c Random-fed blood glucose ( RT2 , RT2_Rip-Cre_200a fl/fl , RT2_141~200cKO , RT2_DKO , n = 11, 7, 11, 13, respectively). Dotted line represents onset of severe hypoglycemia. d Tumor burden in different age classes (box, 25th and 75th percentiles; central line, median; left to right: n = 9, 5, 4, 6, 9, 7, 11, 14 mice). e Representative insulin/glucagon and SV40 immunofluorescence staining of whole pancreas (left; insulin-negative tumors outlined; scale bar = 2 mm) and zoomed-in regions (right; all tumors outlined; scale bar = 100 μm). LN lymph node. f Percent tumors per grade ( n = 5 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO , n = 55, 9, 31 tumors respectively). g Percent insulin-positive and -negative tumors ( n = 5 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO , n = 67, 6, 31 tumors, respectively). h Mean log 2 FC (with 95% confidence intervals) of beta-cell-identity genes in RT2_DKO vs. RT2 islets of 6-week-old mice. i Percent of TUNEL-positive nuclei ( n = 2 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO, n = 17, 15, 17 lesions per group). j Representative H E staining and SV40 immunofluorescence staining of late-stage RT2_DKO liver (scale bar = 2 mm). k Representative images of late-stage RT2 and RT2_DKO mice. l Percent mice with macrometastasis in difference age classes. Data in c , i are plotted as mean ± SD. Significance was evaluated by b Mantel Cox test, c two-tailed t test with Holm−Sidak correction (vs. RT2 ), d , i one-way ANOVA with Dunnett’s multiple comparisons test (vs. RT2 ) and h empirical Bayes method. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001

    Techniques Used: Mouse Assay, Immunofluorescence, Staining, TUNEL Assay, Two Tailed Test

    14) Product Images from "B cell lymphoma in hiv transgenic mice"

    Article Title: B cell lymphoma in hiv transgenic mice

    Journal: Retrovirology

    doi: 10.1186/1742-4690-10-92

    HIV proteins expression in HIV Tg mice. (A) gp120 expression in HIV Tg mice. Protein lysates from mice spleen and lymph node were separated by SDS-PAGE and analyzed by western blot using anti-gp120 antibody as described under “Experimental Procedures”. The mice analyzed are indicated on the top of the figure: WT is the mouse control FVB/N, Tg is an HIV Tg mice without skin lesions, mice number 18, 21, and 22 represent mice at the pre-lymphadenopathy stage while mice number 11, 20 and 35 represent mice at a late stage of lymphadenopathy. The numbers in the right represent the protein molecular weight in KDa. (B) Histogram showing the fold changes in gp120 expression in the lymph node versus the spleen from the same mouse. The relative intensity of gp120 was quantified by densitometry and normalized with the beta actin. (C) Western blot for p17 and nef proteins expression in HIV Tg mice using anti-p17 antibody and HIV-1 Nef antiserum as described under “Experimental Procedures”. The mice analyzed are indicated on the top of the figure: WT are mice wild type control FVB/N, Tg are HIV Tg mice without skin lesions, Tg pre-L are mice at pre-lymphadenopathy stage and Tg-L are mice at the last stage of lymphadenopathy. The protein molecular weights are represented in the right of the figure.
    Figure Legend Snippet: HIV proteins expression in HIV Tg mice. (A) gp120 expression in HIV Tg mice. Protein lysates from mice spleen and lymph node were separated by SDS-PAGE and analyzed by western blot using anti-gp120 antibody as described under “Experimental Procedures”. The mice analyzed are indicated on the top of the figure: WT is the mouse control FVB/N, Tg is an HIV Tg mice without skin lesions, mice number 18, 21, and 22 represent mice at the pre-lymphadenopathy stage while mice number 11, 20 and 35 represent mice at a late stage of lymphadenopathy. The numbers in the right represent the protein molecular weight in KDa. (B) Histogram showing the fold changes in gp120 expression in the lymph node versus the spleen from the same mouse. The relative intensity of gp120 was quantified by densitometry and normalized with the beta actin. (C) Western blot for p17 and nef proteins expression in HIV Tg mice using anti-p17 antibody and HIV-1 Nef antiserum as described under “Experimental Procedures”. The mice analyzed are indicated on the top of the figure: WT are mice wild type control FVB/N, Tg are HIV Tg mice without skin lesions, Tg pre-L are mice at pre-lymphadenopathy stage and Tg-L are mice at the last stage of lymphadenopathy. The protein molecular weights are represented in the right of the figure.

    Techniques Used: Expressing, Mouse Assay, SDS Page, Western Blot, Molecular Weight

    15) Product Images from "PGRMC1 phosphorylation affects cell shape, motility, glycolysis, mitochondrial form and function, and tumor growth"

    Article Title: PGRMC1 phosphorylation affects cell shape, motility, glycolysis, mitochondrial form and function, and tumor growth

    Journal: BMC Molecular and Cell Biology

    doi: 10.1186/s12860-020-00256-3

    MIA PaCa-2 pancreatic cancer cells morphology is affected by PGRMC1 phosphorylation status. a PGRMC1-HA proteins constructed for this figure. TMH: Trans-membrane helix. HA: the C-terminal 3x hemaglutinin tag. b Detection of exogenous PGRMC1 expression levels by western blot (upper panel). Equal loading is controlled by quantifying beta actin (lower panel). The results show three totally independent stably transfected cell lines per plasmid from (A). Open arrow: Exogenous PGRMC1-HA (Ex.). Shaded arrow: endogenous PGRMC1 (End.). Filled arrow: beta actin. The molecular weight ladder is Bio-Rad 1610377 Dual Xtra Standards. c Box plots quantification of replicate gels of (B) with signals normalized to beta actin from the same respective lanes. n = 4 lanes for MP and n = 6 for WT, DM and TM (replicates of respective lines 1–3 per condition). There were no significant differences (ns) except for the exogenous band in MP (ANOVA, post-hoc Dunnet’s T3). d Western blot quantification of HA-tagged exogenous PGRMC1, following B but detecting PGRMC1 with anti-HA antibody. The molecular weight ladder is Abcam ab116028 Prestained Protein Ladder. e PGRMC1 mutant protein expression alters MIA PaCa-2 cell morphology. PGRMC1-HA-expressing stable cells (respective lines 1 from B) or MP cells were stained with a FITC-tagged anti-HA antibody (Anti-HA) and imaged by confocal microscopy. DNA was stained with DAPI. Cells were also imaged in differential interference contrast (DIC) microscopy mode. The respective left panels show merged images of all 3 channels. f The rounded phenotype of double and triple mutant (E) was reversed to elongated phenotype after 125 μM ROCKI addition, but not by addition of DMSO vehicle control
    Figure Legend Snippet: MIA PaCa-2 pancreatic cancer cells morphology is affected by PGRMC1 phosphorylation status. a PGRMC1-HA proteins constructed for this figure. TMH: Trans-membrane helix. HA: the C-terminal 3x hemaglutinin tag. b Detection of exogenous PGRMC1 expression levels by western blot (upper panel). Equal loading is controlled by quantifying beta actin (lower panel). The results show three totally independent stably transfected cell lines per plasmid from (A). Open arrow: Exogenous PGRMC1-HA (Ex.). Shaded arrow: endogenous PGRMC1 (End.). Filled arrow: beta actin. The molecular weight ladder is Bio-Rad 1610377 Dual Xtra Standards. c Box plots quantification of replicate gels of (B) with signals normalized to beta actin from the same respective lanes. n = 4 lanes for MP and n = 6 for WT, DM and TM (replicates of respective lines 1–3 per condition). There were no significant differences (ns) except for the exogenous band in MP (ANOVA, post-hoc Dunnet’s T3). d Western blot quantification of HA-tagged exogenous PGRMC1, following B but detecting PGRMC1 with anti-HA antibody. The molecular weight ladder is Abcam ab116028 Prestained Protein Ladder. e PGRMC1 mutant protein expression alters MIA PaCa-2 cell morphology. PGRMC1-HA-expressing stable cells (respective lines 1 from B) or MP cells were stained with a FITC-tagged anti-HA antibody (Anti-HA) and imaged by confocal microscopy. DNA was stained with DAPI. Cells were also imaged in differential interference contrast (DIC) microscopy mode. The respective left panels show merged images of all 3 channels. f The rounded phenotype of double and triple mutant (E) was reversed to elongated phenotype after 125 μM ROCKI addition, but not by addition of DMSO vehicle control

    Techniques Used: Construct, Expressing, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Molecular Weight, Mutagenesis, Staining, Confocal Microscopy, Microscopy

    16) Product Images from "Daikenchuto (TU‐100) alters murine hepatic and intestinal drug metabolizing enzymes in an in vivo dietary model: effects of gender and withdrawal. Daikenchuto (TU‐100) alters murine hepatic and intestinal drug metabolizing enzymes in an in vivo dietary model: effects of gender and withdrawal"

    Article Title: Daikenchuto (TU‐100) alters murine hepatic and intestinal drug metabolizing enzymes in an in vivo dietary model: effects of gender and withdrawal. Daikenchuto (TU‐100) alters murine hepatic and intestinal drug metabolizing enzymes in an in vivo dietary model: effects of gender and withdrawal

    Journal: Pharmacology Research & Perspectives

    doi: 10.1002/prp2.361

    TU‐100 alters Cyp2b10, Cyp3a11, and Mdr1a protein expression in liver and jejunum. Protein samples were analyzed by 10% SDS‐PAGE and Western blots using specific antibodies. Beta actin was used as a housekeeping gene. (A) Liver tissue from female and male mice fed 1.5% TU‐100 containing AIN‐76A diet for 12 or 24 weeks. (B) Liver tissue from female and male mice fed AIN‐76A diet without TU‐100 for 24 weeks (A/A); 1.5% TU‐100 supplementation for 24 weeks (T/T); 1.5% TU‐100 supplementation for 12 weeks followed by AIN‐76A without TU‐100 for 12 weeks (T/A). (C) Liver tissue from male mice fed AIN‐76A diet without TU‐100, or 0.75, 1.5, or 3.0% TU‐100‐supplemented AIN‐76A diet for 12 weeks. (D) Liver, male mice fed AIN‐76A diet without TU‐100 for 12 or 24 weeks (A/A); 0.75% or 3.0% TU‐100 containing AIN‐76A diet for 12 weeks followed by the diet without TU‐100 for 12 weeks (T/A). (E) Jejunum and liver tissues from females and males fed 1.5% TU‐100 supplemented AIN‐76 diet for 12 weeks followed by the diet without TU‐100 for 12 weeks. (F) Jejunal tissue from female (F) and male (M) mice harvested at 24 weeks of AIN76A diet without TU‐100 (A/A), fed diet with TU‐100 for full 24 weeks (T/T), or fed diet with TU‐100 for 12 weeks followed by 12 weeks without TU‐100 (T/A) were analyzed.
    Figure Legend Snippet: TU‐100 alters Cyp2b10, Cyp3a11, and Mdr1a protein expression in liver and jejunum. Protein samples were analyzed by 10% SDS‐PAGE and Western blots using specific antibodies. Beta actin was used as a housekeeping gene. (A) Liver tissue from female and male mice fed 1.5% TU‐100 containing AIN‐76A diet for 12 or 24 weeks. (B) Liver tissue from female and male mice fed AIN‐76A diet without TU‐100 for 24 weeks (A/A); 1.5% TU‐100 supplementation for 24 weeks (T/T); 1.5% TU‐100 supplementation for 12 weeks followed by AIN‐76A without TU‐100 for 12 weeks (T/A). (C) Liver tissue from male mice fed AIN‐76A diet without TU‐100, or 0.75, 1.5, or 3.0% TU‐100‐supplemented AIN‐76A diet for 12 weeks. (D) Liver, male mice fed AIN‐76A diet without TU‐100 for 12 or 24 weeks (A/A); 0.75% or 3.0% TU‐100 containing AIN‐76A diet for 12 weeks followed by the diet without TU‐100 for 12 weeks (T/A). (E) Jejunum and liver tissues from females and males fed 1.5% TU‐100 supplemented AIN‐76 diet for 12 weeks followed by the diet without TU‐100 for 12 weeks. (F) Jejunal tissue from female (F) and male (M) mice harvested at 24 weeks of AIN76A diet without TU‐100 (A/A), fed diet with TU‐100 for full 24 weeks (T/T), or fed diet with TU‐100 for 12 weeks followed by 12 weeks without TU‐100 (T/A) were analyzed.

    Techniques Used: Expressing, SDS Page, Western Blot, Mouse Assay

    17) Product Images from "Role of Cystathionine Gamma-Lyase in Immediate Renal Impairment and Inflammatory Response in Acute Ischemic Kidney Injury"

    Article Title: Role of Cystathionine Gamma-Lyase in Immediate Renal Impairment and Inflammatory Response in Acute Ischemic Kidney Injury

    Journal: Scientific Reports

    doi: 10.1038/srep27517

    Renal expression of H 2 S-producing enzymes and H 2 S. Gene expression levels of ( A ) cystathionine gamma-lyase ( Cth ), ( B ) cystathionine beta-synthase ( Cbs ) and ( C ) 3-mercaptopyruvate sulfurtransferase ( Mpst ) in sham- and ischemia/reperfusion (I/R)-injured kidneys of wild-type ( Cth +/+ ), heterozygous ( Cth +/− ) and CTH-deficient ( Cth −/− ) mice. Values plotted are mean ± SEM ( n = 4 in sham-operated groups, n = 8 in I/R-injured groups). *P
    Figure Legend Snippet: Renal expression of H 2 S-producing enzymes and H 2 S. Gene expression levels of ( A ) cystathionine gamma-lyase ( Cth ), ( B ) cystathionine beta-synthase ( Cbs ) and ( C ) 3-mercaptopyruvate sulfurtransferase ( Mpst ) in sham- and ischemia/reperfusion (I/R)-injured kidneys of wild-type ( Cth +/+ ), heterozygous ( Cth +/− ) and CTH-deficient ( Cth −/− ) mice. Values plotted are mean ± SEM ( n = 4 in sham-operated groups, n = 8 in I/R-injured groups). *P

    Techniques Used: Expressing, Mouse Assay

    Renal gene expression of cytokines and adhesion molecules. Gene expression levels of ( A ) interleukin ( Il ))1-beta, ( B ) vascular cell adhesion molecule ( Vcam )1, ( C ) tumor necrosis factor alpha ( Tnf ) and ( D ) intercellular adhesion molecule ( Icam )1 in ischemia/reperfusion-injured kidneys of Cth +/+ , Cth +/− , and Cth −/− mice. Values plotted are mean ± SEM (n = 8, 7, and 8 for Cth +/+ , Cth +/− , and Cth −/− mice, respectively). *P
    Figure Legend Snippet: Renal gene expression of cytokines and adhesion molecules. Gene expression levels of ( A ) interleukin ( Il ))1-beta, ( B ) vascular cell adhesion molecule ( Vcam )1, ( C ) tumor necrosis factor alpha ( Tnf ) and ( D ) intercellular adhesion molecule ( Icam )1 in ischemia/reperfusion-injured kidneys of Cth +/+ , Cth +/− , and Cth −/− mice. Values plotted are mean ± SEM (n = 8, 7, and 8 for Cth +/+ , Cth +/− , and Cth −/− mice, respectively). *P

    Techniques Used: Expressing, Mouse Assay

    18) Product Images from "Artemisinin protects human retinal pigment epithelial cells from hydrogen peroxide-induced oxidative damage through activation of ERK/CREB signaling"

    Article Title: Artemisinin protects human retinal pigment epithelial cells from hydrogen peroxide-induced oxidative damage through activation of ERK/CREB signaling

    Journal: Redox Biology

    doi: 10.1016/j.redox.2016.06.002

    Involvement of ERK/CREB signaling in the cytoprotective effect of Artemisinin. (A) D407 cells were pre-treated with 30 µM Artemisinin. The cells were collected at 0, 30, 60, and 120 min. The expression of phosphorylated ERK1/2, total ERK1/2, phosphorylated Akt, total Akt and phosphorylated CREB, total CREB and beta-actin were detected by Western blotting with specific antibodies (A). (B)(C)(D) Quantification of representative protein band from Western blotting. **P
    Figure Legend Snippet: Involvement of ERK/CREB signaling in the cytoprotective effect of Artemisinin. (A) D407 cells were pre-treated with 30 µM Artemisinin. The cells were collected at 0, 30, 60, and 120 min. The expression of phosphorylated ERK1/2, total ERK1/2, phosphorylated Akt, total Akt and phosphorylated CREB, total CREB and beta-actin were detected by Western blotting with specific antibodies (A). (B)(C)(D) Quantification of representative protein band from Western blotting. **P

    Techniques Used: Expressing, Western Blot

    19) Product Images from "Nephroprotective Effects of N-Acetylcysteine Amide against Contrast-Induced Nephropathy through Upregulating Thioredoxin-1, Inhibiting ASK1/p38MAPK Pathway, and Suppressing Oxidative Stress and Apoptosis in Rats"

    Article Title: Nephroprotective Effects of N-Acetylcysteine Amide against Contrast-Induced Nephropathy through Upregulating Thioredoxin-1, Inhibiting ASK1/p38MAPK Pathway, and Suppressing Oxidative Stress and Apoptosis in Rats

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2016/8715185

    NACA blocked CM-induced p38 MAPK activation by inhibiting ASK1 phosphorylation and upregulating Trx1 mRNA and protein expression. (a) Phospho-ASK1 and ASK1 expressions by western blotting ( n = 3 each). (b) Relative densitometry analysis of the ratio of phospho-ASK1 to ASK1. (c) Trx1 expression by western blotting ( n = 3 each). (d) Relative densitometry analysis of the ratio of Trx1 to beta-actin. (e) QPCR analysis for Trx1 mRNA in renal cortex. Data are shown as means ± SD ( n = 3 each). ∗ p
    Figure Legend Snippet: NACA blocked CM-induced p38 MAPK activation by inhibiting ASK1 phosphorylation and upregulating Trx1 mRNA and protein expression. (a) Phospho-ASK1 and ASK1 expressions by western blotting ( n = 3 each). (b) Relative densitometry analysis of the ratio of phospho-ASK1 to ASK1. (c) Trx1 expression by western blotting ( n = 3 each). (d) Relative densitometry analysis of the ratio of Trx1 to beta-actin. (e) QPCR analysis for Trx1 mRNA in renal cortex. Data are shown as means ± SD ( n = 3 each). ∗ p

    Techniques Used: Activation Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    NACA inhibited CM-induced renal tubular cell apoptosis as detected by TUNEL staining and western blot analyses of cleaved caspase 3. CM increased the number of TUNEL-positive renal tubular cells (blue arrows), and pretreatment with NAC or NACA blocked this effect. TUNEL-stained kidney sections (magnifications ×200) from CON rats (a), CIN rats (b), NAC+CIN rats (c), NACA1+CIN rats (d), and NACA2+CIN rats (e). TUNEL-positive cells are marked by blue arrows. (f) Quantitative analysis of TUNEL-positive cell number. (g) Western blot analyses of cleaved capsase-3. (h) Relative densitometry analysis of the ratio of cleaved caspase 3 to beta-actin. Figures are representative of 5 to 8 rats from each group. The values are means ± SD ( n = 5). ∗ p
    Figure Legend Snippet: NACA inhibited CM-induced renal tubular cell apoptosis as detected by TUNEL staining and western blot analyses of cleaved caspase 3. CM increased the number of TUNEL-positive renal tubular cells (blue arrows), and pretreatment with NAC or NACA blocked this effect. TUNEL-stained kidney sections (magnifications ×200) from CON rats (a), CIN rats (b), NAC+CIN rats (c), NACA1+CIN rats (d), and NACA2+CIN rats (e). TUNEL-positive cells are marked by blue arrows. (f) Quantitative analysis of TUNEL-positive cell number. (g) Western blot analyses of cleaved capsase-3. (h) Relative densitometry analysis of the ratio of cleaved caspase 3 to beta-actin. Figures are representative of 5 to 8 rats from each group. The values are means ± SD ( n = 5). ∗ p

    Techniques Used: TUNEL Assay, Staining, Western Blot

    20) Product Images from "Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype"

    Article Title: Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype

    Journal: Molecular Cancer

    doi: 10.1186/s12943-018-0773-5

    Gene expression during neural crest induction in the zebrafish embryo and upon Wnt stimulation in SKMEL28 melanoma cells, and clinical significance. a Heatmaps of real-time qPCR gene expression analysis of the melanoma cell lines SKMEL19, 451 LU, BLM and A375 for AXIN2. TYR, MITF and the four genes INHBA, CYR61, ANGTPL4 and FABP7. Log2 transformed x-fold expression values were used for color-coding. Yellow: upregulated gene expression; blue: downregulated gene expression at 24 h treatment of the melanoma cells; black: not detectable b Western blot analysis to detect protein levels of beta-catenin, PTEN, phospo-Ser473 (AKT) and AKT in the melanoma cell lines A375, SKMEL19, BLM and 451 LU after treatment with 3T3-CM, 3T3-Wnt3a, or 0.5 μM PKF115–584 for 24 h. Beta-actin was used as loading control. Small-hairpin knockdown cells served as samples to investigate the dependence on beta-catenin. c Luciferase reporter assay (Super8xTOPFlash) indicates a 3-fold activation of the canonical Wnt−/β-catenin signaling pathway after stimulation of SKMEL28 cells with Wnt3a-conditioned medium (3T3-Wnt3a). PKF115–584 treatment (0.5 μM for 12 h) significantly inhibited reporter activity. **: p
    Figure Legend Snippet: Gene expression during neural crest induction in the zebrafish embryo and upon Wnt stimulation in SKMEL28 melanoma cells, and clinical significance. a Heatmaps of real-time qPCR gene expression analysis of the melanoma cell lines SKMEL19, 451 LU, BLM and A375 for AXIN2. TYR, MITF and the four genes INHBA, CYR61, ANGTPL4 and FABP7. Log2 transformed x-fold expression values were used for color-coding. Yellow: upregulated gene expression; blue: downregulated gene expression at 24 h treatment of the melanoma cells; black: not detectable b Western blot analysis to detect protein levels of beta-catenin, PTEN, phospo-Ser473 (AKT) and AKT in the melanoma cell lines A375, SKMEL19, BLM and 451 LU after treatment with 3T3-CM, 3T3-Wnt3a, or 0.5 μM PKF115–584 for 24 h. Beta-actin was used as loading control. Small-hairpin knockdown cells served as samples to investigate the dependence on beta-catenin. c Luciferase reporter assay (Super8xTOPFlash) indicates a 3-fold activation of the canonical Wnt−/β-catenin signaling pathway after stimulation of SKMEL28 cells with Wnt3a-conditioned medium (3T3-Wnt3a). PKF115–584 treatment (0.5 μM for 12 h) significantly inhibited reporter activity. **: p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Western Blot, Luciferase, Reporter Assay, Activation Assay, Activity Assay

    Wnt3a induces invasive growth of melanoma cells in organotypic tissue skin reconstructs (TSR). a 451 LU melanoma cells were seeded together with HaCat epidermal cells onto a layer of collagen I embedded human fibroblasts. TSR exposed to Wnt3a conditioned medium (Wnt3a-CM) showed a pronounced invasive morphology in the H E staining (upper pictures) when compared to cells exposed to control medium (3T3-CM). Immunofluorescence stainings for HMB45 (red) and beta-catenin (blue) identified melanoma cells (HMB45+), revealed beta-catenin expression levels and verified the invasion of single 451 LU cells from the epidermis (epi) into the dermal part (der). Nuclei were stained with YO-PRO-1 (green). b Knockdown of beta-catenin (blue) with shRNA (shCTNNB1) reduced the invasion of 451 LU melanoma cells (HMB45+, red) into the dermal part of the TSR
    Figure Legend Snippet: Wnt3a induces invasive growth of melanoma cells in organotypic tissue skin reconstructs (TSR). a 451 LU melanoma cells were seeded together with HaCat epidermal cells onto a layer of collagen I embedded human fibroblasts. TSR exposed to Wnt3a conditioned medium (Wnt3a-CM) showed a pronounced invasive morphology in the H E staining (upper pictures) when compared to cells exposed to control medium (3T3-CM). Immunofluorescence stainings for HMB45 (red) and beta-catenin (blue) identified melanoma cells (HMB45+), revealed beta-catenin expression levels and verified the invasion of single 451 LU cells from the epidermis (epi) into the dermal part (der). Nuclei were stained with YO-PRO-1 (green). b Knockdown of beta-catenin (blue) with shRNA (shCTNNB1) reduced the invasion of 451 LU melanoma cells (HMB45+, red) into the dermal part of the TSR

    Techniques Used: Staining, Immunofluorescence, Expressing, shRNA

    21) Product Images from "Changes in Morphology, Gene Expression and Protein Content in Chondrocytes Cultured on a Random Positioning Machine"

    Article Title: Changes in Morphology, Gene Expression and Protein Content in Chondrocytes Cultured on a Random Positioning Machine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079057

    Immunofluorescence, gene expression and Western blot analysis of beta-tubulin of chondrocytes cultured at 1 g or on the RPM. A–E: Beta-tubulin immunofluorescence of chondrocytes cultured at 1 g or on the RPM for different times (30 min ( A ), 2 h ( B ), 4 h ( C ), 16 h ( D ), 24 h ( E )). Control cells showed a similar distribution of β-tubulin, while chondrocytes exposed to the RPM exhibited a perinuclear accumulation of β-tubulin (orange arrows). F: Gene expression of TUBB6 was significantly elevated after 0.5 h, but did not change significantly during the rest of the 24 h of culturing on the RPM. G: Western blot analysis of ß-tubulin protein: The protein content was significantly elevated after 4 h RPM exposure. After 24 h no change was detectable. Data are given as mean ± standard deviation; #P
    Figure Legend Snippet: Immunofluorescence, gene expression and Western blot analysis of beta-tubulin of chondrocytes cultured at 1 g or on the RPM. A–E: Beta-tubulin immunofluorescence of chondrocytes cultured at 1 g or on the RPM for different times (30 min ( A ), 2 h ( B ), 4 h ( C ), 16 h ( D ), 24 h ( E )). Control cells showed a similar distribution of β-tubulin, while chondrocytes exposed to the RPM exhibited a perinuclear accumulation of β-tubulin (orange arrows). F: Gene expression of TUBB6 was significantly elevated after 0.5 h, but did not change significantly during the rest of the 24 h of culturing on the RPM. G: Western blot analysis of ß-tubulin protein: The protein content was significantly elevated after 4 h RPM exposure. After 24 h no change was detectable. Data are given as mean ± standard deviation; #P

    Techniques Used: Immunofluorescence, Expressing, Western Blot, Cell Culture, Standard Deviation

    Integrity of nuclei counter-stained by Hoechst 33342, distribution of F-Actin, ACTA2 and ACTB gene expression, and Western blot analysis of beta-actin of chondrocytes cultured at 1 g or on the RPM. A–E: Integrity of nuclei counter-stained by Hoechst 33342 and distribution of F-Actin in human chondrocytes cultured at 1 g or on the RPM for different times (30 min ( A ), 2 h ( B ), 4 h ( C ), 16 h ( D ), 24 h ( E )). After 30 min and 2 h stress fibres were visible in RPM samples ( A, B ). After 4 h accumulations of F-actin were visible in the outer cellular membrane ( C, G ). The cells were smaller after 24 h RPM exposure ( F ). H, I: ACTA2 and ACTB gene expression: Early up-regulation of these mRNAs was found after 0.5 h. J: Western blot analysis of beta-actin: Beta-actin clearly increased after 4 h incubation on the RPM. Data are given as mean ± standard deviation; #P
    Figure Legend Snippet: Integrity of nuclei counter-stained by Hoechst 33342, distribution of F-Actin, ACTA2 and ACTB gene expression, and Western blot analysis of beta-actin of chondrocytes cultured at 1 g or on the RPM. A–E: Integrity of nuclei counter-stained by Hoechst 33342 and distribution of F-Actin in human chondrocytes cultured at 1 g or on the RPM for different times (30 min ( A ), 2 h ( B ), 4 h ( C ), 16 h ( D ), 24 h ( E )). After 30 min and 2 h stress fibres were visible in RPM samples ( A, B ). After 4 h accumulations of F-actin were visible in the outer cellular membrane ( C, G ). The cells were smaller after 24 h RPM exposure ( F ). H, I: ACTA2 and ACTB gene expression: Early up-regulation of these mRNAs was found after 0.5 h. J: Western blot analysis of beta-actin: Beta-actin clearly increased after 4 h incubation on the RPM. Data are given as mean ± standard deviation; #P

    Techniques Used: Staining, Expressing, Western Blot, Cell Culture, Incubation, Standard Deviation

    22) Product Images from "Common Effects on Cancer Cells Exerted by a Random Positioning Machine and a 2D Clinostat"

    Article Title: Common Effects on Cancer Cells Exerted by a Random Positioning Machine and a 2D Clinostat

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135157

    Effects of IL-6 and IL-8 on the amount of selected proteins in adherent ML-1 cells grown under normal 1 g -conditions. Western blot analyses and densitometric data are given. A, C: beta-actin, 3d and 7d; B, D: ß 1 -integrin, 3d and 7d; E, G: Ki-67, 3 and 7 days; F, H : Talin-1, 3 and 7 days. IL-6 doses: 0.03 ng/mL; 1 ng/mL; 10 ng/mL and 100 ng/mL. The dose 0.03 ng/mL is the maximal amount of IL-6 released by ML-1 cells in the supernatant and measured by MAP (dark-grey columns). IL-8 doses: 1 ng/mL; 10 ng/mL; 45 ng/mL and 100 ng/mL. The dose 45 ng/mL is the maximal amount of IL-8 released by ML-1 cells in the supernatant and measured by MAP (dark-grey columns).
    Figure Legend Snippet: Effects of IL-6 and IL-8 on the amount of selected proteins in adherent ML-1 cells grown under normal 1 g -conditions. Western blot analyses and densitometric data are given. A, C: beta-actin, 3d and 7d; B, D: ß 1 -integrin, 3d and 7d; E, G: Ki-67, 3 and 7 days; F, H : Talin-1, 3 and 7 days. IL-6 doses: 0.03 ng/mL; 1 ng/mL; 10 ng/mL and 100 ng/mL. The dose 0.03 ng/mL is the maximal amount of IL-6 released by ML-1 cells in the supernatant and measured by MAP (dark-grey columns). IL-8 doses: 1 ng/mL; 10 ng/mL; 45 ng/mL and 100 ng/mL. The dose 45 ng/mL is the maximal amount of IL-8 released by ML-1 cells in the supernatant and measured by MAP (dark-grey columns).

    Techniques Used: Western Blot

    Western blot analyses of A-D: ML-1 cells after a 7-day-exposure on the RPM and CLINO. A : ß-actin, B : ß 1 -integrin, C : talin-1 and D: Ki-67 proteins. Clear differences between the two devices are found for ß-actin. Western blot analyses of E-H: RO82-W-1 cells after a 7-day-exposure on the RPM and CLINO. E: ß-actin, F: ß 1 -integrin, G: talin-1 and H: Ki-67 proteins. There was no change in the beta-actin content of RO82-W-1 cells cultured on the RPM or the CLINO for 7d. The amount of ß 1 -integrin and Ki-67 was comparable to the ML-1 cultures grown under s-μ g . *P
    Figure Legend Snippet: Western blot analyses of A-D: ML-1 cells after a 7-day-exposure on the RPM and CLINO. A : ß-actin, B : ß 1 -integrin, C : talin-1 and D: Ki-67 proteins. Clear differences between the two devices are found for ß-actin. Western blot analyses of E-H: RO82-W-1 cells after a 7-day-exposure on the RPM and CLINO. E: ß-actin, F: ß 1 -integrin, G: talin-1 and H: Ki-67 proteins. There was no change in the beta-actin content of RO82-W-1 cells cultured on the RPM or the CLINO for 7d. The amount of ß 1 -integrin and Ki-67 was comparable to the ML-1 cultures grown under s-μ g . *P

    Techniques Used: Western Blot, Cell Culture

    23) Product Images from "Diet-induced alteration of fatty acid synthase in prostate cancer progression"

    Article Title: Diet-induced alteration of fatty acid synthase in prostate cancer progression

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2015.42

    The expression of FASN and its related signal pathways in LNCaP xenograft mice under HFD or LFD conditions. LNCaP xenograft mice were generated, and those with palpable tumours were randomly assigned to HFD and LFD groups (12 mice per group). After the 14-week diet experiments, the tumour and serum samples were separated. ( a , b ) mRNA expression of FASN and SREBP-1 in xenograft tumours was quantified by quantitative reverse transcription–PCR and the relative mRNA expression ratio was compared against beta-actin in the two diet groups. * P
    Figure Legend Snippet: The expression of FASN and its related signal pathways in LNCaP xenograft mice under HFD or LFD conditions. LNCaP xenograft mice were generated, and those with palpable tumours were randomly assigned to HFD and LFD groups (12 mice per group). After the 14-week diet experiments, the tumour and serum samples were separated. ( a , b ) mRNA expression of FASN and SREBP-1 in xenograft tumours was quantified by quantitative reverse transcription–PCR and the relative mRNA expression ratio was compared against beta-actin in the two diet groups. * P

    Techniques Used: Expressing, Mouse Assay, Generated, Polymerase Chain Reaction

    24) Product Images from "Potential Use of 1-25-dihydroxyvitamin D in the Diagnosis and Treatment of Papillary Thyroid Cancer"

    Article Title: Potential Use of 1-25-dihydroxyvitamin D in the Diagnosis and Treatment of Papillary Thyroid Cancer

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.909544

    Knockdown of cathelicidin antimicrobial peptide (CAMP) expression can reverse the proliferation and apoptosis caused by 1,25(OH)2D3. ( A ) Relative expression of CAMP after transfecting CAMP siRNA into K1 and IHH4 cells was analyzed by qRT-PCR and normalized to GAPDH expression. ( B ) Expression of CAMP protein was measured, and beta-actin protein was used for normalization after treatment with only 1,25(OH)2D3 or CAMP siRNA plus 1,25(OH)2D3 for 48 hours. ( C ) CAMP siRNA reduces the inhibition of cell proliferation caused by 1,25(OH)2D3. ( D ) CAMP siRNA reverses the apoptosis caused by 1,25(OH)2D3. The 1,25(OH)2D3 concentration in these experiments was 100 μM.
    Figure Legend Snippet: Knockdown of cathelicidin antimicrobial peptide (CAMP) expression can reverse the proliferation and apoptosis caused by 1,25(OH)2D3. ( A ) Relative expression of CAMP after transfecting CAMP siRNA into K1 and IHH4 cells was analyzed by qRT-PCR and normalized to GAPDH expression. ( B ) Expression of CAMP protein was measured, and beta-actin protein was used for normalization after treatment with only 1,25(OH)2D3 or CAMP siRNA plus 1,25(OH)2D3 for 48 hours. ( C ) CAMP siRNA reduces the inhibition of cell proliferation caused by 1,25(OH)2D3. ( D ) CAMP siRNA reverses the apoptosis caused by 1,25(OH)2D3. The 1,25(OH)2D3 concentration in these experiments was 100 μM.

    Techniques Used: Expressing, Quantitative RT-PCR, Inhibition, Concentration Assay

    25) Product Images from "Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype"

    Article Title: Wnt-signaling enhances neural crest migration of melanoma cells and induces an invasive phenotype

    Journal: Molecular Cancer

    doi: 10.1186/s12943-018-0773-5

    Gene expression during neural crest induction in the zebrafish embryo and upon Wnt stimulation in SKMEL28 melanoma cells, and clinical significance. a Heatmaps of real-time qPCR gene expression analysis of the melanoma cell lines SKMEL19, 451 LU, BLM and A375 for AXIN2. TYR, MITF and the four genes INHBA, CYR61, ANGTPL4 and FABP7. Log2 transformed x-fold expression values were used for color-coding. Yellow: upregulated gene expression; blue: downregulated gene expression at 24 h treatment of the melanoma cells; black: not detectable b Western blot analysis to detect protein levels of beta-catenin, PTEN, phospo-Ser473 (AKT) and AKT in the melanoma cell lines A375, SKMEL19, BLM and 451 LU after treatment with 3T3-CM, 3T3-Wnt3a, or 0.5 μM PKF115–584 for 24 h. Beta-actin was used as loading control. Small-hairpin knockdown cells served as samples to investigate the dependence on beta-catenin. c Luciferase reporter assay (Super8xTOPFlash) indicates a 3-fold activation of the canonical Wnt−/β-catenin signaling pathway after stimulation of SKMEL28 cells with Wnt3a-conditioned medium (3T3-Wnt3a). PKF115–584 treatment (0.5 μM for 12 h) significantly inhibited reporter activity. **: p
    Figure Legend Snippet: Gene expression during neural crest induction in the zebrafish embryo and upon Wnt stimulation in SKMEL28 melanoma cells, and clinical significance. a Heatmaps of real-time qPCR gene expression analysis of the melanoma cell lines SKMEL19, 451 LU, BLM and A375 for AXIN2. TYR, MITF and the four genes INHBA, CYR61, ANGTPL4 and FABP7. Log2 transformed x-fold expression values were used for color-coding. Yellow: upregulated gene expression; blue: downregulated gene expression at 24 h treatment of the melanoma cells; black: not detectable b Western blot analysis to detect protein levels of beta-catenin, PTEN, phospo-Ser473 (AKT) and AKT in the melanoma cell lines A375, SKMEL19, BLM and 451 LU after treatment with 3T3-CM, 3T3-Wnt3a, or 0.5 μM PKF115–584 for 24 h. Beta-actin was used as loading control. Small-hairpin knockdown cells served as samples to investigate the dependence on beta-catenin. c Luciferase reporter assay (Super8xTOPFlash) indicates a 3-fold activation of the canonical Wnt−/β-catenin signaling pathway after stimulation of SKMEL28 cells with Wnt3a-conditioned medium (3T3-Wnt3a). PKF115–584 treatment (0.5 μM for 12 h) significantly inhibited reporter activity. **: p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Western Blot, Luciferase, Reporter Assay, Activation Assay, Activity Assay

    Wnt3a induces invasive growth of melanoma cells in organotypic tissue skin reconstructs (TSR). a 451 LU melanoma cells were seeded together with HaCat epidermal cells onto a layer of collagen I embedded human fibroblasts. TSR exposed to Wnt3a conditioned medium (Wnt3a-CM) showed a pronounced invasive morphology in the H E staining (upper pictures) when compared to cells exposed to control medium (3T3-CM). Immunofluorescence stainings for HMB45 (red) and beta-catenin (blue) identified melanoma cells (HMB45+), revealed beta-catenin expression levels and verified the invasion of single 451 LU cells from the epidermis (epi) into the dermal part (der). Nuclei were stained with YO-PRO-1 (green). b Knockdown of beta-catenin (blue) with shRNA (shCTNNB1) reduced the invasion of 451 LU melanoma cells (HMB45+, red) into the dermal part of the TSR
    Figure Legend Snippet: Wnt3a induces invasive growth of melanoma cells in organotypic tissue skin reconstructs (TSR). a 451 LU melanoma cells were seeded together with HaCat epidermal cells onto a layer of collagen I embedded human fibroblasts. TSR exposed to Wnt3a conditioned medium (Wnt3a-CM) showed a pronounced invasive morphology in the H E staining (upper pictures) when compared to cells exposed to control medium (3T3-CM). Immunofluorescence stainings for HMB45 (red) and beta-catenin (blue) identified melanoma cells (HMB45+), revealed beta-catenin expression levels and verified the invasion of single 451 LU cells from the epidermis (epi) into the dermal part (der). Nuclei were stained with YO-PRO-1 (green). b Knockdown of beta-catenin (blue) with shRNA (shCTNNB1) reduced the invasion of 451 LU melanoma cells (HMB45+, red) into the dermal part of the TSR

    Techniques Used: Staining, Immunofluorescence, Expressing, shRNA

    26) Product Images from "Potential Use of 1-25-dihydroxyvitamin D in the Diagnosis and Treatment of Papillary Thyroid Cancer"

    Article Title: Potential Use of 1-25-dihydroxyvitamin D in the Diagnosis and Treatment of Papillary Thyroid Cancer

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.909544

    Knockdown of cathelicidin antimicrobial peptide (CAMP) expression can reverse the proliferation and apoptosis caused by 1,25(OH)2D3. ( A ) Relative expression of CAMP after transfecting CAMP siRNA into K1 and IHH4 cells was analyzed by qRT-PCR and normalized to GAPDH expression. ( B ) Expression of CAMP protein was measured, and beta-actin protein was used for normalization after treatment with only 1,25(OH)2D3 or CAMP siRNA plus 1,25(OH)2D3 for 48 hours. ( C ) CAMP siRNA reduces the inhibition of cell proliferation caused by 1,25(OH)2D3. ( D ) CAMP siRNA reverses the apoptosis caused by 1,25(OH)2D3. The 1,25(OH)2D3 concentration in these experiments was 100 μM.
    Figure Legend Snippet: Knockdown of cathelicidin antimicrobial peptide (CAMP) expression can reverse the proliferation and apoptosis caused by 1,25(OH)2D3. ( A ) Relative expression of CAMP after transfecting CAMP siRNA into K1 and IHH4 cells was analyzed by qRT-PCR and normalized to GAPDH expression. ( B ) Expression of CAMP protein was measured, and beta-actin protein was used for normalization after treatment with only 1,25(OH)2D3 or CAMP siRNA plus 1,25(OH)2D3 for 48 hours. ( C ) CAMP siRNA reduces the inhibition of cell proliferation caused by 1,25(OH)2D3. ( D ) CAMP siRNA reverses the apoptosis caused by 1,25(OH)2D3. The 1,25(OH)2D3 concentration in these experiments was 100 μM.

    Techniques Used: Expressing, Quantitative RT-PCR, Inhibition, Concentration Assay

    27) Product Images from "Genetic dissection of the miR-200–Zeb1 axis reveals its importance in tumor differentiation and invasion"

    Article Title: Genetic dissection of the miR-200–Zeb1 axis reveals its importance in tumor differentiation and invasion

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07130-z

    Mutation of miR-200 sites in Zeb1 is sufficient to phenocopy miR-200 ablation. a Murine Zeb1 3′UTR, miR-200 site conservation, and mutated miR-200 site sequences. b Percent survival of RT2 mice with Zeb1 mutation ( RT2 , RT2_Zeb1 200H , RT2_Zeb1 200M , n = 42, 45, 26, respectively). c Random-fed blood glucose ( RT2 , RT2_Zeb1 200H , RT2_Zeb1 200M , n = 11, 11, 10, respectively). Dotted line represents onset of severe hypoglycemia. d Tumor burden in different age classes (box, 25th and 75th percentiles; central line, median; from left to right: n = 9, 4, 4, 4, 5, 11, 12 mice). e Representative insulin/glucagon and SV40 immunofluorescence staining of whole pancreas (left; insulin-negative tumors outlined; scale bar = 2 mm) and zoomed-in regions (right; all tumors outlined; scale bar = 100 μm). LN lymph node. f Percent tumors per grade ( n = 5 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 55, 17, 16 tumors, respectively). g Percent insulin-positive and -negative tumors ( n = 5 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 67, 12, 15 tumors, respectively). h Mean log 2 FC (with 95% confidence intervals) of beta-cell-identity genes in RT2_Zeb1 200M vs. RT2 islets of 6-week-old mice. i Percent TUNEL-positive nuclei ( n = 2 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 17, 21, 13 lesions, respectively). j Representative H E and SV40 immunofluorescence staining of late-stage RT2_Zeb1 200M liver (scale bar = 2 mm). k Representative images of late-stage RT2_Zeb1 200H and RT2_Zeb1 200M mice. l Percent of mice with macrometastasis in difference age classes. c , i Data are plotted as mean ± SD. Significance was evaluated by b Mantel Cox test, c two-tailed t test with Holm−Sidak correction (vs. RT2 ), d , i one-way ANOVA with Dunnett’s multiple comparisons (vs. RT2 ) and h empirical Bayes method. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001
    Figure Legend Snippet: Mutation of miR-200 sites in Zeb1 is sufficient to phenocopy miR-200 ablation. a Murine Zeb1 3′UTR, miR-200 site conservation, and mutated miR-200 site sequences. b Percent survival of RT2 mice with Zeb1 mutation ( RT2 , RT2_Zeb1 200H , RT2_Zeb1 200M , n = 42, 45, 26, respectively). c Random-fed blood glucose ( RT2 , RT2_Zeb1 200H , RT2_Zeb1 200M , n = 11, 11, 10, respectively). Dotted line represents onset of severe hypoglycemia. d Tumor burden in different age classes (box, 25th and 75th percentiles; central line, median; from left to right: n = 9, 4, 4, 4, 5, 11, 12 mice). e Representative insulin/glucagon and SV40 immunofluorescence staining of whole pancreas (left; insulin-negative tumors outlined; scale bar = 2 mm) and zoomed-in regions (right; all tumors outlined; scale bar = 100 μm). LN lymph node. f Percent tumors per grade ( n = 5 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 55, 17, 16 tumors, respectively). g Percent insulin-positive and -negative tumors ( n = 5 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 67, 12, 15 tumors, respectively). h Mean log 2 FC (with 95% confidence intervals) of beta-cell-identity genes in RT2_Zeb1 200M vs. RT2 islets of 6-week-old mice. i Percent TUNEL-positive nuclei ( n = 2 mice per group; RT2 , early-stage RT2_Zeb1 200M , late-stage RT2_Zeb1 200M , n = 17, 21, 13 lesions, respectively). j Representative H E and SV40 immunofluorescence staining of late-stage RT2_Zeb1 200M liver (scale bar = 2 mm). k Representative images of late-stage RT2_Zeb1 200H and RT2_Zeb1 200M mice. l Percent of mice with macrometastasis in difference age classes. c , i Data are plotted as mean ± SD. Significance was evaluated by b Mantel Cox test, c two-tailed t test with Holm−Sidak correction (vs. RT2 ), d , i one-way ANOVA with Dunnett’s multiple comparisons (vs. RT2 ) and h empirical Bayes method. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001

    Techniques Used: Mutagenesis, Mouse Assay, Immunofluorescence, Staining, TUNEL Assay, Two Tailed Test

    miR-200 ablation promotes tumor growth, malignancy, and invasion. a Diagram of murine miR-200 superfamily (seed regions boxed). b Percent survival of RT2 mice with one or both miR-200 genomic clusters ablated ( RT2 , RT2_Rip-Cre_200a fl/fl , RT2_141~200cKO , RT2_DKO , n = 42, 8, 43, 36, respectively). c Random-fed blood glucose ( RT2 , RT2_Rip-Cre_200a fl/fl , RT2_141~200cKO , RT2_DKO , n = 11, 7, 11, 13, respectively). Dotted line represents onset of severe hypoglycemia. d Tumor burden in different age classes (box, 25th and 75th percentiles; central line, median; left to right: n = 9, 5, 4, 6, 9, 7, 11, 14 mice). e Representative insulin/glucagon and SV40 immunofluorescence staining of whole pancreas (left; insulin-negative tumors outlined; scale bar = 2 mm) and zoomed-in regions (right; all tumors outlined; scale bar = 100 μm). LN lymph node. f Percent tumors per grade ( n = 5 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO , n = 55, 9, 31 tumors respectively). g Percent insulin-positive and -negative tumors ( n = 5 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO , n = 67, 6, 31 tumors, respectively). h Mean log 2 FC (with 95% confidence intervals) of beta-cell-identity genes in RT2_DKO vs. RT2 islets of 6-week-old mice. i Percent of TUNEL-positive nuclei ( n = 2 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO, n = 17, 15, 17 lesions per group). j Representative H E staining and SV40 immunofluorescence staining of late-stage RT2_DKO liver (scale bar = 2 mm). k Representative images of late-stage RT2 and RT2_DKO mice. l Percent mice with macrometastasis in difference age classes. Data in c , i are plotted as mean ± SD. Significance was evaluated by b Mantel Cox test, c two-tailed t test with Holm−Sidak correction (vs. RT2 ), d , i one-way ANOVA with Dunnett’s multiple comparisons test (vs. RT2 ) and h empirical Bayes method. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001
    Figure Legend Snippet: miR-200 ablation promotes tumor growth, malignancy, and invasion. a Diagram of murine miR-200 superfamily (seed regions boxed). b Percent survival of RT2 mice with one or both miR-200 genomic clusters ablated ( RT2 , RT2_Rip-Cre_200a fl/fl , RT2_141~200cKO , RT2_DKO , n = 42, 8, 43, 36, respectively). c Random-fed blood glucose ( RT2 , RT2_Rip-Cre_200a fl/fl , RT2_141~200cKO , RT2_DKO , n = 11, 7, 11, 13, respectively). Dotted line represents onset of severe hypoglycemia. d Tumor burden in different age classes (box, 25th and 75th percentiles; central line, median; left to right: n = 9, 5, 4, 6, 9, 7, 11, 14 mice). e Representative insulin/glucagon and SV40 immunofluorescence staining of whole pancreas (left; insulin-negative tumors outlined; scale bar = 2 mm) and zoomed-in regions (right; all tumors outlined; scale bar = 100 μm). LN lymph node. f Percent tumors per grade ( n = 5 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO , n = 55, 9, 31 tumors respectively). g Percent insulin-positive and -negative tumors ( n = 5 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO , n = 67, 6, 31 tumors, respectively). h Mean log 2 FC (with 95% confidence intervals) of beta-cell-identity genes in RT2_DKO vs. RT2 islets of 6-week-old mice. i Percent of TUNEL-positive nuclei ( n = 2 mice per group; RT2 , early-stage RT2_DKO , late-stage RT2_DKO, n = 17, 15, 17 lesions per group). j Representative H E staining and SV40 immunofluorescence staining of late-stage RT2_DKO liver (scale bar = 2 mm). k Representative images of late-stage RT2 and RT2_DKO mice. l Percent mice with macrometastasis in difference age classes. Data in c , i are plotted as mean ± SD. Significance was evaluated by b Mantel Cox test, c two-tailed t test with Holm−Sidak correction (vs. RT2 ), d , i one-way ANOVA with Dunnett’s multiple comparisons test (vs. RT2 ) and h empirical Bayes method. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001

    Techniques Used: Mouse Assay, Immunofluorescence, Staining, TUNEL Assay, Two Tailed Test

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    Cell Signaling Technology Inc rabbit anti human β actin antibody
    Ornithine transcarbamylase (OTC) is expressed in both hMSCs and T-ALL cells and BCT-10 treatment could suppress OTC expression significant in T-ALL cells but only modestly on MSCs. ( a ) Basal expression of OTC in hTertMSCs, hMSCs from 2 healthy donors, CCRF-CEM, Jurkat and MOLT-4. Cells were harvested and respective proteins were extracted from the corresponding cell lines. SDS-PAGE was performed to separate the proteins. OTC and <t>β-actin</t> (internal control) expression were probed by the corresponding antibodies and then visualized by enzyme coupled luminescence (ECL). ( b ) Expression of OTC in hMSCs after BCT-100 treatment. hTertMSCs or hMSCs from a healthy donor were cultured in different doses of BCT-100 (0 U/ml, 0.5 U/ml, 1 U/ml and 2 U/ml) for 36 hours and protein was extracted from the cell culture. SDS-PAGE followed by Western blotting was performed. The expression of OTC and β-actin (internal control) of hTertMSCs is presented in the figure. ( c ), ( d ) and ( e ) Expression of OTC in CCRF-CEM, Jurkat or MOLT-4 after BCT-100 treatment respectively. CCRF-CEM, Jurkat and MOLT-4 were cultured in different doses of BCT-100 (0 U/ml, 0.5 U/ml, 1 U/ml and 2 U/ml) for 36 hours and protein was extracted. SDS-PAGE followed by Western blotting was performed. The expression of OTC and β-actin (internal control) of CCRF-CEM, Jurkat or MOLT-4 is presented in the figures. The most representative results are presented.
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    84
    Cell Signaling Technology Inc anti actin b
    RING1A mediates platinum-induced monoubiquitination of γH2AX. (A) OVCAR5 cells were untreated (U) or treated with 12 μM cisplatin (IC50 dose) for 1, 3, 8 and 16 hours or with 2 mM H 2 O 2 for 30 mins (used as a negative control). Cell lysates were analyzed by western blot. Graph depicts mean ± SEM of densitometric analysis of indicated proteins relative to <t>Actin</t> B at the indicated time points (N=3). (B) OVCAR5 cells infected with empty vector (EV) or 2 different RING1A shRNAs were untreated (U) or treated (T) with 12 μM cisplatin for 8 hours. Data is presented as in (A). OVCAR5 cells were either untreated (U) or treated with 12 μM cisplatin (T) alone or in combination with DMSO or 10 μM PRT4165 for 8 hours. Data is presented as in (A). OVCAR5 cells were treated 12 μM cisplatin for 8 hours. Immunofluorescence analysis was performed for RING1A (red) and the damage marker γH2AX (green). Merge image shows overlap of γH2AX and RING1A. White arrows indicate examples of RING1A foci that co-localize with γH2AX. Graph displays mean percentage of cells with ≥ 4 γH2AX and RING1A co-localized foci ± SEM (N=3). Scale bar = 5 μm. A representative RGB profile of untreated and cisplatin treated cell showing RING1A colocalization with γH2AX. Blue arrows point to foci which colocalize. (E) OVCAR5 cells were treated as in (D). Immunofluorescence analysis was performed for H2AK119ub1 (Red) and γH2AX (green). Merge image shows overlap of H2AK119ub1 and γH2AX. White arrows indicate examples of H2AK119 foci that co-localize with γH2AX. Graph displays mean percentage of cells with ≥ 4 γH2AX and H2AK119ub1 co-localized foci ± SEM (N=3). Scale bar = 5 μm. (F) OVCAR5 cells infected with empty vector (EV) or 2 different RING1A shRNAs were untreated or treated with 12 μM cisplatin for 8 hours and immunofluorescence was performed as in (E). Graph displays mean percentage of cells with ≥ 4 γH2AX and H2AK119ub1 co-localized foci ± SEM (N=3). Statistical significance was calculated using Student’s t-test. For all U versus T, P-values *
    Anti Actin B, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse anti β actin
    CCS is the necessary Cu chaperone for MEK1. a , Immunoblot detection of biotinylated MEK1, ATP7A, SOD1, MYC-BirA, MYC-BirA-ATOX1, or MYC-BirA-CCS from strepavadin pulldowns and whole cell extracts from HEK-HT cells stably expressing MYC-BirA, MYC-BirA-ATOX1 , or MYC-BirA-CCS treated with media with (+) or without (-) biotin for 48 hours. n=2 biologically independent experiments. b,c , Immunoblot detection of phosphorylated (P)-ERK1/2, total (T)-ERK1/2, P-MEK1/2, T-MEK1/2, CCS, or <t>β-ACTIN</t> from HEK-HT cells stably expressing doxycycline inducible shRNA against control (-) or CCS ( #1 or #2 ) treated with doxycycline for 24 hours followed by b , vehicle (VEH) or 1µM CuCl 2 for 20 minutes or c , VEH or 0.1ng/mL EGF for 15 minutes. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments. d , Immunoblot detection of P-ERK1/2, T-ERK1/2, P-MEK1/2, T-MEK1/2, MYC, or β-ACTIN from HEK-HT cells stably expressing doxycycline inducible shRNA against control (-) or CCS ( #2 ) reconstituted with MYC-CCS WT (WT), MYC-CCS Domain 1 Mutant (D1), MYC-CCS Domain 3 Mutant (D3), or MYC-CCS Domain 1+3 Mutant (D1+D3) treated with doxycycline for 24 hours. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments.
    Mouse Anti β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti β actin
    Immunofluorescence detection of iNOS in activated J774-A1 macrophages infected with T. gondii . (A) Detection of iNOS (green) in non-infected (Control) and in T. gondii (red) infected cells (DAPI - blue) at 2, 6, and 24 h post-infection. Scale bar = 10 μm. (B) Analysis of the proportion of iNOS positive or negative macrophages in non-infected (Control) and T. gondii infected cells at 2 and 24 h post-infection. Mean ± SEM ( n = 4 experiments, each with 8 replicates). (C) Western blot detection of iNOS expression in non-infected (Control) and T. gondii infected (Infected) cells. <t>β-actin</t> was used as loading control. (D) Densitometry of western blots normalized to β-actin at 2 h post-infection. Mean ± SD ( n = 3 experiments, each with 1 replicate). ∗ P ≤ 0.05, two-way ANOVA with Tukey post-test, n.s (not significant).
    Rabbit Anti β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Ornithine transcarbamylase (OTC) is expressed in both hMSCs and T-ALL cells and BCT-10 treatment could suppress OTC expression significant in T-ALL cells but only modestly on MSCs. ( a ) Basal expression of OTC in hTertMSCs, hMSCs from 2 healthy donors, CCRF-CEM, Jurkat and MOLT-4. Cells were harvested and respective proteins were extracted from the corresponding cell lines. SDS-PAGE was performed to separate the proteins. OTC and β-actin (internal control) expression were probed by the corresponding antibodies and then visualized by enzyme coupled luminescence (ECL). ( b ) Expression of OTC in hMSCs after BCT-100 treatment. hTertMSCs or hMSCs from a healthy donor were cultured in different doses of BCT-100 (0 U/ml, 0.5 U/ml, 1 U/ml and 2 U/ml) for 36 hours and protein was extracted from the cell culture. SDS-PAGE followed by Western blotting was performed. The expression of OTC and β-actin (internal control) of hTertMSCs is presented in the figure. ( c ), ( d ) and ( e ) Expression of OTC in CCRF-CEM, Jurkat or MOLT-4 after BCT-100 treatment respectively. CCRF-CEM, Jurkat and MOLT-4 were cultured in different doses of BCT-100 (0 U/ml, 0.5 U/ml, 1 U/ml and 2 U/ml) for 36 hours and protein was extracted. SDS-PAGE followed by Western blotting was performed. The expression of OTC and β-actin (internal control) of CCRF-CEM, Jurkat or MOLT-4 is presented in the figures. The most representative results are presented.

    Journal: Experimental Hematology & Oncology

    Article Title: Vincristine could partly suppress stromal support to T-ALL blasts during pegylated arginase I treatment

    doi: 10.1186/2162-3619-2-11

    Figure Lengend Snippet: Ornithine transcarbamylase (OTC) is expressed in both hMSCs and T-ALL cells and BCT-10 treatment could suppress OTC expression significant in T-ALL cells but only modestly on MSCs. ( a ) Basal expression of OTC in hTertMSCs, hMSCs from 2 healthy donors, CCRF-CEM, Jurkat and MOLT-4. Cells were harvested and respective proteins were extracted from the corresponding cell lines. SDS-PAGE was performed to separate the proteins. OTC and β-actin (internal control) expression were probed by the corresponding antibodies and then visualized by enzyme coupled luminescence (ECL). ( b ) Expression of OTC in hMSCs after BCT-100 treatment. hTertMSCs or hMSCs from a healthy donor were cultured in different doses of BCT-100 (0 U/ml, 0.5 U/ml, 1 U/ml and 2 U/ml) for 36 hours and protein was extracted from the cell culture. SDS-PAGE followed by Western blotting was performed. The expression of OTC and β-actin (internal control) of hTertMSCs is presented in the figure. ( c ), ( d ) and ( e ) Expression of OTC in CCRF-CEM, Jurkat or MOLT-4 after BCT-100 treatment respectively. CCRF-CEM, Jurkat and MOLT-4 were cultured in different doses of BCT-100 (0 U/ml, 0.5 U/ml, 1 U/ml and 2 U/ml) for 36 hours and protein was extracted. SDS-PAGE followed by Western blotting was performed. The expression of OTC and β-actin (internal control) of CCRF-CEM, Jurkat or MOLT-4 is presented in the figures. The most representative results are presented.

    Article Snippet: Rabbit anti-human β-actin antibody was purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, SDS Page, Cell Culture, Western Blot

    RING1A mediates platinum-induced monoubiquitination of γH2AX. (A) OVCAR5 cells were untreated (U) or treated with 12 μM cisplatin (IC50 dose) for 1, 3, 8 and 16 hours or with 2 mM H 2 O 2 for 30 mins (used as a negative control). Cell lysates were analyzed by western blot. Graph depicts mean ± SEM of densitometric analysis of indicated proteins relative to Actin B at the indicated time points (N=3). (B) OVCAR5 cells infected with empty vector (EV) or 2 different RING1A shRNAs were untreated (U) or treated (T) with 12 μM cisplatin for 8 hours. Data is presented as in (A). OVCAR5 cells were either untreated (U) or treated with 12 μM cisplatin (T) alone or in combination with DMSO or 10 μM PRT4165 for 8 hours. Data is presented as in (A). OVCAR5 cells were treated 12 μM cisplatin for 8 hours. Immunofluorescence analysis was performed for RING1A (red) and the damage marker γH2AX (green). Merge image shows overlap of γH2AX and RING1A. White arrows indicate examples of RING1A foci that co-localize with γH2AX. Graph displays mean percentage of cells with ≥ 4 γH2AX and RING1A co-localized foci ± SEM (N=3). Scale bar = 5 μm. A representative RGB profile of untreated and cisplatin treated cell showing RING1A colocalization with γH2AX. Blue arrows point to foci which colocalize. (E) OVCAR5 cells were treated as in (D). Immunofluorescence analysis was performed for H2AK119ub1 (Red) and γH2AX (green). Merge image shows overlap of H2AK119ub1 and γH2AX. White arrows indicate examples of H2AK119 foci that co-localize with γH2AX. Graph displays mean percentage of cells with ≥ 4 γH2AX and H2AK119ub1 co-localized foci ± SEM (N=3). Scale bar = 5 μm. (F) OVCAR5 cells infected with empty vector (EV) or 2 different RING1A shRNAs were untreated or treated with 12 μM cisplatin for 8 hours and immunofluorescence was performed as in (E). Graph displays mean percentage of cells with ≥ 4 γH2AX and H2AK119ub1 co-localized foci ± SEM (N=3). Statistical significance was calculated using Student’s t-test. For all U versus T, P-values *

    Journal: bioRxiv

    Article Title: Platinum-Induced Ubiquitination of Phosphorylated H2AX by RING1A is Mediated by Replication Protein A in Ovarian Cancer

    doi: 10.1101/2020.04.30.070185

    Figure Lengend Snippet: RING1A mediates platinum-induced monoubiquitination of γH2AX. (A) OVCAR5 cells were untreated (U) or treated with 12 μM cisplatin (IC50 dose) for 1, 3, 8 and 16 hours or with 2 mM H 2 O 2 for 30 mins (used as a negative control). Cell lysates were analyzed by western blot. Graph depicts mean ± SEM of densitometric analysis of indicated proteins relative to Actin B at the indicated time points (N=3). (B) OVCAR5 cells infected with empty vector (EV) or 2 different RING1A shRNAs were untreated (U) or treated (T) with 12 μM cisplatin for 8 hours. Data is presented as in (A). OVCAR5 cells were either untreated (U) or treated with 12 μM cisplatin (T) alone or in combination with DMSO or 10 μM PRT4165 for 8 hours. Data is presented as in (A). OVCAR5 cells were treated 12 μM cisplatin for 8 hours. Immunofluorescence analysis was performed for RING1A (red) and the damage marker γH2AX (green). Merge image shows overlap of γH2AX and RING1A. White arrows indicate examples of RING1A foci that co-localize with γH2AX. Graph displays mean percentage of cells with ≥ 4 γH2AX and RING1A co-localized foci ± SEM (N=3). Scale bar = 5 μm. A representative RGB profile of untreated and cisplatin treated cell showing RING1A colocalization with γH2AX. Blue arrows point to foci which colocalize. (E) OVCAR5 cells were treated as in (D). Immunofluorescence analysis was performed for H2AK119ub1 (Red) and γH2AX (green). Merge image shows overlap of H2AK119ub1 and γH2AX. White arrows indicate examples of H2AK119 foci that co-localize with γH2AX. Graph displays mean percentage of cells with ≥ 4 γH2AX and H2AK119ub1 co-localized foci ± SEM (N=3). Scale bar = 5 μm. (F) OVCAR5 cells infected with empty vector (EV) or 2 different RING1A shRNAs were untreated or treated with 12 μM cisplatin for 8 hours and immunofluorescence was performed as in (E). Graph displays mean percentage of cells with ≥ 4 γH2AX and H2AK119ub1 co-localized foci ± SEM (N=3). Statistical significance was calculated using Student’s t-test. For all U versus T, P-values *

    Article Snippet: Antibodies For western blot of endogenous proteins, anti-γH2AX (Cell Signaling Technology (CST), MA #9718, 1:1000), anti-Actin B (CST, MA #4970, 1:1000), anti-p-ATM S1981 (CST, MA #13050, 1:1000), anti-total ATM (CST, MA #2873, 1:1000), anti-total H2AX (CST, MA #2595, 1:1000), anti-RING1A (CST, MA #13069, 1:1000), anti-RING1B (Santa Cruz (SC), CA sc-101109, 1:1000), anti-H2AK119ub1 (CST, MA #8240,1:1000), anti-XPC (SC, CA sc-74410, 1:1000), anti-lamin B (SC, CA sc-6216, 1:1000; SC, CA sc-374015, 1:1000), anti-XPA (SC, CA sc-28353, 1:1000), anti-pRPA32 S33 (Bethyl Laboratories, TX A300-246A-M, 1:1000), anti-RPA32 (CST, MA #2208,1:1000), anti-phospho-Chk1 S345 (CST, MA #2348, 1:1000) and anti-total Chk1 (CST, MA #2360, 1:1000) antibodies were used.

    Techniques: Negative Control, Western Blot, Infection, Plasmid Preparation, Immunofluorescence, Marker

    CCS is the necessary Cu chaperone for MEK1. a , Immunoblot detection of biotinylated MEK1, ATP7A, SOD1, MYC-BirA, MYC-BirA-ATOX1, or MYC-BirA-CCS from strepavadin pulldowns and whole cell extracts from HEK-HT cells stably expressing MYC-BirA, MYC-BirA-ATOX1 , or MYC-BirA-CCS treated with media with (+) or without (-) biotin for 48 hours. n=2 biologically independent experiments. b,c , Immunoblot detection of phosphorylated (P)-ERK1/2, total (T)-ERK1/2, P-MEK1/2, T-MEK1/2, CCS, or β-ACTIN from HEK-HT cells stably expressing doxycycline inducible shRNA against control (-) or CCS ( #1 or #2 ) treated with doxycycline for 24 hours followed by b , vehicle (VEH) or 1µM CuCl 2 for 20 minutes or c , VEH or 0.1ng/mL EGF for 15 minutes. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments. d , Immunoblot detection of P-ERK1/2, T-ERK1/2, P-MEK1/2, T-MEK1/2, MYC, or β-ACTIN from HEK-HT cells stably expressing doxycycline inducible shRNA against control (-) or CCS ( #2 ) reconstituted with MYC-CCS WT (WT), MYC-CCS Domain 1 Mutant (D1), MYC-CCS Domain 3 Mutant (D3), or MYC-CCS Domain 1+3 Mutant (D1+D3) treated with doxycycline for 24 hours. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments.

    Journal: bioRxiv

    Article Title: Structural and molecular determinants of CCS-mediated copper activation of MEK1/2

    doi: 10.1101/2020.05.01.072124

    Figure Lengend Snippet: CCS is the necessary Cu chaperone for MEK1. a , Immunoblot detection of biotinylated MEK1, ATP7A, SOD1, MYC-BirA, MYC-BirA-ATOX1, or MYC-BirA-CCS from strepavadin pulldowns and whole cell extracts from HEK-HT cells stably expressing MYC-BirA, MYC-BirA-ATOX1 , or MYC-BirA-CCS treated with media with (+) or without (-) biotin for 48 hours. n=2 biologically independent experiments. b,c , Immunoblot detection of phosphorylated (P)-ERK1/2, total (T)-ERK1/2, P-MEK1/2, T-MEK1/2, CCS, or β-ACTIN from HEK-HT cells stably expressing doxycycline inducible shRNA against control (-) or CCS ( #1 or #2 ) treated with doxycycline for 24 hours followed by b , vehicle (VEH) or 1µM CuCl 2 for 20 minutes or c , VEH or 0.1ng/mL EGF for 15 minutes. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments. d , Immunoblot detection of P-ERK1/2, T-ERK1/2, P-MEK1/2, T-MEK1/2, MYC, or β-ACTIN from HEK-HT cells stably expressing doxycycline inducible shRNA against control (-) or CCS ( #2 ) reconstituted with MYC-CCS WT (WT), MYC-CCS Domain 1 Mutant (D1), MYC-CCS Domain 3 Mutant (D3), or MYC-CCS Domain 1+3 Mutant (D1+D3) treated with doxycycline for 24 hours. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments.

    Article Snippet: Equal amount of lysates were resolved by SDS-PAGE using standard techniques, and protein was detected with the following primary antibodies: mouse anti-ATP7A (1:1000; 376467, Santa Cruz), mouse anti-β -ACTIN (1:10000; 3700, Cell Signaling), rabbit anti-biotin (1:1000; 5597, Cell Signaling), mouse anti-CCS (1:1000; 55561, Santa Cruz), rabbit anti-phospho(Thr202/Tyr204)-ERK1/2 (1:1000; 9101, Cell Signaling), mouse anti-ERK1/2 (1:1000; 9107, Cell Signaling), rabbit anti-phospho(Ser217/221)-MEK1/2 (1:1000; 9154S, Cell Signaling), mouse anti-MEK1/2 (1:1000; 4694S, Cell Signaling), mouse anti-MEK1 (1:1000; 2352, Cell Signaling), mouse anti-MYC (1:1000; 227S, Cell Signaling), and mouse anti-SOD1 (1:1000; MA1-105, ThermoFisher Scientific) followed by detection with one of the horseradish peroxidase conjugated secondary antibodies: goat anti-rabbit IgG (1:2000; 7074, Cell Signaling) or goat anti-mouse (1:2000; 7076, Cell Signaling) using SignalFire (Cell Signaling) or SignalFire Elite ECL (Cell Signaling) detection reagents.

    Techniques: Stable Transfection, Expressing, shRNA, Mutagenesis

    Treatment with small molecular inhibitor of CCS blunts MAPK pathway activation at the level of MEK1/2. a , Immunoblot detection of phosphorylated (P)-ERK1/2, total (T)-ERK1/2, or β-ACTIN from HEK-HT cells treated with vehicle (0) or increasing concentrations of DCAC50 for 24 hours. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments. b,c , Immunoblot detection of P-ERK1/2, T-ERK1/2, P-MEK1/2, T-MEK1/2, or β-ACTIN from HEK-HT cells treated with vehicle (-) or 10µM DCAC50 for 24 hours followed stimulation with b , 1µM CuCl 2 or c , 0.1ng/mL EGF for indicated time. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments. d , Model of CCS-mediated Cu activation of MEK1/2.

    Journal: bioRxiv

    Article Title: Structural and molecular determinants of CCS-mediated copper activation of MEK1/2

    doi: 10.1101/2020.05.01.072124

    Figure Lengend Snippet: Treatment with small molecular inhibitor of CCS blunts MAPK pathway activation at the level of MEK1/2. a , Immunoblot detection of phosphorylated (P)-ERK1/2, total (T)-ERK1/2, or β-ACTIN from HEK-HT cells treated with vehicle (0) or increasing concentrations of DCAC50 for 24 hours. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments. b,c , Immunoblot detection of P-ERK1/2, T-ERK1/2, P-MEK1/2, T-MEK1/2, or β-ACTIN from HEK-HT cells treated with vehicle (-) or 10µM DCAC50 for 24 hours followed stimulation with b , 1µM CuCl 2 or c , 0.1ng/mL EGF for indicated time. Quantification: Fold change P-ERK1/2/T-ERK1/2 normalized to control, VEH. n=3 biologically independent experiments. d , Model of CCS-mediated Cu activation of MEK1/2.

    Article Snippet: Equal amount of lysates were resolved by SDS-PAGE using standard techniques, and protein was detected with the following primary antibodies: mouse anti-ATP7A (1:1000; 376467, Santa Cruz), mouse anti-β -ACTIN (1:10000; 3700, Cell Signaling), rabbit anti-biotin (1:1000; 5597, Cell Signaling), mouse anti-CCS (1:1000; 55561, Santa Cruz), rabbit anti-phospho(Thr202/Tyr204)-ERK1/2 (1:1000; 9101, Cell Signaling), mouse anti-ERK1/2 (1:1000; 9107, Cell Signaling), rabbit anti-phospho(Ser217/221)-MEK1/2 (1:1000; 9154S, Cell Signaling), mouse anti-MEK1/2 (1:1000; 4694S, Cell Signaling), mouse anti-MEK1 (1:1000; 2352, Cell Signaling), mouse anti-MYC (1:1000; 227S, Cell Signaling), and mouse anti-SOD1 (1:1000; MA1-105, ThermoFisher Scientific) followed by detection with one of the horseradish peroxidase conjugated secondary antibodies: goat anti-rabbit IgG (1:2000; 7074, Cell Signaling) or goat anti-mouse (1:2000; 7076, Cell Signaling) using SignalFire (Cell Signaling) or SignalFire Elite ECL (Cell Signaling) detection reagents.

    Techniques: Activation Assay

    Immunofluorescence detection of iNOS in activated J774-A1 macrophages infected with T. gondii . (A) Detection of iNOS (green) in non-infected (Control) and in T. gondii (red) infected cells (DAPI - blue) at 2, 6, and 24 h post-infection. Scale bar = 10 μm. (B) Analysis of the proportion of iNOS positive or negative macrophages in non-infected (Control) and T. gondii infected cells at 2 and 24 h post-infection. Mean ± SEM ( n = 4 experiments, each with 8 replicates). (C) Western blot detection of iNOS expression in non-infected (Control) and T. gondii infected (Infected) cells. β-actin was used as loading control. (D) Densitometry of western blots normalized to β-actin at 2 h post-infection. Mean ± SD ( n = 3 experiments, each with 1 replicate). ∗ P ≤ 0.05, two-way ANOVA with Tukey post-test, n.s (not significant).

    Journal: Frontiers in Microbiology

    Article Title: Inhibition of Nitric Oxide Production in Activated Macrophages Caused by Toxoplasma gondii Infection Occurs by Distinct Mechanisms in Different Mouse Macrophage Cell Lines

    doi: 10.3389/fmicb.2018.01936

    Figure Lengend Snippet: Immunofluorescence detection of iNOS in activated J774-A1 macrophages infected with T. gondii . (A) Detection of iNOS (green) in non-infected (Control) and in T. gondii (red) infected cells (DAPI - blue) at 2, 6, and 24 h post-infection. Scale bar = 10 μm. (B) Analysis of the proportion of iNOS positive or negative macrophages in non-infected (Control) and T. gondii infected cells at 2 and 24 h post-infection. Mean ± SEM ( n = 4 experiments, each with 8 replicates). (C) Western blot detection of iNOS expression in non-infected (Control) and T. gondii infected (Infected) cells. β-actin was used as loading control. (D) Densitometry of western blots normalized to β-actin at 2 h post-infection. Mean ± SD ( n = 3 experiments, each with 1 replicate). ∗ P ≤ 0.05, two-way ANOVA with Tukey post-test, n.s (not significant).

    Article Snippet: Samples were diluted 4:1 in 5× Laemmli buffer containing 10 mM of dithiothreitol (Sigma-Aldrich, United States), boiled for 5 min, resolved in 8% polyacrylamide gels (Bio-Rad Laboratories, Inc., United States) by SDS–PAGE, and transferred to nitrocellulose membranes Amersham Protran 0.45 NC (GE Healthcare Life Sciences, United States) for 1 h. Membranes were blocked overnight at 4°C with 5% fat-free milk in PBS 0.1% Tween 20 (Sigma-Aldrich, United States), probed for 1 h with anti-iNOS mouse monoclonal antibody (Santa Cruz Biotechnology, United States) dilute 1:1000 and rabbit anti-β-actin (Cell Signaling Technology, United States) diluted 1:1000 in 5% fat-free milk in PBS 0.1% Tween 20.

    Techniques: Immunofluorescence, Infection, Western Blot, Expressing

    Immunofluorescence detection of iNOS in activated RAW 264.7 macrophages infected with T. gondii . (A) Detection of iNOS (green) in non-infected (Control) and in T. gondii (red) infected cells (DAPI - blue) at 2, 6, and 24 h post-infection. Scale bar - 10 μm. (B) Analysis of the proportion of iNOS positive or negative macrophages in non-infected (Control) and T. gondii infected cells at 2 and 24 h post-infection. Mean ± SEM ( n = 4 experiments, each with 8 replicates). (C) Western blot detection of iNOS expression in non-infected (Control) and T. gondii - infected (Infected) cells at different time intervals post-infection. β-actin was used as loading control. (D) Densitometry of western bolts normalized to β-actin at 2 h post-infection. Mean ± SD ( n = 3 experiments, each with 1 replicate), n.s (not significant).

    Journal: Frontiers in Microbiology

    Article Title: Inhibition of Nitric Oxide Production in Activated Macrophages Caused by Toxoplasma gondii Infection Occurs by Distinct Mechanisms in Different Mouse Macrophage Cell Lines

    doi: 10.3389/fmicb.2018.01936

    Figure Lengend Snippet: Immunofluorescence detection of iNOS in activated RAW 264.7 macrophages infected with T. gondii . (A) Detection of iNOS (green) in non-infected (Control) and in T. gondii (red) infected cells (DAPI - blue) at 2, 6, and 24 h post-infection. Scale bar - 10 μm. (B) Analysis of the proportion of iNOS positive or negative macrophages in non-infected (Control) and T. gondii infected cells at 2 and 24 h post-infection. Mean ± SEM ( n = 4 experiments, each with 8 replicates). (C) Western blot detection of iNOS expression in non-infected (Control) and T. gondii - infected (Infected) cells at different time intervals post-infection. β-actin was used as loading control. (D) Densitometry of western bolts normalized to β-actin at 2 h post-infection. Mean ± SD ( n = 3 experiments, each with 1 replicate), n.s (not significant).

    Article Snippet: Samples were diluted 4:1 in 5× Laemmli buffer containing 10 mM of dithiothreitol (Sigma-Aldrich, United States), boiled for 5 min, resolved in 8% polyacrylamide gels (Bio-Rad Laboratories, Inc., United States) by SDS–PAGE, and transferred to nitrocellulose membranes Amersham Protran 0.45 NC (GE Healthcare Life Sciences, United States) for 1 h. Membranes were blocked overnight at 4°C with 5% fat-free milk in PBS 0.1% Tween 20 (Sigma-Aldrich, United States), probed for 1 h with anti-iNOS mouse monoclonal antibody (Santa Cruz Biotechnology, United States) dilute 1:1000 and rabbit anti-β-actin (Cell Signaling Technology, United States) diluted 1:1000 in 5% fat-free milk in PBS 0.1% Tween 20.

    Techniques: Immunofluorescence, Infection, Western Blot, Expressing