anti-actin antibody Santa Cruz Biotechnology Search Results


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  • 95
    Santa Cruz Biotechnology anti β actin
    Pulmonary muscarinic receptors function and expression in allergic AIRmin and AIRmax mice. AIRmin or AIRmax mice were sensitized and challenged with OVA as in  Figure 4 . Control group consisted of nonmanipulated animals. The experiments were performed 24 h after the last OVA challenge. Respiratory pattern of allergic AIRmin (a and c) or AIRmax (b and d) to inhaled MCh was evaluated in the presence or absence of gallamine. Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh. Area under the curve was obtained from Penh values (c and d). Gene (e and g) or protein (f and h) expression of the M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from OVA-sensitized AIRmin and AIRmax mice. The real-time PCR was carried out using  β -actin gene expression as internal control for normalization of M3R (e) and M2R (g) mRNA transcription levels. In Western blot analysis the density of M3R (f) and M2R (h) protein expression was normalized to actin expression in each sample. Data are expressed as mean ± SEM of four mice per group and are representative of two experiments. Western blots data were quantified by densitometry using the ImageJ software (NIH). Statistical analyses of Student's  t  test for (a), (b), (e), (f), (g), and (h). Statistical analyses of ANOVA following Tukey HSD for (c) and (d). * P
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    Santa Cruz Biotechnology mouse anti β actin
    Loss of LIN28A/B attenuates mTOR signaling and limits supporting cell proliferation and hair cell formation in cochlear organoid culture. Cochlear organoid cultures were established from UBC-CreERT2; Lin28a f/f ; Lin28b f/f mice and Lin28a f/f ; Lin28b f/f littermates’ stage P2. Cultures received 4-hydroxytamoxifen (TM) or vehicle control DMSO at plating. SOX2 (green) marks supporting cells/ pro-sensory cells, Hoechst (blue) staining marks cell nuclei. Bars in (D) and (G) represent mean± SD, otherwise individual data points and their mean ± SD were plotted. (A-G) Loss of Lin28a/b inhibits cell proliferation and hair cell production in organoid culture. ( A ) Representative BF images of control organoids and Lin28a/b dKO organoids at 7 days of expansion. ( B ) Diameter of control and Lin28a/b dKO organoids in ( A ) (n=7 animals per group, from 2 independent experiments). ( C ) Organoid forming efficiency in control and Lin28a/b dKO cultures (n=7 animals per group, from 2 independent experiments). ( D ) RT-qPCR analysis of Lin28a, Lin28b and Hmga2 mRNA expression in Lin28a/b dKO organoids (red bar) compared to control organoids (blue bar) at 7 days of expansion (n=5-6 animals per group, from 2 independent experiments). ( E ) Cell proliferation in control and Lin28a/b dKO organoids. A single EdU pulse was given at 7 days of expansion and EdU incorporation (red) was analyzed 1 hour later. ( F ) EdU incorporation in ( E ) (n=7 animals per group, from 2 independent experiments). ( G ) RT-qPCR analysis of Atoh1, Myo7a and Pou4f3 mRNA expression in Lin28a/b dKO organoids (red bar) compared to control organoids (blue bar) at 10 days of differentiation (n=4 animals per group, from 2 independent experiments). (H) Immunoblots for LIN28B, p-Akt, p-S6 and <t>β-actin</t> using protein lysates of acutely isolated control and Lin28a/b dKO cochlear epithelia, stage P5. ( I - L ) Loss of Lin28a/b attenuates mTOR signaling in cochlear organoids. ( I ) Immunostaining for p-S6 protein (red) in control and Lin28a/b dKO organoids at 7 days of expansion. ( J ) Percentage of p-S6 + cells in (I) (n=5 animals per group, from 2 independent experiments). ( K ) Immunoblots for p-Akt, p-4EBP1, 4EBP1, mLIN28B and β-actin (loading control) using protein lysates of control and Lin28a/b dKO organoids after 7 days of expansion. ( L ) Normalized p-Akt, p-4EBP1 and m-LIN28B protein levels in control and Lin28a/b dKO organoids in ( K ) (n=3 animals per group, from 1 representative experiment, 2 independent experiments). 2-way ANOVA with Tukey’s correction was used to calculate p-values in ( B ), ( C ) and ( J ). 2-tailed, unpaired Student’s t-test was used to calculate p-values in ( D ), ( F ), ( G ) and ( L ). Note that the individual data points in ( B ), ( F ) and ( J ) represent the average values per animal.
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    88
    Santa Cruz Biotechnology goat anti beta actin
    Nucleocytoplasmic Distribution of SpYY1 in S. purpuratus ova. Ova of S. purpuratus were fractionated into nuclear and cytoplasmic extracts using previously described methods. Whole-cell (W) as well as nuclear (N) and cytoplasmic (C) fractions were analyzed by SDS-PAGE followed by Western blotting. Antibodies used are indicated to the left of the panel, αYY1, Rabbit anti- Xenopus YY1; αH3, Goat anti-Histone H3 antibody, nuclear marker; αActin, Goat <t>anti-Beta-Actin</t> antibody, cytoplasmic marker.
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    Santa Cruz Biotechnology goat anti β actin
    Mice fed high-fat diet show steatosis and inflammation of the liver, and impairment of the autophagic process. A: The body weights of mice at the start of feeding or at 12 wk after feeding with normal chow (Cont.) or high-fat diet (HFD) are presented in the left panel. The serum ALT levels after 12 wk of feeding are shown in the right panel; B: Histology of the liver of control and HFD mice is shown in the left and the right panel, respectively (Hematoxylin and Eosin staining); C: Non-alcoholic steatohepatitis activity scores (NAS) of control and HFD mice; D: Immunoblotting analyses of phosphorylated JNK, p62, LC3, Rubicon, and <t>β-actin.</t> Protein samples were prepared from the liver tissue of each of the control and HFD mice. All of the above experiments were repeated three times and representative results are shown. The quantitative data are presented as the means ± SD.
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    92
    Santa Cruz Biotechnology rabbit anti β actin
    Identification of 14-3-3 isoforms expressed in GV and GVBD mouse oocytes. (A) the mRNA levels of 14-3-3 isoforms at GV stage of mouse oocytes. mRNA of 120 mouse oocytes of GV stage were extracted (described in the“ Materials and Methods ” Section). RT-PCR products using primers for specific 14-3-3 isoforms are observed in ethidium bromide-stained agarose gel. ε, β, γ, η, σ, τ and ζ represent seven 14-3-3 isoforms. (B) the mRNA levels of 14-3-3ε at GV and GVBD stage of mouse oocytes. Line GV: mouse oocytes at GV stage. Line GVBD: mouse oocytes at GVBD stage. (C) A protein band of approximately 28 kDa from 200 mouse oocytes at GV or GVBD stage is detectable by Western blot with an anti-14-3-3ε antibody (upper panel). In contrast an <t>anti-β-actin</t> antibody detected a low molecular weight band of approximately 43 kDa (lower panel). (D) Western blot analysis 14-3-3β protein expression with an anti-14-3-3β antibody from 300 mouse oocytes at GV or GVBD stage, A protein band of approximately 29 kDa (upper panel). Line Brain: mouse brain protein as positive control. Shown is a representative of three independent experiments.
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    Santa Cruz Biotechnology anti α sma
    Expression of E-cadherin, CK, <t>α-SMA,</t> vimentin in HK-2 cells by immunochemistry. (A–C)The HK-2 cells under 30 mM glucose(B) or 30 mM glucose+cont siRNA(C) showed a loss of CK and E-cadherin and an increase of α-SMA and vimentin expression compared to that of the cells under 5.5 mM glucose condition(A).(D–F)These changes were prevented by exposed to 30 mM glucose+p38siRNA for 24 h(D) or 48 h(E) or+AP-1 inhibitor(F).
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    85
    Santa Cruz Biotechnology rabbit anti alpha actin
    Expression of chymase, tryptase and carboxypeptidase A were altered during tumor progression. Western blots of cell lysate from control, phases I, II and III of tumor progression. The levels of mMCP-4 are constant in the 3 phases. mMCP-5 is only expressed during tumorigenesis. The levels of mMCP-6 increase progressively during the three phases. mMCP-7 was not detected in control animals, begging to expression in phase I and remained unchanged in phases II and III. The expression of mMC-CPA is almost not detectable in controls, but increases in phases I and II, and remains unchanged in phase III. <t>Alpha-actin</t> was used as a loading control. Mean optical density of blots presenting the data of the mean values ± SEM of 3 independent experiments. *P
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    91
    Santa Cruz Biotechnology goat polyclonal anti actin
    HT-1080 cells containing the infectious HTLV-1 ACH.p30 II mutant provirus, defective for p30 II production, exhibit increased genomic and mitochondrial DNA-damage as compared to the wildtype ACH provirus. (A and B) The parental HT-1080 cell-line and the HT1080/HTLV-1 ACH.wt and ACH.p30 II mutant proviral clones were stained using a Click-iT Alexa Fluor 594-TUNEL kit (Molecular Probes; red signal) and a rabbit <t>polyclonal</t> primary antibody that recognizes the Ser139-phosphorylated histone variant H2A.X (Anti-Phospho-Ser139-H2A.X; green signal) which localizes at sites of genomic DNA-damage. The chemical uncoupler CCCP and DNAse I were included as positive controls. The cell nuclei were stained with the fluorescent dye, Hoechst 33342 (Molecular Probes; blue signal). The arrows in the enlarged inset panels at right indicate Phospho-Ser139-H2A.X foci in the merged images. The data in B represent the mean ± standard deviation (error bars) from three independent experiments. (C and D) HT-1080 cells and the HT-1080/HTLV-1 ACH.wt and ACH.p30 II mutant proviral clones were stained using a fluorescent TUNEL kit (red signal) and a rabbit polyclonal Anti-TOM20 primary antibody (green signal) to detect mitochondrial DNA-damage resulting from oxidative stress. The cell nuclei were stained with Hoechst 33342. Positive control samples were treated with either CCCP or DNAse I. The arrows in the enlarged inset panels at right indicate sites of mitochondrial DNA-damage. The graph in C shows the relative numbers of TUNEL/TOM20-positive foci per cell; and the data represent the mean ± standard deviation (error bars) from three independent experiments. (E and F) The ability of the HTLV-1 p30 II protein to protect against Tax/HBZ-induced cytotoxicity was determined by either (E) transfecting HT-1080 cells with Tax or HBZ expression constructs and then transducing them with lentiviral-HTLV-1 p30 II (HA) particles or an empty lentiviral vector, or (F) cotransfecting the cells with expression constructs for Tax, HBZ, and/or p30 II (HA). Certain samples were repeatedly cotransfected with a siRNA- tigar oligonucleotide or scrRNA control using HiPerFect transfection reagent. The cells in E were also treated with staurosporine (100 nM) as a positive control to induce apoptosis. Apoptotic cells were detected by staining them with Annexin V-FITC (green signal) and propidium iodide (PI; red signal); and the relative percentages of Annexin V-FITC +/− PI-positive cells were quantified by fluorescence-microscopy, as compared to the total numbers of cells visualized using a DIC phase-contrast filter. All the data is representative of at least three independent experiments; and the data in F represent the mean of the experiments ± standard deviation (error bars).
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    Santa Cruz Biotechnology mouse monoclonal anti beta actin
    Parasite burden and COX-2 expression in C. callosus after infection with T. gondii and treatment with COX-2 inhibitors. Females were infected orally with 50 cysts of T. gondii (ME49 strain), treated or untreated with meloxicam (0.5 mg/kg) or celecoxib (5 mg/kg) daily, euthanized after 40 days of infection and treatment, and the brains collected and analyzed by real-time PCR to quantify the parasite burden (A) or Western blotting to detect COX-2 and <t>beta-actin</t> expressions (B) . The data are shown as T. gondii DNA (TgDNA) concentration in 100 ng/μL total DNA (A) . Differences between groups were analyzed by one-way ANOVA with the Bonferroni multiple comparison post hoc test. Significant differences in relation to untreated/infected animals (control) ( ∗ P
    Mouse Monoclonal Anti Beta Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology monoclonal anti β actin antibody
    Senescence-associated gene expression in WI-38 and IDH4 fibroblasts. (A) Northern blot analysis of expression of the genes indicated using early-passage (Young; ≈28 pdl) and late-passage (senescent [Sen.]; ≈60 pdl) WI-38 cells, as well as young (dex-treated) and senescent (7 days after removing dex) IDH4 cells. (B) Stabilities of cyclin A, cyclin B1, c-fos, and <t>β-actin</t> mRNAs in IDH4 cells that were either dex-treated [Young (+dex)] or cultured without dex for 7 days [Senescent (−dex)] were assessed after the addition of 2 μg of actinomycin D/ml; preparation of RNA at the times indicated; measurement of cyclin A, cyclin B1, c-fos, and β-actin mRNA Northern blot signals; normalizing them to 18S rRNA; and plotting them on a logarithmic scale (bottom). Dashed horizontal lines, 50% of untreated. The data represent the means ± standard errors of the means of four independent experiments.
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    90
    Santa Cruz Biotechnology anti actin hrp
    Senescence-associated gene expression in WI-38 and IDH4 fibroblasts. (A) Northern blot analysis of expression of the genes indicated using early-passage (Young; ≈28 pdl) and late-passage (senescent [Sen.]; ≈60 pdl) WI-38 cells, as well as young (dex-treated) and senescent (7 days after removing dex) IDH4 cells. (B) Stabilities of cyclin A, cyclin B1, c-fos, and <t>β-actin</t> mRNAs in IDH4 cells that were either dex-treated [Young (+dex)] or cultured without dex for 7 days [Senescent (−dex)] were assessed after the addition of 2 μg of actinomycin D/ml; preparation of RNA at the times indicated; measurement of cyclin A, cyclin B1, c-fos, and β-actin mRNA Northern blot signals; normalizing them to 18S rRNA; and plotting them on a logarithmic scale (bottom). Dashed horizontal lines, 50% of untreated. The data represent the means ± standard errors of the means of four independent experiments.
    Anti Actin Hrp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pulmonary muscarinic receptors function and expression in allergic AIRmin and AIRmax mice. AIRmin or AIRmax mice were sensitized and challenged with OVA as in  Figure 4 . Control group consisted of nonmanipulated animals. The experiments were performed 24 h after the last OVA challenge. Respiratory pattern of allergic AIRmin (a and c) or AIRmax (b and d) to inhaled MCh was evaluated in the presence or absence of gallamine. Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh. Area under the curve was obtained from Penh values (c and d). Gene (e and g) or protein (f and h) expression of the M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from OVA-sensitized AIRmin and AIRmax mice. The real-time PCR was carried out using  β -actin gene expression as internal control for normalization of M3R (e) and M2R (g) mRNA transcription levels. In Western blot analysis the density of M3R (f) and M2R (h) protein expression was normalized to actin expression in each sample. Data are expressed as mean ± SEM of four mice per group and are representative of two experiments. Western blots data were quantified by densitometry using the ImageJ software (NIH). Statistical analyses of Student's  t  test for (a), (b), (e), (f), (g), and (h). Statistical analyses of ANOVA following Tukey HSD for (c) and (d). * P

    Journal: BioMed Research International

    Article Title: Role of M2 Muscarinic Receptor in the Airway Response to Methacholine of Mice Selected for Minimal or Maximal Acute Inflammatory Response

    doi: 10.1155/2013/805627

    Figure Lengend Snippet: Pulmonary muscarinic receptors function and expression in allergic AIRmin and AIRmax mice. AIRmin or AIRmax mice were sensitized and challenged with OVA as in Figure 4 . Control group consisted of nonmanipulated animals. The experiments were performed 24 h after the last OVA challenge. Respiratory pattern of allergic AIRmin (a and c) or AIRmax (b and d) to inhaled MCh was evaluated in the presence or absence of gallamine. Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh. Area under the curve was obtained from Penh values (c and d). Gene (e and g) or protein (f and h) expression of the M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from OVA-sensitized AIRmin and AIRmax mice. The real-time PCR was carried out using β -actin gene expression as internal control for normalization of M3R (e) and M2R (g) mRNA transcription levels. In Western blot analysis the density of M3R (f) and M2R (h) protein expression was normalized to actin expression in each sample. Data are expressed as mean ± SEM of four mice per group and are representative of two experiments. Western blots data were quantified by densitometry using the ImageJ software (NIH). Statistical analyses of Student's t test for (a), (b), (e), (f), (g), and (h). Statistical analyses of ANOVA following Tukey HSD for (c) and (d). * P

    Article Snippet: The following polyclonal rabbit antisera were used: anti-M1 (1 : 200), anti-M2 (1 : 100), anti-M3 (1 : 100), and anti-β -actin (1 : 400) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). β -actin protein expression was used as an internal standard for relative quantification of muscarinic receptors expression levels.

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Software

    Respiratory pattern and expression of muscarinic receptors in AIRmin and AIRmax mice. Respiratory pattern was determined in awake, unrestrained mice by noninvasive whole-body barometric plethysmography. (a) Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh (3, 6, 12, and 25 mg/mL) and (b) provocative concentration of aerosol MCh at a 200% increase (PC200) over baseline values. Gene (c and e) or protein (d and f) expression of M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from AIRmin and AIRmax mice. Real-time PCR was carried out using  β -actin gene expression as internal control for normalization of M3R (c) and M2R (e) mRNA transcription levels. All PCR reactions were quantitative reactions made by real-time PCR. In Western blot analysis the density of M3R (d) and M2R (f) protein expression was nor aerosol at a malized to actin expression in each sample. Western blots were quantified by densitometry using the ImageJ software (NIH). Real-time PCR and Western-blot analyses were performed using pooled lungs from 5 mice. Data are expressed as mean ± SEM of five mice per group and are representative of three experiments; * P

    Journal: BioMed Research International

    Article Title: Role of M2 Muscarinic Receptor in the Airway Response to Methacholine of Mice Selected for Minimal or Maximal Acute Inflammatory Response

    doi: 10.1155/2013/805627

    Figure Lengend Snippet: Respiratory pattern and expression of muscarinic receptors in AIRmin and AIRmax mice. Respiratory pattern was determined in awake, unrestrained mice by noninvasive whole-body barometric plethysmography. (a) Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh (3, 6, 12, and 25 mg/mL) and (b) provocative concentration of aerosol MCh at a 200% increase (PC200) over baseline values. Gene (c and e) or protein (d and f) expression of M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from AIRmin and AIRmax mice. Real-time PCR was carried out using β -actin gene expression as internal control for normalization of M3R (c) and M2R (e) mRNA transcription levels. All PCR reactions were quantitative reactions made by real-time PCR. In Western blot analysis the density of M3R (d) and M2R (f) protein expression was nor aerosol at a malized to actin expression in each sample. Western blots were quantified by densitometry using the ImageJ software (NIH). Real-time PCR and Western-blot analyses were performed using pooled lungs from 5 mice. Data are expressed as mean ± SEM of five mice per group and are representative of three experiments; * P

    Article Snippet: The following polyclonal rabbit antisera were used: anti-M1 (1 : 200), anti-M2 (1 : 100), anti-M3 (1 : 100), and anti-β -actin (1 : 400) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). β -actin protein expression was used as an internal standard for relative quantification of muscarinic receptors expression levels.

    Techniques: Expressing, Mouse Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction, Software

    HCV infection affects intracellular localization, expression levels, and activities of focal adhesion molecules. ( A ) Immunofluorescence of paxillin ( green ), and alpha-actinin ( red ) by confocal microscopy in Ctrl and HCV Huh7.5.1 cells after 24 hrs from plating. DAPI ( blue ) was included to stain the nuclei. Magnification bar: 30 µ. ( B ) Paxillin and alpha-actinin protein expression levels observed 24 hrs after plating. ( C ) Immunohistochemical analysis of paxillin expression in HCCs and control livers (×200 magnification). ( D ) Total FAK and tyrosine 397 phosphorylated FAK were observed 24 hrs after plating. Immunoblots are representative of at least four independent experiments. ( E ) Immunohistochemical analysis of tyrosine 397 phosphorylated FAK expression in HCCs and control livers (×200 magnification). In left histograms densitometric analysis is reported as fold changes in protein levels respect to the control cells considered as 1 after normalization against beta-actin (as loading control). *P

    Journal: PLoS ONE

    Article Title: Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells

    doi: 10.1371/journal.pone.0044147

    Figure Lengend Snippet: HCV infection affects intracellular localization, expression levels, and activities of focal adhesion molecules. ( A ) Immunofluorescence of paxillin ( green ), and alpha-actinin ( red ) by confocal microscopy in Ctrl and HCV Huh7.5.1 cells after 24 hrs from plating. DAPI ( blue ) was included to stain the nuclei. Magnification bar: 30 µ. ( B ) Paxillin and alpha-actinin protein expression levels observed 24 hrs after plating. ( C ) Immunohistochemical analysis of paxillin expression in HCCs and control livers (×200 magnification). ( D ) Total FAK and tyrosine 397 phosphorylated FAK were observed 24 hrs after plating. Immunoblots are representative of at least four independent experiments. ( E ) Immunohistochemical analysis of tyrosine 397 phosphorylated FAK expression in HCCs and control livers (×200 magnification). In left histograms densitometric analysis is reported as fold changes in protein levels respect to the control cells considered as 1 after normalization against beta-actin (as loading control). *P

    Article Snippet: Antibodies Antibodies used: anti-core protein monoclonal antibody (Affinity BioReagents, Inc., Golden, CO); anti-NS3, anti-FAK, anti-paxillin, anti-alpha-actinin, anti-alpha-smooth muscle actin, anti-beta-actin and anti-phosphotyrosine monoclonal antibodies (Santa Cruz Biotech.); peroxidase-conjugated goat anti rabbit and anti-mouse IgG (Sigma-Aldrich Inc.); FITC-conjugated anti-mouse and TRIC-conjugated anti-rabbit (Sigma-Aldrich Inc.).

    Techniques: Infection, Expressing, Immunofluorescence, Confocal Microscopy, Staining, Immunohistochemistry, Western Blot

    HCV affects proliferation, anchorage-independent growth, adhesion and migration of Huh7.5.1 cells. ( A ) RT-PCR for the 5′UTR in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). ( B ) Protein expression levels of HCV core ( upper panel ), HCV NS3 ( middle panel ) and beta-actin (loading control, lower panel ) in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). Immunoblots are representative of at least five independent experiments. ( C ) Proliferation rate was evaluated as incorporation of BrdU performed at three different time points: 0, 6 and 24 hrs. Quantitative data of the analysis of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD ( bars ) of five independent experiments. *P

    Journal: PLoS ONE

    Article Title: Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells

    doi: 10.1371/journal.pone.0044147

    Figure Lengend Snippet: HCV affects proliferation, anchorage-independent growth, adhesion and migration of Huh7.5.1 cells. ( A ) RT-PCR for the 5′UTR in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). ( B ) Protein expression levels of HCV core ( upper panel ), HCV NS3 ( middle panel ) and beta-actin (loading control, lower panel ) in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). Immunoblots are representative of at least five independent experiments. ( C ) Proliferation rate was evaluated as incorporation of BrdU performed at three different time points: 0, 6 and 24 hrs. Quantitative data of the analysis of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD ( bars ) of five independent experiments. *P

    Article Snippet: Antibodies Antibodies used: anti-core protein monoclonal antibody (Affinity BioReagents, Inc., Golden, CO); anti-NS3, anti-FAK, anti-paxillin, anti-alpha-actinin, anti-alpha-smooth muscle actin, anti-beta-actin and anti-phosphotyrosine monoclonal antibodies (Santa Cruz Biotech.); peroxidase-conjugated goat anti rabbit and anti-mouse IgG (Sigma-Aldrich Inc.); FITC-conjugated anti-mouse and TRIC-conjugated anti-rabbit (Sigma-Aldrich Inc.).

    Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Infection, Expressing, Western Blot, BrdU Incorporation Assay

    The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Combination of Berberine with Resveratrol Improves the Lipid-Lowering Efficacy

    doi: 10.3390/ijms19123903

    Figure Lengend Snippet: The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Article Snippet: Goat antibodies directed against LDLR (C-7, sc-18823), HRP-labeled anti-goat IgG (sc-2354) and anti-β-actin antibody (sc-8432) were from Santa Cruz Biotechnology (Heidelberg, German).

    Techniques: Expressing, Cell Culture, Concentration Assay, Western Blot

    Effects of MEKS on the activations of apoptosis-related proteins. MDA-MB-231 cells were treated with 0, 25, or 50 μg/ml of MEKS for 4 h or 30 nM paclitaxel for 24 h (positive control). Protein expressions of cleaved caspase 3 (C), 8 (B) and 9 (A), cleaved PARP (D) and beta-actin (the loading control) were detected by Western blotting

    Journal: Pharmacognosy Magazine

    Article Title: Anti-cancer effects of Kochia scoparia fruit in human breast cancer cells

    doi: 10.4103/0973-1296.139812

    Figure Lengend Snippet: Effects of MEKS on the activations of apoptosis-related proteins. MDA-MB-231 cells were treated with 0, 25, or 50 μg/ml of MEKS for 4 h or 30 nM paclitaxel for 24 h (positive control). Protein expressions of cleaved caspase 3 (C), 8 (B) and 9 (A), cleaved PARP (D) and beta-actin (the loading control) were detected by Western blotting

    Article Snippet: The antibodies targeting cleaved caspase 3, cleaved caspase 8, cleaved caspase 9 and cleaved Poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA, USA), while anti-beta actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Multiple Displacement Amplification, Positive Control, Western Blot

    GLO1−419C > A polymorphism genotyping, argpyrimidine and oxidative stress indices in LNCaP and PC3 cells. (A) GLO1 −419C > A homozygous wild type (CC) LNCaP and homozygous mutant type (AA) PC3 cells; (B) Argpyrimidine (AP) intracellular levels and densitometric analysis from Western blot detection. Western blot was obtained by using a mAb mouse anti-AP. The blot was stripped of the bound Ab and re-probed with mouse anti-β-actin, to confirm equal loading. The Western blot shown is representative of three separate experiments. (C) Reactive oxygen species (ROS), malondialdehyde (MDA) and reduced glutathione (GSH) intracellular levels. Histograms indicate means ± SD of three different cultures each of one was tested in quadruplicate and expressed as fold change.**P

    Journal: PLoS ONE

    Article Title: Glyoxalase 1-419C > A Variant Is Associated with Oxidative Stress: Implications in Prostate Cancer Progression

    doi: 10.1371/journal.pone.0074014

    Figure Lengend Snippet: GLO1−419C > A polymorphism genotyping, argpyrimidine and oxidative stress indices in LNCaP and PC3 cells. (A) GLO1 −419C > A homozygous wild type (CC) LNCaP and homozygous mutant type (AA) PC3 cells; (B) Argpyrimidine (AP) intracellular levels and densitometric analysis from Western blot detection. Western blot was obtained by using a mAb mouse anti-AP. The blot was stripped of the bound Ab and re-probed with mouse anti-β-actin, to confirm equal loading. The Western blot shown is representative of three separate experiments. (C) Reactive oxygen species (ROS), malondialdehyde (MDA) and reduced glutathione (GSH) intracellular levels. Histograms indicate means ± SD of three different cultures each of one was tested in quadruplicate and expressed as fold change.**P

    Article Snippet: An appropriate dilution of mouse anti-MG-AGE (Arg-Pyrimidine, AP) mAb (Antibodies-online, GmbH) and anti-β-actin mAb (Santa Cruz Biotechnology) were used.

    Techniques: Mutagenesis, Western Blot, Multiple Displacement Amplification

    Effect of PQQ on antioxidant proteins of tibiae in Bmi1 -/- mice. (A) Representative tibiae western blots for expression of BCL-2, SOD1, SOD2, prdx I, pxdx IV. β-actin was used as loading control for western blot in WT mice, BKO mice and BKO+PQQ mice respectively. BCL-2 (B), SOD1 (C), SOD2 (D), prdx I (E), prdx IV (F) proteins levels relative to β-actin protein levels were assessed by densitometric analysis and expressed relative to levels of WT mice. Each value is the mean ± SEM of determinations in six animals of the same groups. *, P

    Journal: American Journal of Translational Research

    Article Title: Effect and mechanism of pyrroloquinoline quinone on anti-osteoporosis in Bmi-1 knockout mice-Anti-oxidant effect of pyrroloquinoline quinone

    doi:

    Figure Lengend Snippet: Effect of PQQ on antioxidant proteins of tibiae in Bmi1 -/- mice. (A) Representative tibiae western blots for expression of BCL-2, SOD1, SOD2, prdx I, pxdx IV. β-actin was used as loading control for western blot in WT mice, BKO mice and BKO+PQQ mice respectively. BCL-2 (B), SOD1 (C), SOD2 (D), prdx I (E), prdx IV (F) proteins levels relative to β-actin protein levels were assessed by densitometric analysis and expressed relative to levels of WT mice. Each value is the mean ± SEM of determinations in six animals of the same groups. *, P

    Article Snippet: Western blot was carried out as described previously (28) using antibodies against P16 (goat anti-mouse, M-156, Santa Cruz Biotechnology, USA), P19 (goat anti-mouse, Santa Cruz Biotechnology, USA), P21 (goat anti-mouse, M19, Santa Cruz Biotechnology, USA), P27 (goat anti-mouse, Zymed Laboratories, Santa Cruz, CA, USA), P53 (goat anti-mouse, Cell Signal, China), SOD1 and SOD2 (goat anti-rabbit, Abcam, UK), prdx I (goat anti-rabbit, Abcam, UK), prdx IV (goat anti-rabbit, Abcam, UK), Caspase-3 (goat anti-rabbit, Cell Signal, China), γH2AX (goat anti-mouse, Santa Cruz, CA, USA), and β-actin (goat anti-rabbit, Santa Cruz Biotechnology, USA).

    Techniques: Mouse Assay, Western Blot, Expressing

    Effect of PQQ on DNA damage and cell apoptosis of tibiae in Bmi1 -/- mice. (A) Representative tibiae western blot for expression of γH2AX and Caspase-3. β-actin was used as loading control for western blot in WT mice, BKO mice and BKO+PQQ mice respectively; γH2AX (B) and Caspase-3 (C) proteins levels relative to β-actin protein levels were assessed by densitometric analysis and expressed relative to levels of WT mice. Each value is the mean ± SEM of determinations in six animals of the same groups. *, P

    Journal: American Journal of Translational Research

    Article Title: Effect and mechanism of pyrroloquinoline quinone on anti-osteoporosis in Bmi-1 knockout mice-Anti-oxidant effect of pyrroloquinoline quinone

    doi:

    Figure Lengend Snippet: Effect of PQQ on DNA damage and cell apoptosis of tibiae in Bmi1 -/- mice. (A) Representative tibiae western blot for expression of γH2AX and Caspase-3. β-actin was used as loading control for western blot in WT mice, BKO mice and BKO+PQQ mice respectively; γH2AX (B) and Caspase-3 (C) proteins levels relative to β-actin protein levels were assessed by densitometric analysis and expressed relative to levels of WT mice. Each value is the mean ± SEM of determinations in six animals of the same groups. *, P

    Article Snippet: Western blot was carried out as described previously (28) using antibodies against P16 (goat anti-mouse, M-156, Santa Cruz Biotechnology, USA), P19 (goat anti-mouse, Santa Cruz Biotechnology, USA), P21 (goat anti-mouse, M19, Santa Cruz Biotechnology, USA), P27 (goat anti-mouse, Zymed Laboratories, Santa Cruz, CA, USA), P53 (goat anti-mouse, Cell Signal, China), SOD1 and SOD2 (goat anti-rabbit, Abcam, UK), prdx I (goat anti-rabbit, Abcam, UK), prdx IV (goat anti-rabbit, Abcam, UK), Caspase-3 (goat anti-rabbit, Cell Signal, China), γH2AX (goat anti-mouse, Santa Cruz, CA, USA), and β-actin (goat anti-rabbit, Santa Cruz Biotechnology, USA).

    Techniques: Mouse Assay, Western Blot, Expressing

    Effect of PQQ on cell cycle proteins of tibiae in Bmi1 -/- mice. (A) Representative tibiae western blot for expression of P16, P19, P21, P27 and P53. β-actin was used as loading control for western blot in WT mice, BKO mice and BKO+PQQ mice respectively. P16 (B), P19 (C), P21 (D), P27 (E) and P53 (F) proteins levels relative to β-actin protein levels were assessed by densitometric analysis and expressed relative to levels of WT mice. Each value is the mean ± SEM of determinations in six animals of the same groups. *, P

    Journal: American Journal of Translational Research

    Article Title: Effect and mechanism of pyrroloquinoline quinone on anti-osteoporosis in Bmi-1 knockout mice-Anti-oxidant effect of pyrroloquinoline quinone

    doi:

    Figure Lengend Snippet: Effect of PQQ on cell cycle proteins of tibiae in Bmi1 -/- mice. (A) Representative tibiae western blot for expression of P16, P19, P21, P27 and P53. β-actin was used as loading control for western blot in WT mice, BKO mice and BKO+PQQ mice respectively. P16 (B), P19 (C), P21 (D), P27 (E) and P53 (F) proteins levels relative to β-actin protein levels were assessed by densitometric analysis and expressed relative to levels of WT mice. Each value is the mean ± SEM of determinations in six animals of the same groups. *, P

    Article Snippet: Western blot was carried out as described previously (28) using antibodies against P16 (goat anti-mouse, M-156, Santa Cruz Biotechnology, USA), P19 (goat anti-mouse, Santa Cruz Biotechnology, USA), P21 (goat anti-mouse, M19, Santa Cruz Biotechnology, USA), P27 (goat anti-mouse, Zymed Laboratories, Santa Cruz, CA, USA), P53 (goat anti-mouse, Cell Signal, China), SOD1 and SOD2 (goat anti-rabbit, Abcam, UK), prdx I (goat anti-rabbit, Abcam, UK), prdx IV (goat anti-rabbit, Abcam, UK), Caspase-3 (goat anti-rabbit, Cell Signal, China), γH2AX (goat anti-mouse, Santa Cruz, CA, USA), and β-actin (goat anti-rabbit, Santa Cruz Biotechnology, USA).

    Techniques: Mouse Assay, Western Blot, Expressing

    Loss of LIN28A/B attenuates mTOR signaling and limits supporting cell proliferation and hair cell formation in cochlear organoid culture. Cochlear organoid cultures were established from UBC-CreERT2; Lin28a f/f ; Lin28b f/f mice and Lin28a f/f ; Lin28b f/f littermates’ stage P2. Cultures received 4-hydroxytamoxifen (TM) or vehicle control DMSO at plating. SOX2 (green) marks supporting cells/ pro-sensory cells, Hoechst (blue) staining marks cell nuclei. Bars in (D) and (G) represent mean± SD, otherwise individual data points and their mean ± SD were plotted. (A-G) Loss of Lin28a/b inhibits cell proliferation and hair cell production in organoid culture. ( A ) Representative BF images of control organoids and Lin28a/b dKO organoids at 7 days of expansion. ( B ) Diameter of control and Lin28a/b dKO organoids in ( A ) (n=7 animals per group, from 2 independent experiments). ( C ) Organoid forming efficiency in control and Lin28a/b dKO cultures (n=7 animals per group, from 2 independent experiments). ( D ) RT-qPCR analysis of Lin28a, Lin28b and Hmga2 mRNA expression in Lin28a/b dKO organoids (red bar) compared to control organoids (blue bar) at 7 days of expansion (n=5-6 animals per group, from 2 independent experiments). ( E ) Cell proliferation in control and Lin28a/b dKO organoids. A single EdU pulse was given at 7 days of expansion and EdU incorporation (red) was analyzed 1 hour later. ( F ) EdU incorporation in ( E ) (n=7 animals per group, from 2 independent experiments). ( G ) RT-qPCR analysis of Atoh1, Myo7a and Pou4f3 mRNA expression in Lin28a/b dKO organoids (red bar) compared to control organoids (blue bar) at 10 days of differentiation (n=4 animals per group, from 2 independent experiments). (H) Immunoblots for LIN28B, p-Akt, p-S6 and β-actin using protein lysates of acutely isolated control and Lin28a/b dKO cochlear epithelia, stage P5. ( I - L ) Loss of Lin28a/b attenuates mTOR signaling in cochlear organoids. ( I ) Immunostaining for p-S6 protein (red) in control and Lin28a/b dKO organoids at 7 days of expansion. ( J ) Percentage of p-S6 + cells in (I) (n=5 animals per group, from 2 independent experiments). ( K ) Immunoblots for p-Akt, p-4EBP1, 4EBP1, mLIN28B and β-actin (loading control) using protein lysates of control and Lin28a/b dKO organoids after 7 days of expansion. ( L ) Normalized p-Akt, p-4EBP1 and m-LIN28B protein levels in control and Lin28a/b dKO organoids in ( K ) (n=3 animals per group, from 1 representative experiment, 2 independent experiments). 2-way ANOVA with Tukey’s correction was used to calculate p-values in ( B ), ( C ) and ( J ). 2-tailed, unpaired Student’s t-test was used to calculate p-values in ( D ), ( F ), ( G ) and ( L ). Note that the individual data points in ( B ), ( F ) and ( J ) represent the average values per animal.

    Journal: bioRxiv

    Article Title: LIN28B controls the regenerative capacity of neonatal murine auditory supporting cells through activation of mTOR signaling

    doi: 10.1101/2020.05.31.126193

    Figure Lengend Snippet: Loss of LIN28A/B attenuates mTOR signaling and limits supporting cell proliferation and hair cell formation in cochlear organoid culture. Cochlear organoid cultures were established from UBC-CreERT2; Lin28a f/f ; Lin28b f/f mice and Lin28a f/f ; Lin28b f/f littermates’ stage P2. Cultures received 4-hydroxytamoxifen (TM) or vehicle control DMSO at plating. SOX2 (green) marks supporting cells/ pro-sensory cells, Hoechst (blue) staining marks cell nuclei. Bars in (D) and (G) represent mean± SD, otherwise individual data points and their mean ± SD were plotted. (A-G) Loss of Lin28a/b inhibits cell proliferation and hair cell production in organoid culture. ( A ) Representative BF images of control organoids and Lin28a/b dKO organoids at 7 days of expansion. ( B ) Diameter of control and Lin28a/b dKO organoids in ( A ) (n=7 animals per group, from 2 independent experiments). ( C ) Organoid forming efficiency in control and Lin28a/b dKO cultures (n=7 animals per group, from 2 independent experiments). ( D ) RT-qPCR analysis of Lin28a, Lin28b and Hmga2 mRNA expression in Lin28a/b dKO organoids (red bar) compared to control organoids (blue bar) at 7 days of expansion (n=5-6 animals per group, from 2 independent experiments). ( E ) Cell proliferation in control and Lin28a/b dKO organoids. A single EdU pulse was given at 7 days of expansion and EdU incorporation (red) was analyzed 1 hour later. ( F ) EdU incorporation in ( E ) (n=7 animals per group, from 2 independent experiments). ( G ) RT-qPCR analysis of Atoh1, Myo7a and Pou4f3 mRNA expression in Lin28a/b dKO organoids (red bar) compared to control organoids (blue bar) at 10 days of differentiation (n=4 animals per group, from 2 independent experiments). (H) Immunoblots for LIN28B, p-Akt, p-S6 and β-actin using protein lysates of acutely isolated control and Lin28a/b dKO cochlear epithelia, stage P5. ( I - L ) Loss of Lin28a/b attenuates mTOR signaling in cochlear organoids. ( I ) Immunostaining for p-S6 protein (red) in control and Lin28a/b dKO organoids at 7 days of expansion. ( J ) Percentage of p-S6 + cells in (I) (n=5 animals per group, from 2 independent experiments). ( K ) Immunoblots for p-Akt, p-4EBP1, 4EBP1, mLIN28B and β-actin (loading control) using protein lysates of control and Lin28a/b dKO organoids after 7 days of expansion. ( L ) Normalized p-Akt, p-4EBP1 and m-LIN28B protein levels in control and Lin28a/b dKO organoids in ( K ) (n=3 animals per group, from 1 representative experiment, 2 independent experiments). 2-way ANOVA with Tukey’s correction was used to calculate p-values in ( B ), ( C ) and ( J ). 2-tailed, unpaired Student’s t-test was used to calculate p-values in ( D ), ( F ), ( G ) and ( L ). Note that the individual data points in ( B ), ( F ) and ( J ) represent the average values per animal.

    Article Snippet: The following primary antibodies were used: rabbit anti-Akt (Cell Signaling, no. 4691, 1:1000), rabbit anti-p-Akt (Ser473) (Cell Signaling, no. 4060, 1:1000), rabbit anti-p-4E-BP1 (Thr37/46) (Cell Signaling, no. 2855, 1:1000), rabbit anti-4E-BP1 (Cell Signaling, no. 9644, 1:2000), rabbit anti-human LIN28B (Cell Signaling, no. 4196, 1:2000), rabbit anti-mouse LIN28B (Cell Signaling, no. 5422, 1:500), rabbit anti-p-S6 (Ser240/244) (Cell Signaling, no. 5364, 1:500), mouse anti-β-actin (Santa Cruz Biotechnology, no. sc-47778, 1:500).

    Techniques: Mouse Assay, Staining, Quantitative RT-PCR, Expressing, Western Blot, Isolation, Immunostaining

    DXR treatment increases the levels of HMGB1 in osteosarcoma cells. (A) MG63 cells were treated with 0.1, 0.5 or 1.0 µg/ml DXR for 24 h, and the expression of HMGB1 was detected by western blot analysis. (B) MG63 cells were treated with 0.5 µg/ml DXR for 24, 48 and 72 h, and HMGB1 expression was again evaluated by western blotting. β-actin was used as an internal reference gene. DXR, doxorubicin; HMGB1, high-mobility group protein 1.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Necrosis of osteosarcoma cells induces the production and release of high-mobility group box 1 protein

    doi: 10.3892/etm.2017.5415

    Figure Lengend Snippet: DXR treatment increases the levels of HMGB1 in osteosarcoma cells. (A) MG63 cells were treated with 0.1, 0.5 or 1.0 µg/ml DXR for 24 h, and the expression of HMGB1 was detected by western blot analysis. (B) MG63 cells were treated with 0.5 µg/ml DXR for 24, 48 and 72 h, and HMGB1 expression was again evaluated by western blotting. β-actin was used as an internal reference gene. DXR, doxorubicin; HMGB1, high-mobility group protein 1.

    Article Snippet: The primary antibodies used were mouse monoclonal anti-HMGB1 (cat. no. ab77302; Abcam, Cambridge, UK), mouse monoclonal anti-β-actin (C-2, cat. no. sc-8432; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), while the secondary antibody was goat anti-mouse immunoglobulin G-horseradish peroxidase (cat. no. sc-2005; Santa Cruz Biotechnology, Inc.,).

    Techniques: Expressing, Western Blot

    Soluble ESAT-6:CFP-10 reduces surface levels of β2M-associated HLA class I molecules. (A) PMA-differentiated THP-1 macrophages were treated with 12.5 µM of either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein for 2 hours. Cells were stained with (W6/32) mAb followed by FITC conjugated anti-mouse secondary Ab. Expression of surface β2M conjugated HLA class I molecules was studied by flow cytometry. Isotype-matched Ab was used as control. (B) Median fluorescence intensities of different experimental groups of Figure 7A were calculated and the results are shown as mean ± SD of 3 different experiments. (C) THP-1 macrophages were either left untreated (control) or treated with 12.5 µM of ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10. After 2 hours, cell were harvested and lysates were prepared. Equal amount of protein from each experimental group was incubated with W6/32 mAb bound to protein A/G agarose. Isotype matched Ab was used as control. Pulled-down complexes (Lanes 5–8) were resolved on a 15% glycine SDS-PAGE and transferred onto a nitrocellulose membrane which was probed with anti-β2M Ab. About 10% of the lysate was used as input controls (Lanes 1–4, upper panel). Equal loading in the input samples was also confirmed by probing the input controls with anti-β-actin Ab (Lanes 1–4, lower panel).

    Journal: PLoS Pathogens

    Article Title: The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

    doi: 10.1371/journal.ppat.1004446

    Figure Lengend Snippet: Soluble ESAT-6:CFP-10 reduces surface levels of β2M-associated HLA class I molecules. (A) PMA-differentiated THP-1 macrophages were treated with 12.5 µM of either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein for 2 hours. Cells were stained with (W6/32) mAb followed by FITC conjugated anti-mouse secondary Ab. Expression of surface β2M conjugated HLA class I molecules was studied by flow cytometry. Isotype-matched Ab was used as control. (B) Median fluorescence intensities of different experimental groups of Figure 7A were calculated and the results are shown as mean ± SD of 3 different experiments. (C) THP-1 macrophages were either left untreated (control) or treated with 12.5 µM of ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10. After 2 hours, cell were harvested and lysates were prepared. Equal amount of protein from each experimental group was incubated with W6/32 mAb bound to protein A/G agarose. Isotype matched Ab was used as control. Pulled-down complexes (Lanes 5–8) were resolved on a 15% glycine SDS-PAGE and transferred onto a nitrocellulose membrane which was probed with anti-β2M Ab. About 10% of the lysate was used as input controls (Lanes 1–4, upper panel). Equal loading in the input samples was also confirmed by probing the input controls with anti-β-actin Ab (Lanes 1–4, lower panel).

    Article Snippet: Recombinant His-tagged proteins were detected with rabbit anti-His Ab (Abcam Inc, USA), β2M with rabbit anti-β2M Ab (Abcam Inc, USA) and β-actin with mouse anti-β-actin Ab (Santa Cruz Biotechnology, USA).

    Techniques: Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence, Incubation, SDS Page

    Skeletal muscle atrophy is ameliorated in the SMAΔ7/ Sam68 −/− mice. (A) Hematoxylin and eosin staining of triceps muscle sections from 10-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Bar, 100 µm. (B) Frequency distribution of muscle fiber areas (at least 2,000 for each genotype) of the experimental animals described in A. The colored lines and dotted line (non-SMA mice) represent the median value for each genotype: non-SMA mice ( n = 3) median size = 531 µm 2 and fiber size (mean ± SEM) = 568.76 ± 5.8 µm 2 ; non-SMA/ Sam68 −/− mice ( n = 3) median size = 557 µm 2 and fiber size (mean ± SEM) = 586.60 ± 5.7 µm 2 ; SMAΔ7/ Sam68 +/+ median size = 327 µm 2 and fiber size (mean ± SEM) = 342.32 ± 3.1 µm 2 ; and SMAΔ7/ Sam68 −/− median size = 486 µm 2 and fiber size (mean ± SEM) = 503.59 ± 4.6 µm 2 . Statistical analysis of the median values among groups was performed by one-way ANOVA test followed by Dunn’s multiple comparison posttest. (C) qPCR analysis of the expression of atrophy-related genes in quadriceps and lower leg muscles of mice described in B. Values (mean ± SD; n = 4) were normalized with Actin mRNA. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest. *, P

    Journal: The Journal of Cell Biology

    Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

    doi: 10.1083/jcb.201502059

    Figure Lengend Snippet: Skeletal muscle atrophy is ameliorated in the SMAΔ7/ Sam68 −/− mice. (A) Hematoxylin and eosin staining of triceps muscle sections from 10-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Bar, 100 µm. (B) Frequency distribution of muscle fiber areas (at least 2,000 for each genotype) of the experimental animals described in A. The colored lines and dotted line (non-SMA mice) represent the median value for each genotype: non-SMA mice ( n = 3) median size = 531 µm 2 and fiber size (mean ± SEM) = 568.76 ± 5.8 µm 2 ; non-SMA/ Sam68 −/− mice ( n = 3) median size = 557 µm 2 and fiber size (mean ± SEM) = 586.60 ± 5.7 µm 2 ; SMAΔ7/ Sam68 +/+ median size = 327 µm 2 and fiber size (mean ± SEM) = 342.32 ± 3.1 µm 2 ; and SMAΔ7/ Sam68 −/− median size = 486 µm 2 and fiber size (mean ± SEM) = 503.59 ± 4.6 µm 2 . Statistical analysis of the median values among groups was performed by one-way ANOVA test followed by Dunn’s multiple comparison posttest. (C) qPCR analysis of the expression of atrophy-related genes in quadriceps and lower leg muscles of mice described in B. Values (mean ± SD; n = 4) were normalized with Actin mRNA. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest. *, P

    Article Snippet: The primary antibodies (1:1,000) were rabbit anti-SAM68, mouse anti-ACTIN, mouse anti-GAPDH (all from Santa Cruz Biotechnology, Inc.), mouse anti-U2AF65 (Sigma-Aldrich), and mouse anti-SMN (BD).

    Techniques: Mouse Assay, Staining, Real-time Polymerase Chain Reaction, Expressing

    Sam68 deletion rescues SMN2 splicing and expression in SMAΔ7 tissues. (A) qPCR analysis of exon 7–containing SMN2 transgene transcripts normalized to constant exon 6 in tissues of 10-dpp SMAΔ7/ Sam68 +/+ and SMAΔ7/ Sam68 −/− mice. Bar graph represents mean ± SD. n = 3. The p-value was determined by two-tailed t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) Western blot analysis of SMN protein expression in the indicated tissues of 10-dpp SMAΔ7/ Sam68 +/+ (wt) or SMAΔ7/ Sam68 −/− (ko) mice. Actin was used as a loading control. Quantification of the SMN band is shown at the bottom. Each point value represents the mean ± SD. n = 2. Cereb, cerebellum; ko, knockout; S.cord, spinal cord; wt, wild type.

    Journal: The Journal of Cell Biology

    Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

    doi: 10.1083/jcb.201502059

    Figure Lengend Snippet: Sam68 deletion rescues SMN2 splicing and expression in SMAΔ7 tissues. (A) qPCR analysis of exon 7–containing SMN2 transgene transcripts normalized to constant exon 6 in tissues of 10-dpp SMAΔ7/ Sam68 +/+ and SMAΔ7/ Sam68 −/− mice. Bar graph represents mean ± SD. n = 3. The p-value was determined by two-tailed t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) Western blot analysis of SMN protein expression in the indicated tissues of 10-dpp SMAΔ7/ Sam68 +/+ (wt) or SMAΔ7/ Sam68 −/− (ko) mice. Actin was used as a loading control. Quantification of the SMN band is shown at the bottom. Each point value represents the mean ± SD. n = 2. Cereb, cerebellum; ko, knockout; S.cord, spinal cord; wt, wild type.

    Article Snippet: The primary antibodies (1:1,000) were rabbit anti-SAM68, mouse anti-ACTIN, mouse anti-GAPDH (all from Santa Cruz Biotechnology, Inc.), mouse anti-U2AF65 (Sigma-Aldrich), and mouse anti-SMN (BD).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Two Tailed Test, Western Blot, Knock-Out

    ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 β−Actin copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value

    Journal: Scientific Reports

    Article Title: An allelic variant in the intergenic region between ERAP1 and ERAP2 correlates with an inverse expression of the two genes

    doi: 10.1038/s41598-018-28799-8

    Figure Lengend Snippet: ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 β−Actin copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value

    Article Snippet: The membranes were incubated ON with mouse anti-ERAP1 mAb antibody (clone B-10, sc-271823 SantaCruz), mouse anti-ERAP2 mAb (clone 3F5, MAB 3830 R & D Systems) and mouse anti-β-Actin mAb (clone C4, sc-477778 SantaCruz).

    Techniques: Inhibition

    Akt activity plays an important role in FN expression and TamR cell growth. (A) TamS cells were transfected with adenoviral vectors and CA-Akt for 24 h and then further incubated in serum-free medium for 24 h. (B) TamR cells were treated with 0.5 μM AKT IV for 24 h. (C) After serum starvation for 24 h, MDA-MB231 cells were treated with or without 1 μM AKT IV for 24 h under serum-free conditions. The protein levels of FN, p-Akt, and β-actin were analyzed by Western blotting. (D) TamR cells were seeded on 6-well soft agar plates with or without 1 μM AKT IV and incubated for 2 weeks. After 2 weeks, viable colonies were stained with 0.01% crystal violet. (E) TamR cells were treated with or without 1 μM AKT IV for the indicated time periods, after which cells were counted using a Countess TM Automated Cell Counter. (F) Schematic model of this study. Results are representative of three independent experiments. Data are presented as means ± SEMs. *P

    Journal: BMB Reports

    Article Title: Fibronectin expression is upregulated by PI-3K/Akt activation in tamoxifen-resistant breast cancer cells

    doi: 10.5483/BMBRep.2017.50.12.096

    Figure Lengend Snippet: Akt activity plays an important role in FN expression and TamR cell growth. (A) TamS cells were transfected with adenoviral vectors and CA-Akt for 24 h and then further incubated in serum-free medium for 24 h. (B) TamR cells were treated with 0.5 μM AKT IV for 24 h. (C) After serum starvation for 24 h, MDA-MB231 cells were treated with or without 1 μM AKT IV for 24 h under serum-free conditions. The protein levels of FN, p-Akt, and β-actin were analyzed by Western blotting. (D) TamR cells were seeded on 6-well soft agar plates with or without 1 μM AKT IV and incubated for 2 weeks. After 2 weeks, viable colonies were stained with 0.01% crystal violet. (E) TamR cells were treated with or without 1 μM AKT IV for the indicated time periods, after which cells were counted using a Countess TM Automated Cell Counter. (F) Schematic model of this study. Results are representative of three independent experiments. Data are presented as means ± SEMs. *P

    Article Snippet: AKT IV, secondary HRP-conjugated antibodies, and mouse monoclonal anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Activity Assay, Expressing, Transfection, Incubation, Multiple Displacement Amplification, Western Blot, Staining

    Akt activity is increased in tamoxifen-resistant breast cancer cells. (A) The levels of p- and t-Akt, JNK, and STAT3 were analyzed by Western blotting. (B, C) After serum starvation, MCF7 cells were treated with 10 nM PMA for the indicated times. (C) FN and β-actin expression in cell culture media and whole cell lysates were analyzed via Western blotting. Results are representative of three independent experiments. Con, Control.

    Journal: BMB Reports

    Article Title: Fibronectin expression is upregulated by PI-3K/Akt activation in tamoxifen-resistant breast cancer cells

    doi: 10.5483/BMBRep.2017.50.12.096

    Figure Lengend Snippet: Akt activity is increased in tamoxifen-resistant breast cancer cells. (A) The levels of p- and t-Akt, JNK, and STAT3 were analyzed by Western blotting. (B, C) After serum starvation, MCF7 cells were treated with 10 nM PMA for the indicated times. (C) FN and β-actin expression in cell culture media and whole cell lysates were analyzed via Western blotting. Results are representative of three independent experiments. Con, Control.

    Article Snippet: AKT IV, secondary HRP-conjugated antibodies, and mouse monoclonal anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Activity Assay, Western Blot, Expressing, Cell Culture

    Polyubiquitinated p53 species detected in U2OS cells titrated with LMP1 . Western blot detection of transfected LMP1, endogenous p53 protein levels and detection of ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP1 (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increased polyubiquitinated p53 protein was seen as high molecular weight aggregates in LMP1 expressing cells.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: Polyubiquitinated p53 species detected in U2OS cells titrated with LMP1 . Western blot detection of transfected LMP1, endogenous p53 protein levels and detection of ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP1 (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increased polyubiquitinated p53 protein was seen as high molecular weight aggregates in LMP1 expressing cells.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Western Blot, Transfection, Immunoprecipitation, Molecular Weight, Expressing

    Dominant-negative Akt (DN-Akt) does not restore p53 protein levels . Western blot detection of LMP1, Dominant-negative Akt (DN-Akt) and p53 protein levels in U2OS cells transfected with fixed amount of LMP1 (0.5 μg) and titrated with DN-Akt (0.05 - 4.0 μg). β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increasing amounts of DN-Akt (detected by anti-Akt antibodies) did not rescue reduction of p53 protein levels by LMP1.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: Dominant-negative Akt (DN-Akt) does not restore p53 protein levels . Western blot detection of LMP1, Dominant-negative Akt (DN-Akt) and p53 protein levels in U2OS cells transfected with fixed amount of LMP1 (0.5 μg) and titrated with DN-Akt (0.05 - 4.0 μg). β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increasing amounts of DN-Akt (detected by anti-Akt antibodies) did not rescue reduction of p53 protein levels by LMP1.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Dominant Negative Mutation, Western Blot, Transfection

    LMP1, but not LMP2A or EBNA1, reduces p53 protein levels . (A) Western blot analysis of the effects of LMP1, LMP2A and EBNA1 on the expression levels of endogenous p53 protein in U2OS cells. Transfection of LMP1 reduced the level of p53 protein. (B) Co-transfection of LMP1 abolished p53 protein level in HONE1 NPC cells transiently transfected with p53. A slight reduction in the exogenous p53 protein level in LMP2A co-transfected in HONE1 NPC cells setting but not in U2OS cells in (A). ( C ) EBV-negative HONE1 and EBV-positive HONE Akata (HA) cells were transfected with p53 construct. p53 protein levels were determined in the Actinomycin-D induced and uninduced state. EBV-positive HA NPC cells have lower levels of p53 protein in comparison with EBV-negative HONE NPC cells. β-actin was served as a loading control in all the three experiments. Representative blots were quantitated using Image J.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: LMP1, but not LMP2A or EBNA1, reduces p53 protein levels . (A) Western blot analysis of the effects of LMP1, LMP2A and EBNA1 on the expression levels of endogenous p53 protein in U2OS cells. Transfection of LMP1 reduced the level of p53 protein. (B) Co-transfection of LMP1 abolished p53 protein level in HONE1 NPC cells transiently transfected with p53. A slight reduction in the exogenous p53 protein level in LMP2A co-transfected in HONE1 NPC cells setting but not in U2OS cells in (A). ( C ) EBV-negative HONE1 and EBV-positive HONE Akata (HA) cells were transfected with p53 construct. p53 protein levels were determined in the Actinomycin-D induced and uninduced state. EBV-positive HA NPC cells have lower levels of p53 protein in comparison with EBV-negative HONE NPC cells. β-actin was served as a loading control in all the three experiments. Representative blots were quantitated using Image J.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Western Blot, Expressing, Transfection, Cotransfection, Construct

    Only monoubiquitinated p53 species detected in U2OS titrated with LMP2A . Western blot detection of transfected LMP2A-HA, endogenous p53 protein levels and ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP2A-HA (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. No polyubiquitinated p53 protein was seen in LMP2A expressing cells.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: Only monoubiquitinated p53 species detected in U2OS titrated with LMP2A . Western blot detection of transfected LMP2A-HA, endogenous p53 protein levels and ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP2A-HA (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. No polyubiquitinated p53 protein was seen in LMP2A expressing cells.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Western Blot, Transfection, Immunoprecipitation, Expressing

    PI3K-Akt inhibitor LY294002 does not restore p53 protein levels . (A) Western blot analysis of the effects of PI3K inhibitor LY294002 on the endogenous p53 protein levels in U2OS cells. The cells were treated overnight, with 25 μM LY294002 or vehicle alone (DMSO). Treatment by LY294002 did not rescue the reduction of p53 protein levels by LMP1. (B) Western blot analysis of the effects of PI3K inhibitor LY294002 on the transfected p53 protein levels in CNE1-LMP1 stable cell line. The cells were treated overnight with 25 μM LY294002 or vehicle alone (DMSO). β-actin was served as a loading control in both the experiments. Representative blots were quantitated using Image J. Treatment with LY294002 did not rescue the reduction of p53 protein levels by LMP1.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: PI3K-Akt inhibitor LY294002 does not restore p53 protein levels . (A) Western blot analysis of the effects of PI3K inhibitor LY294002 on the endogenous p53 protein levels in U2OS cells. The cells were treated overnight, with 25 μM LY294002 or vehicle alone (DMSO). Treatment by LY294002 did not rescue the reduction of p53 protein levels by LMP1. (B) Western blot analysis of the effects of PI3K inhibitor LY294002 on the transfected p53 protein levels in CNE1-LMP1 stable cell line. The cells were treated overnight with 25 μM LY294002 or vehicle alone (DMSO). β-actin was served as a loading control in both the experiments. Representative blots were quantitated using Image J. Treatment with LY294002 did not rescue the reduction of p53 protein levels by LMP1.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Western Blot, Transfection, Stable Transfection

    Nucleocytoplasmic Distribution of SpYY1 in S. purpuratus ova. Ova of S. purpuratus were fractionated into nuclear and cytoplasmic extracts using previously described methods. Whole-cell (W) as well as nuclear (N) and cytoplasmic (C) fractions were analyzed by SDS-PAGE followed by Western blotting. Antibodies used are indicated to the left of the panel, αYY1, Rabbit anti- Xenopus YY1; αH3, Goat anti-Histone H3 antibody, nuclear marker; αActin, Goat anti-Beta-Actin antibody, cytoplasmic marker.

    Journal: Scientific Reports

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus

    doi: 10.1038/s41598-018-26264-0

    Figure Lengend Snippet: Nucleocytoplasmic Distribution of SpYY1 in S. purpuratus ova. Ova of S. purpuratus were fractionated into nuclear and cytoplasmic extracts using previously described methods. Whole-cell (W) as well as nuclear (N) and cytoplasmic (C) fractions were analyzed by SDS-PAGE followed by Western blotting. Antibodies used are indicated to the left of the panel, αYY1, Rabbit anti- Xenopus YY1; αH3, Goat anti-Histone H3 antibody, nuclear marker; αActin, Goat anti-Beta-Actin antibody, cytoplasmic marker.

    Article Snippet: Other antibodies used were as follows: Rabbit anti-HIS6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat. #SC-803) at 1:5000; mouse anti-HA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat. #SC-7392) at 1:5000; rabbit anti-IκB-α (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat. #SC-847) at 1:1000; mouse anti-PCNA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat. #SC-56) at 1:5000; goat anti-Histone H3 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat. #SC-8654) at 1:1000, goat anti-beta-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat. #SC-1615) at 1:1000, goat anti-rabbit HRP conjugate (Bio-Rad Inc., Mississauga, Ontario, Canada, Cat. #170–6515) at 1:5000; mouse anti-goat HRP conjugate (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat. #SC-2354) at 1:5000, and goat anti-mouse HRP conjugate (Bio-Rad Inc., Mississauga, Ontario, Canada, Cat. #170-6516) at 1:5000.

    Techniques: SDS Page, Western Blot, Marker

    Mice fed high-fat diet show steatosis and inflammation of the liver, and impairment of the autophagic process. A: The body weights of mice at the start of feeding or at 12 wk after feeding with normal chow (Cont.) or high-fat diet (HFD) are presented in the left panel. The serum ALT levels after 12 wk of feeding are shown in the right panel; B: Histology of the liver of control and HFD mice is shown in the left and the right panel, respectively (Hematoxylin and Eosin staining); C: Non-alcoholic steatohepatitis activity scores (NAS) of control and HFD mice; D: Immunoblotting analyses of phosphorylated JNK, p62, LC3, Rubicon, and β-actin. Protein samples were prepared from the liver tissue of each of the control and HFD mice. All of the above experiments were repeated three times and representative results are shown. The quantitative data are presented as the means ± SD.

    Journal: World Journal of Gastroenterology

    Article Title: c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

    doi: 10.3748/wjg.v22.i28.6509

    Figure Lengend Snippet: Mice fed high-fat diet show steatosis and inflammation of the liver, and impairment of the autophagic process. A: The body weights of mice at the start of feeding or at 12 wk after feeding with normal chow (Cont.) or high-fat diet (HFD) are presented in the left panel. The serum ALT levels after 12 wk of feeding are shown in the right panel; B: Histology of the liver of control and HFD mice is shown in the left and the right panel, respectively (Hematoxylin and Eosin staining); C: Non-alcoholic steatohepatitis activity scores (NAS) of control and HFD mice; D: Immunoblotting analyses of phosphorylated JNK, p62, LC3, Rubicon, and β-actin. Protein samples were prepared from the liver tissue of each of the control and HFD mice. All of the above experiments were repeated three times and representative results are shown. The quantitative data are presented as the means ± SD.

    Article Snippet: Antibodies and reagents: The antibodies used in this study were obtained from the following sources: anti-p62 (1:1000; #5114), anti-LC3 (1:1000; #4108), anti-Rubicon (1:1000; #8465), anti-cleaved caspase-3 (1:1000; #9661), and rabbit anti-phospho-JNK (1:1000; #9251) were obtained from Cell Signaling Technology, Tokyo, Japan; mouse anti-C/EBP homologous protein (CHOP) (1:500; sc-575), mouse anti-phospho-c-Jun (1:1000; sc-822), and goat anti-β-actin (1:1000; sc-1616) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, United States).

    Techniques: Mouse Assay, Staining, Activity Assay

    c-Jun N-terminal kinase inhibitor, Caspase-9 inhibitor, and Pan-Caspase inhibitor ameliorate palmitate-induced cell death, and Caspase-9 inhibition attenuates the decrease of Rubicon protein. A: AML12 cells were incubated with 800 μmol/L PA for 10 h in the presence or absence of either 30 μmol/L SP600125, 20 μmol/L LEHD-fmk, or 20 μmol/L QVD-OPh. Untreated AML12 cells were used as the control. PA cytotoxicity was evaluated by cell proliferation assay. Living cells are presented as the ratio of the absorbance at the indicated conditions to that of AML12 without PA treatment; B: Whole cell lysates were prepared from AML12 cells treated with PA (800 μmol/L) for 10 h in the presence or absence of either 30 μmol/L SP600125, 20 μmol/L z-LEHD-fmk, or 20 μmol/L QVD-OPh. Immunoblot analysis of CHOP and Rubicon. β-actin was used as the loading control; C: The size of the AML12 cells after PA treatment (800 μmol/L) for 10 h was compared to that of untreated AML12 cells using the Image J software program. z-LEHD-fmk and/or siRubicon were used for Caspase-9 inhibition or Rubicon knockdown. The difference in cell size under each treatment condition is presented as the ratio to the cell size of the respective controls. All of the above experiments were repeated three times and representative results are shown. The quantitative data are presented as the mean ± SD; a P

    Journal: World Journal of Gastroenterology

    Article Title: c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

    doi: 10.3748/wjg.v22.i28.6509

    Figure Lengend Snippet: c-Jun N-terminal kinase inhibitor, Caspase-9 inhibitor, and Pan-Caspase inhibitor ameliorate palmitate-induced cell death, and Caspase-9 inhibition attenuates the decrease of Rubicon protein. A: AML12 cells were incubated with 800 μmol/L PA for 10 h in the presence or absence of either 30 μmol/L SP600125, 20 μmol/L LEHD-fmk, or 20 μmol/L QVD-OPh. Untreated AML12 cells were used as the control. PA cytotoxicity was evaluated by cell proliferation assay. Living cells are presented as the ratio of the absorbance at the indicated conditions to that of AML12 without PA treatment; B: Whole cell lysates were prepared from AML12 cells treated with PA (800 μmol/L) for 10 h in the presence or absence of either 30 μmol/L SP600125, 20 μmol/L z-LEHD-fmk, or 20 μmol/L QVD-OPh. Immunoblot analysis of CHOP and Rubicon. β-actin was used as the loading control; C: The size of the AML12 cells after PA treatment (800 μmol/L) for 10 h was compared to that of untreated AML12 cells using the Image J software program. z-LEHD-fmk and/or siRubicon were used for Caspase-9 inhibition or Rubicon knockdown. The difference in cell size under each treatment condition is presented as the ratio to the cell size of the respective controls. All of the above experiments were repeated three times and representative results are shown. The quantitative data are presented as the mean ± SD; a P

    Article Snippet: Antibodies and reagents: The antibodies used in this study were obtained from the following sources: anti-p62 (1:1000; #5114), anti-LC3 (1:1000; #4108), anti-Rubicon (1:1000; #8465), anti-cleaved caspase-3 (1:1000; #9661), and rabbit anti-phospho-JNK (1:1000; #9251) were obtained from Cell Signaling Technology, Tokyo, Japan; mouse anti-C/EBP homologous protein (CHOP) (1:500; sc-575), mouse anti-phospho-c-Jun (1:1000; sc-822), and goat anti-β-actin (1:1000; sc-1616) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, United States).

    Techniques: Inhibition, Incubation, Proliferation Assay, Software

    Palmitate induces expression of both p62 and Rubicon, and PA-induced Rubicon expression is mediated by c-Jun N-terminal kinase phosphorylation. A: Whole cell lysates were prepared from AML12 cells treated with PA (400 or 800 μmol/L) for 4 h in the presence or absence of SP600125. Immunoblotting analyses were performed for p62, LC3, and Rubicon. β-actin was used as the loading control; B: AML12 cells were incubated with 800 μmol/L PA for 4 h in the presence or absence of 30 μmol/L SP600125. p62 antibody was used as the primary antibody, and Alexa Fluor ® 488-labeled secondary antibody was used for detecting p62 antibody. Samples were visualized using an EVOS microscope; C: Total RNA was prepared from AML12 cells treated with PA (800 μmol/L) for 4 h in the presence or absence of 30 μmol/L SP600125. Vehicle-treated cells were used as the control (CT). Both p62 and Rubicon mRNA were quantified by RT-qPCR, normalized to Actin, and expressed as the fold-change vs control cells without SP600125. All of the above experiments were repeated three times and representative results are shown. The quantitative data are presented as the mean ± SD; a P

    Journal: World Journal of Gastroenterology

    Article Title: c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

    doi: 10.3748/wjg.v22.i28.6509

    Figure Lengend Snippet: Palmitate induces expression of both p62 and Rubicon, and PA-induced Rubicon expression is mediated by c-Jun N-terminal kinase phosphorylation. A: Whole cell lysates were prepared from AML12 cells treated with PA (400 or 800 μmol/L) for 4 h in the presence or absence of SP600125. Immunoblotting analyses were performed for p62, LC3, and Rubicon. β-actin was used as the loading control; B: AML12 cells were incubated with 800 μmol/L PA for 4 h in the presence or absence of 30 μmol/L SP600125. p62 antibody was used as the primary antibody, and Alexa Fluor ® 488-labeled secondary antibody was used for detecting p62 antibody. Samples were visualized using an EVOS microscope; C: Total RNA was prepared from AML12 cells treated with PA (800 μmol/L) for 4 h in the presence or absence of 30 μmol/L SP600125. Vehicle-treated cells were used as the control (CT). Both p62 and Rubicon mRNA were quantified by RT-qPCR, normalized to Actin, and expressed as the fold-change vs control cells without SP600125. All of the above experiments were repeated three times and representative results are shown. The quantitative data are presented as the mean ± SD; a P

    Article Snippet: Antibodies and reagents: The antibodies used in this study were obtained from the following sources: anti-p62 (1:1000; #5114), anti-LC3 (1:1000; #4108), anti-Rubicon (1:1000; #8465), anti-cleaved caspase-3 (1:1000; #9661), and rabbit anti-phospho-JNK (1:1000; #9251) were obtained from Cell Signaling Technology, Tokyo, Japan; mouse anti-C/EBP homologous protein (CHOP) (1:500; sc-575), mouse anti-phospho-c-Jun (1:1000; sc-822), and goat anti-β-actin (1:1000; sc-1616) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, United States).

    Techniques: Expressing, Incubation, Labeling, Microscopy, Quantitative RT-PCR

    Identification of 14-3-3 isoforms expressed in GV and GVBD mouse oocytes. (A) the mRNA levels of 14-3-3 isoforms at GV stage of mouse oocytes. mRNA of 120 mouse oocytes of GV stage were extracted (described in the“ Materials and Methods ” Section). RT-PCR products using primers for specific 14-3-3 isoforms are observed in ethidium bromide-stained agarose gel. ε, β, γ, η, σ, τ and ζ represent seven 14-3-3 isoforms. (B) the mRNA levels of 14-3-3ε at GV and GVBD stage of mouse oocytes. Line GV: mouse oocytes at GV stage. Line GVBD: mouse oocytes at GVBD stage. (C) A protein band of approximately 28 kDa from 200 mouse oocytes at GV or GVBD stage is detectable by Western blot with an anti-14-3-3ε antibody (upper panel). In contrast an anti-β-actin antibody detected a low molecular weight band of approximately 43 kDa (lower panel). (D) Western blot analysis 14-3-3β protein expression with an anti-14-3-3β antibody from 300 mouse oocytes at GV or GVBD stage, A protein band of approximately 29 kDa (upper panel). Line Brain: mouse brain protein as positive control. Shown is a representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: The Role of 14-3-3? Interaction with Phosphorylated Cdc25B at Its Ser321 in the Release of the Mouse Oocyte from Prophase I Arrest

    doi: 10.1371/journal.pone.0053633

    Figure Lengend Snippet: Identification of 14-3-3 isoforms expressed in GV and GVBD mouse oocytes. (A) the mRNA levels of 14-3-3 isoforms at GV stage of mouse oocytes. mRNA of 120 mouse oocytes of GV stage were extracted (described in the“ Materials and Methods ” Section). RT-PCR products using primers for specific 14-3-3 isoforms are observed in ethidium bromide-stained agarose gel. ε, β, γ, η, σ, τ and ζ represent seven 14-3-3 isoforms. (B) the mRNA levels of 14-3-3ε at GV and GVBD stage of mouse oocytes. Line GV: mouse oocytes at GV stage. Line GVBD: mouse oocytes at GVBD stage. (C) A protein band of approximately 28 kDa from 200 mouse oocytes at GV or GVBD stage is detectable by Western blot with an anti-14-3-3ε antibody (upper panel). In contrast an anti-β-actin antibody detected a low molecular weight band of approximately 43 kDa (lower panel). (D) Western blot analysis 14-3-3β protein expression with an anti-14-3-3β antibody from 300 mouse oocytes at GV or GVBD stage, A protein band of approximately 29 kDa (upper panel). Line Brain: mouse brain protein as positive control. Shown is a representative of three independent experiments.

    Article Snippet: The blots were probed overnight at 4°C in 1% milk in TBST with the following antibodies: rabbit anti-14-3-3ε (1∶1000) (Abcam); rabbit anti-14-3-3β (1∶800) (cellsignaling); rabbit anti-Myc (1∶1000) (Clontech); rabbit anti-HA (1∶800) (Sigma); Tyr(P)15 of Cdc2 (1∶500; Santa Cruz Biotechnology) and rabbit anti-β-actin (1∶400) (Santa Cruz Biotechnology).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Western Blot, Molecular Weight, Expressing, Positive Control

    Knockdown of 14-3-3ε by siRNA causes partial meiotic resumption mouse oocytes. (A) 120 GV-oocytes microinjected with 14-3-3ε siRNA or control siRNA (5pl of 20 µM) were collected 24 h after microinjection. Oocytes microinjected with control siRNA and no injection served as control. mRNA of 14-3-3ε was detected by RT-PCR using primers specific for 14-3-3ε. (B) Protein expression of 14-3-3ε was examined by western blot with an anti-14-3-3ε antibody. β-actin used as endogenous internal reference demonstrate 14-3-3ε siRNA specificity and equal loading. (C) at the indicated times, the percentages of germinal vesicle breakdown (GVBD) or MII or death were counted in cultured mouse oocytes after 14-3-3ε siRNA or control siRNA microinjection. GVBD, 24 h; MII or death, 30 h. The total number of oocytes undergoing meiotic maturation or death is given on top of bar graph from three independent experiments. (D) H1 kinase activity in oocytes injected with 14-3-3ε siRNA or control siRNA or no injection groups. Each value was expressed as mean±S.D of at least three independent experiments. (E) Protein extracts from oocytes microinjected with 14-3-3ε siRNA or control siRNA and no injection groups were incubated with histone H1 and [γ -32 P] ATP. The protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and incorporation of 32 P into histone H1 was visualized by autoradiography. (F) Western analysis of phosphorylation status of Cdc2-Tyr15. GV oocytes injected with 14-3-3ε siRNA or control siRNA were collected 24 h after microinjection. The collected oocytes were immunoblotted with anti-pTyr15 of Cdc2 antibody. (G) Co-localization of Endogenous Cdc25B and 14-3-3ε in mouse oocytes injected with 14-3-3ε siRNA or control siRNA. red fluorescent Cdc25B signals and green fluorescent 14-3-3ε signals were co-localized in the cytoplasm in oocytes injected with control siRNA, while red fluorescent Cdc25B signals were translocated from the cytoplasm to the nucleus in oocytes injected with14-3-3ε siRNA. Scale bar = 20 µm.

    Journal: PLoS ONE

    Article Title: The Role of 14-3-3? Interaction with Phosphorylated Cdc25B at Its Ser321 in the Release of the Mouse Oocyte from Prophase I Arrest

    doi: 10.1371/journal.pone.0053633

    Figure Lengend Snippet: Knockdown of 14-3-3ε by siRNA causes partial meiotic resumption mouse oocytes. (A) 120 GV-oocytes microinjected with 14-3-3ε siRNA or control siRNA (5pl of 20 µM) were collected 24 h after microinjection. Oocytes microinjected with control siRNA and no injection served as control. mRNA of 14-3-3ε was detected by RT-PCR using primers specific for 14-3-3ε. (B) Protein expression of 14-3-3ε was examined by western blot with an anti-14-3-3ε antibody. β-actin used as endogenous internal reference demonstrate 14-3-3ε siRNA specificity and equal loading. (C) at the indicated times, the percentages of germinal vesicle breakdown (GVBD) or MII or death were counted in cultured mouse oocytes after 14-3-3ε siRNA or control siRNA microinjection. GVBD, 24 h; MII or death, 30 h. The total number of oocytes undergoing meiotic maturation or death is given on top of bar graph from three independent experiments. (D) H1 kinase activity in oocytes injected with 14-3-3ε siRNA or control siRNA or no injection groups. Each value was expressed as mean±S.D of at least three independent experiments. (E) Protein extracts from oocytes microinjected with 14-3-3ε siRNA or control siRNA and no injection groups were incubated with histone H1 and [γ -32 P] ATP. The protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and incorporation of 32 P into histone H1 was visualized by autoradiography. (F) Western analysis of phosphorylation status of Cdc2-Tyr15. GV oocytes injected with 14-3-3ε siRNA or control siRNA were collected 24 h after microinjection. The collected oocytes were immunoblotted with anti-pTyr15 of Cdc2 antibody. (G) Co-localization of Endogenous Cdc25B and 14-3-3ε in mouse oocytes injected with 14-3-3ε siRNA or control siRNA. red fluorescent Cdc25B signals and green fluorescent 14-3-3ε signals were co-localized in the cytoplasm in oocytes injected with control siRNA, while red fluorescent Cdc25B signals were translocated from the cytoplasm to the nucleus in oocytes injected with14-3-3ε siRNA. Scale bar = 20 µm.

    Article Snippet: The blots were probed overnight at 4°C in 1% milk in TBST with the following antibodies: rabbit anti-14-3-3ε (1∶1000) (Abcam); rabbit anti-14-3-3β (1∶800) (cellsignaling); rabbit anti-Myc (1∶1000) (Clontech); rabbit anti-HA (1∶800) (Sigma); Tyr(P)15 of Cdc2 (1∶500; Santa Cruz Biotechnology) and rabbit anti-β-actin (1∶400) (Santa Cruz Biotechnology).

    Techniques: Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Cell Culture, Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Autoradiography

    Expression of E-cadherin, CK, α-SMA, vimentin in HK-2 cells by immunochemistry. (A–C)The HK-2 cells under 30 mM glucose(B) or 30 mM glucose+cont siRNA(C) showed a loss of CK and E-cadherin and an increase of α-SMA and vimentin expression compared to that of the cells under 5.5 mM glucose condition(A).(D–F)These changes were prevented by exposed to 30 mM glucose+p38siRNA for 24 h(D) or 48 h(E) or+AP-1 inhibitor(F).

    Journal: PLoS ONE

    Article Title: The Role of the p38 MAPK Signaling Pathway in High Glucose-Induced Epithelial-Mesenchymal Transition of Cultured Human Renal Tubular Epithelial Cells

    doi: 10.1371/journal.pone.0022806

    Figure Lengend Snippet: Expression of E-cadherin, CK, α-SMA, vimentin in HK-2 cells by immunochemistry. (A–C)The HK-2 cells under 30 mM glucose(B) or 30 mM glucose+cont siRNA(C) showed a loss of CK and E-cadherin and an increase of α-SMA and vimentin expression compared to that of the cells under 5.5 mM glucose condition(A).(D–F)These changes were prevented by exposed to 30 mM glucose+p38siRNA for 24 h(D) or 48 h(E) or+AP-1 inhibitor(F).

    Article Snippet: Anti-E-cadherin, anti-α-SMA, anti-vimentin, anti-CK and anti-TGF-β1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (USA), and -Gingerol was purchased from Wako Ltd. (Japan).

    Techniques: Expressing

    Expression of E-cadherin, CK, α-SMA, vimentin in HK-2 cells by Western blot and RT-PCR analysis. (A–B)E-cadherin expression(A1,A2) and α-SMA(B1,B2) were analyzed by Western blot of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 30 mM glucose (lane 2), 30 mM glucose+Cont siRNA(lane 3), 30 mM glucose+p38 siRNA for 24 h(lane 4), 30 mM glucose+p38 siRNA for 48 h(lane 5).(C–D)mRNA of CK (C1,C2) and vimentin(D1,D2) were analyzed by RT-PCR of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 30 mM glucose (lane 2), 30 mM glucose+Cont siRNA(lane 3), 30 mM glucose+p38 siRNA for 24 h(lane 4), 30 mM glucose+p38 siRNA for 48 h(lane 5).Values represent the mean ± SD, a P

    Journal: PLoS ONE

    Article Title: The Role of the p38 MAPK Signaling Pathway in High Glucose-Induced Epithelial-Mesenchymal Transition of Cultured Human Renal Tubular Epithelial Cells

    doi: 10.1371/journal.pone.0022806

    Figure Lengend Snippet: Expression of E-cadherin, CK, α-SMA, vimentin in HK-2 cells by Western blot and RT-PCR analysis. (A–B)E-cadherin expression(A1,A2) and α-SMA(B1,B2) were analyzed by Western blot of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 30 mM glucose (lane 2), 30 mM glucose+Cont siRNA(lane 3), 30 mM glucose+p38 siRNA for 24 h(lane 4), 30 mM glucose+p38 siRNA for 48 h(lane 5).(C–D)mRNA of CK (C1,C2) and vimentin(D1,D2) were analyzed by RT-PCR of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 30 mM glucose (lane 2), 30 mM glucose+Cont siRNA(lane 3), 30 mM glucose+p38 siRNA for 24 h(lane 4), 30 mM glucose+p38 siRNA for 48 h(lane 5).Values represent the mean ± SD, a P

    Article Snippet: Anti-E-cadherin, anti-α-SMA, anti-vimentin, anti-CK and anti-TGF-β1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (USA), and -Gingerol was purchased from Wako Ltd. (Japan).

    Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    Expression of E-cadherin, CK, α-SMA, vimentin in HK-2 cells by Western blot. (A)α-SMA, (B)E-caderin, (C)CK, (D)vimentin were measured by Western blot of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 5.5 mM glucose with p38 siRNA (lane 2), 5.5 mM glucose with AP-1 inhibitor (lane 3), 5.5 mM glucose+TGF-β1 (10 ng/ml) (lane 4), 5.5 mM glucose+TGF-β1(10 ng/ml) with p38 siRNA (lane 5), 5.5 mM glucose+TGF-β1(10 ng/ml) with the AP-1 inhibitor (lane 6). a P

    Journal: PLoS ONE

    Article Title: The Role of the p38 MAPK Signaling Pathway in High Glucose-Induced Epithelial-Mesenchymal Transition of Cultured Human Renal Tubular Epithelial Cells

    doi: 10.1371/journal.pone.0022806

    Figure Lengend Snippet: Expression of E-cadherin, CK, α-SMA, vimentin in HK-2 cells by Western blot. (A)α-SMA, (B)E-caderin, (C)CK, (D)vimentin were measured by Western blot of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 5.5 mM glucose with p38 siRNA (lane 2), 5.5 mM glucose with AP-1 inhibitor (lane 3), 5.5 mM glucose+TGF-β1 (10 ng/ml) (lane 4), 5.5 mM glucose+TGF-β1(10 ng/ml) with p38 siRNA (lane 5), 5.5 mM glucose+TGF-β1(10 ng/ml) with the AP-1 inhibitor (lane 6). a P

    Article Snippet: Anti-E-cadherin, anti-α-SMA, anti-vimentin, anti-CK and anti-TGF-β1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (USA), and -Gingerol was purchased from Wako Ltd. (Japan).

    Techniques: Expressing, Western Blot, Cell Culture

    Expression of E-cadherin, CK, α-SMA, vimentin in HK-2 cells by Western blot and RT-PCR analysis. (A–B)α-SMA(A) and E-cadherin(B) were detected by Western blot of the HK-2 cells incubated in 5.5 mM glucose (lane 1), 30 mM glucose(lane 2), 30 mM glucose+AP-1 inhibitor(lane 3).(C–D)mRNA of CK(C) and vimentin(D) was measured by RT-PCR of the HK-2 cells incubated in 5.5 mM glucose (lane 1), 30 mM glucose(lane 2), 30 mM glucose+AP-1 inhibitor(lane 3). Values represent the mean ± SD. a P

    Journal: PLoS ONE

    Article Title: The Role of the p38 MAPK Signaling Pathway in High Glucose-Induced Epithelial-Mesenchymal Transition of Cultured Human Renal Tubular Epithelial Cells

    doi: 10.1371/journal.pone.0022806

    Figure Lengend Snippet: Expression of E-cadherin, CK, α-SMA, vimentin in HK-2 cells by Western blot and RT-PCR analysis. (A–B)α-SMA(A) and E-cadherin(B) were detected by Western blot of the HK-2 cells incubated in 5.5 mM glucose (lane 1), 30 mM glucose(lane 2), 30 mM glucose+AP-1 inhibitor(lane 3).(C–D)mRNA of CK(C) and vimentin(D) was measured by RT-PCR of the HK-2 cells incubated in 5.5 mM glucose (lane 1), 30 mM glucose(lane 2), 30 mM glucose+AP-1 inhibitor(lane 3). Values represent the mean ± SD. a P

    Article Snippet: Anti-E-cadherin, anti-α-SMA, anti-vimentin, anti-CK and anti-TGF-β1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (USA), and -Gingerol was purchased from Wako Ltd. (Japan).

    Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation

    Expression of chymase, tryptase and carboxypeptidase A were altered during tumor progression. Western blots of cell lysate from control, phases I, II and III of tumor progression. The levels of mMCP-4 are constant in the 3 phases. mMCP-5 is only expressed during tumorigenesis. The levels of mMCP-6 increase progressively during the three phases. mMCP-7 was not detected in control animals, begging to expression in phase I and remained unchanged in phases II and III. The expression of mMC-CPA is almost not detectable in controls, but increases in phases I and II, and remains unchanged in phase III. Alpha-actin was used as a loading control. Mean optical density of blots presenting the data of the mean values ± SEM of 3 independent experiments. *P

    Journal: PLoS ONE

    Article Title: Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression

    doi: 10.1371/journal.pone.0040790

    Figure Lengend Snippet: Expression of chymase, tryptase and carboxypeptidase A were altered during tumor progression. Western blots of cell lysate from control, phases I, II and III of tumor progression. The levels of mMCP-4 are constant in the 3 phases. mMCP-5 is only expressed during tumorigenesis. The levels of mMCP-6 increase progressively during the three phases. mMCP-7 was not detected in control animals, begging to expression in phase I and remained unchanged in phases II and III. The expression of mMC-CPA is almost not detectable in controls, but increases in phases I and II, and remains unchanged in phase III. Alpha-actin was used as a loading control. Mean optical density of blots presenting the data of the mean values ± SEM of 3 independent experiments. *P

    Article Snippet: Rabbit anti-integrin αV, rabbit anti-integrin β3, goat anti-mouse mMCP-7, rat anti-mouse mMC-CPA and rabbit anti-alpha actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).The secondary antibodies goat anti-rabbit HRP and donkey anti-rat HRP were purchased from JacksonImmunoResearch (West Grove, PA).

    Techniques: Expressing, Western Blot

    HT-1080 cells containing the infectious HTLV-1 ACH.p30 II mutant provirus, defective for p30 II production, exhibit increased genomic and mitochondrial DNA-damage as compared to the wildtype ACH provirus. (A and B) The parental HT-1080 cell-line and the HT1080/HTLV-1 ACH.wt and ACH.p30 II mutant proviral clones were stained using a Click-iT Alexa Fluor 594-TUNEL kit (Molecular Probes; red signal) and a rabbit polyclonal primary antibody that recognizes the Ser139-phosphorylated histone variant H2A.X (Anti-Phospho-Ser139-H2A.X; green signal) which localizes at sites of genomic DNA-damage. The chemical uncoupler CCCP and DNAse I were included as positive controls. The cell nuclei were stained with the fluorescent dye, Hoechst 33342 (Molecular Probes; blue signal). The arrows in the enlarged inset panels at right indicate Phospho-Ser139-H2A.X foci in the merged images. The data in B represent the mean ± standard deviation (error bars) from three independent experiments. (C and D) HT-1080 cells and the HT-1080/HTLV-1 ACH.wt and ACH.p30 II mutant proviral clones were stained using a fluorescent TUNEL kit (red signal) and a rabbit polyclonal Anti-TOM20 primary antibody (green signal) to detect mitochondrial DNA-damage resulting from oxidative stress. The cell nuclei were stained with Hoechst 33342. Positive control samples were treated with either CCCP or DNAse I. The arrows in the enlarged inset panels at right indicate sites of mitochondrial DNA-damage. The graph in C shows the relative numbers of TUNEL/TOM20-positive foci per cell; and the data represent the mean ± standard deviation (error bars) from three independent experiments. (E and F) The ability of the HTLV-1 p30 II protein to protect against Tax/HBZ-induced cytotoxicity was determined by either (E) transfecting HT-1080 cells with Tax or HBZ expression constructs and then transducing them with lentiviral-HTLV-1 p30 II (HA) particles or an empty lentiviral vector, or (F) cotransfecting the cells with expression constructs for Tax, HBZ, and/or p30 II (HA). Certain samples were repeatedly cotransfected with a siRNA- tigar oligonucleotide or scrRNA control using HiPerFect transfection reagent. The cells in E were also treated with staurosporine (100 nM) as a positive control to induce apoptosis. Apoptotic cells were detected by staining them with Annexin V-FITC (green signal) and propidium iodide (PI; red signal); and the relative percentages of Annexin V-FITC +/− PI-positive cells were quantified by fluorescence-microscopy, as compared to the total numbers of cells visualized using a DIC phase-contrast filter. All the data is representative of at least three independent experiments; and the data in F represent the mean of the experiments ± standard deviation (error bars).

    Journal: Virology

    Article Title: The TP53-Induced Glycolysis and Apoptosis Regulator mediates cooperation between HTLV-1 p30II and the retroviral oncoproteins Tax and HBZ and is highly expressed in an in vivo xenograft model of HTLV-1-induced lymphoma

    doi: 10.1016/j.virol.2018.05.007

    Figure Lengend Snippet: HT-1080 cells containing the infectious HTLV-1 ACH.p30 II mutant provirus, defective for p30 II production, exhibit increased genomic and mitochondrial DNA-damage as compared to the wildtype ACH provirus. (A and B) The parental HT-1080 cell-line and the HT1080/HTLV-1 ACH.wt and ACH.p30 II mutant proviral clones were stained using a Click-iT Alexa Fluor 594-TUNEL kit (Molecular Probes; red signal) and a rabbit polyclonal primary antibody that recognizes the Ser139-phosphorylated histone variant H2A.X (Anti-Phospho-Ser139-H2A.X; green signal) which localizes at sites of genomic DNA-damage. The chemical uncoupler CCCP and DNAse I were included as positive controls. The cell nuclei were stained with the fluorescent dye, Hoechst 33342 (Molecular Probes; blue signal). The arrows in the enlarged inset panels at right indicate Phospho-Ser139-H2A.X foci in the merged images. The data in B represent the mean ± standard deviation (error bars) from three independent experiments. (C and D) HT-1080 cells and the HT-1080/HTLV-1 ACH.wt and ACH.p30 II mutant proviral clones were stained using a fluorescent TUNEL kit (red signal) and a rabbit polyclonal Anti-TOM20 primary antibody (green signal) to detect mitochondrial DNA-damage resulting from oxidative stress. The cell nuclei were stained with Hoechst 33342. Positive control samples were treated with either CCCP or DNAse I. The arrows in the enlarged inset panels at right indicate sites of mitochondrial DNA-damage. The graph in C shows the relative numbers of TUNEL/TOM20-positive foci per cell; and the data represent the mean ± standard deviation (error bars) from three independent experiments. (E and F) The ability of the HTLV-1 p30 II protein to protect against Tax/HBZ-induced cytotoxicity was determined by either (E) transfecting HT-1080 cells with Tax or HBZ expression constructs and then transducing them with lentiviral-HTLV-1 p30 II (HA) particles or an empty lentiviral vector, or (F) cotransfecting the cells with expression constructs for Tax, HBZ, and/or p30 II (HA). Certain samples were repeatedly cotransfected with a siRNA- tigar oligonucleotide or scrRNA control using HiPerFect transfection reagent. The cells in E were also treated with staurosporine (100 nM) as a positive control to induce apoptosis. Apoptotic cells were detected by staining them with Annexin V-FITC (green signal) and propidium iodide (PI; red signal); and the relative percentages of Annexin V-FITC +/− PI-positive cells were quantified by fluorescence-microscopy, as compared to the total numbers of cells visualized using a DIC phase-contrast filter. All the data is representative of at least three independent experiments; and the data in F represent the mean of the experiments ± standard deviation (error bars).

    Article Snippet: The following primary antibodies were used throughout: goat polyclonal Anti-HTLV-1 Tax (vC-12; Santa Cruz Biotechnology), goat polyclonal Anti-Actin (I-19; Santa Cruz Biotechnology), mouse monoclonal Anti-Beclin-1 (E-8; Santa Cruz Biotechnology), goat polyclonal Anti-TIM23 (C-19; Santa Cruz Biotechnology), rabbit polyclonal Anti-TOM20 (FL-145; Santa Cruz Biotechnology), mouse monoclonal Anti-SQSTM1 (D-3; Santa Cruz Biotechnology), rabbit polyclonal Anti-Phospho-Ser139-H2A.X (Santa Cruz Biotechnology), rabbit polyclonal Anti-GFP (FL; Santa Cruz Biotechnology), Anti-HTLV-1 p19Gag (Zeptometrix), mouse monoclonal Anti-TIGAR (G-2; Santa Cruz Biotechnology), rabbit polyclonal Anti-Human TIGAR (described in ), rabbit polyclonal Anti-Human Ki67 (huKi67, H-300; Santa Cruz Biotechnology), mouse monoclonal Anti-FLAG M2 (Sigma-Aldrich), mouse monoclonal Anti-c-Myc (9E10; Santa Cruz Biotechnology), mouse monoclonal Anti-CD31/PECAM1 (H-3; Santa Cruz Biotechnology), mouse monoclonal Anti-Flk1/VEGFR2 (A-3; Santa Cruz Biotechnology), mouse monoclonal Anti-HIF-1α (28b; Santa Cruz Biotechnology), and mouse monoclonal Anti-VEGF (C-1; Santa Cruz Biotechnology).

    Techniques: Mutagenesis, Clone Assay, Staining, TUNEL Assay, Variant Assay, Standard Deviation, Positive Control, Expressing, Construct, Plasmid Preparation, Transfection, Fluorescence, Microscopy

    Parasite burden and COX-2 expression in C. callosus after infection with T. gondii and treatment with COX-2 inhibitors. Females were infected orally with 50 cysts of T. gondii (ME49 strain), treated or untreated with meloxicam (0.5 mg/kg) or celecoxib (5 mg/kg) daily, euthanized after 40 days of infection and treatment, and the brains collected and analyzed by real-time PCR to quantify the parasite burden (A) or Western blotting to detect COX-2 and beta-actin expressions (B) . The data are shown as T. gondii DNA (TgDNA) concentration in 100 ng/μL total DNA (A) . Differences between groups were analyzed by one-way ANOVA with the Bonferroni multiple comparison post hoc test. Significant differences in relation to untreated/infected animals (control) ( ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Cyclooxygenase (COX)-2 Inhibitors Reduce Toxoplasma gondii Infection and Upregulate the Pro-inflammatory Immune Response in Calomys callosus Rodents and Human Monocyte Cell Line

    doi: 10.3389/fmicb.2019.00225

    Figure Lengend Snippet: Parasite burden and COX-2 expression in C. callosus after infection with T. gondii and treatment with COX-2 inhibitors. Females were infected orally with 50 cysts of T. gondii (ME49 strain), treated or untreated with meloxicam (0.5 mg/kg) or celecoxib (5 mg/kg) daily, euthanized after 40 days of infection and treatment, and the brains collected and analyzed by real-time PCR to quantify the parasite burden (A) or Western blotting to detect COX-2 and beta-actin expressions (B) . The data are shown as T. gondii DNA (TgDNA) concentration in 100 ng/μL total DNA (A) . Differences between groups were analyzed by one-way ANOVA with the Bonferroni multiple comparison post hoc test. Significant differences in relation to untreated/infected animals (control) ( ∗ P

    Article Snippet: Next, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Scientific, Rockford, IL, United States), blocked with 4% skimmed milk in Tris-buffered saline solution (TBS: 25 mM TRIS and 0.15 M NaCl, pH 7.4) for 1 h, and incubated overnight with goat polyclonal anti-COX-2 (1:100, R & D Systems, Minneapolis, MN, United States) or mouse monoclonal anti-beta-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, United States) in TBS.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay

    Number of tachyzoites and COX-2 expression in THP-1 cells treated with COX-2 inhibitors and/or PGE 2 . THP-1 cells were seeded in 96-well plates (3 × 10 4 cells/200 μL) for 24 h, infected with T. gondii tachyzoites (2F1 clone, RH strain), treated or untreated with meloxicam or celecoxib for additional 24 h, and submitted to T. gondii intracellular proliferation assay by β-galactosidase colorimetric reaction (A) . In parallel, THP-1 cells (1 × 10 6 cells/2000 μL in 6-well plates) were infected or not and treated or not with meloxicam or celecoxib in RPMI medium during 24 h. Then, THP-1 cells were lysed and submitted to Western blotting for COX-2 and beta-actin detections (B) . After, THP-1 cells (3 × 10 4 cells/200 μL in 96-well plates) were infected (2F1 clone) and treated with several concentrations of PGE 2 for additional 24 h (C) , or THP-1 cells were infected and treated with meloxicam or celecoxib plus PGE 2 for 24 h (D) , and all conditions submitted to T. gondii intracellular proliferation assay by β-galactosidase colorimetric reaction. DMSO (0.022%) was used to exclude any effect of DMSO in the parasitism. Data are shown as mean ± SEM of the number of tachyzoites from two independents experiments with eight replicates. Differences between groups were analyzed by one-way ANOVA with the Bonferroni multiple comparison post hoc test. Significant differences in relation to untreated/infected cells (medium) or infected/DMSO-treated cells ( ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Cyclooxygenase (COX)-2 Inhibitors Reduce Toxoplasma gondii Infection and Upregulate the Pro-inflammatory Immune Response in Calomys callosus Rodents and Human Monocyte Cell Line

    doi: 10.3389/fmicb.2019.00225

    Figure Lengend Snippet: Number of tachyzoites and COX-2 expression in THP-1 cells treated with COX-2 inhibitors and/or PGE 2 . THP-1 cells were seeded in 96-well plates (3 × 10 4 cells/200 μL) for 24 h, infected with T. gondii tachyzoites (2F1 clone, RH strain), treated or untreated with meloxicam or celecoxib for additional 24 h, and submitted to T. gondii intracellular proliferation assay by β-galactosidase colorimetric reaction (A) . In parallel, THP-1 cells (1 × 10 6 cells/2000 μL in 6-well plates) were infected or not and treated or not with meloxicam or celecoxib in RPMI medium during 24 h. Then, THP-1 cells were lysed and submitted to Western blotting for COX-2 and beta-actin detections (B) . After, THP-1 cells (3 × 10 4 cells/200 μL in 96-well plates) were infected (2F1 clone) and treated with several concentrations of PGE 2 for additional 24 h (C) , or THP-1 cells were infected and treated with meloxicam or celecoxib plus PGE 2 for 24 h (D) , and all conditions submitted to T. gondii intracellular proliferation assay by β-galactosidase colorimetric reaction. DMSO (0.022%) was used to exclude any effect of DMSO in the parasitism. Data are shown as mean ± SEM of the number of tachyzoites from two independents experiments with eight replicates. Differences between groups were analyzed by one-way ANOVA with the Bonferroni multiple comparison post hoc test. Significant differences in relation to untreated/infected cells (medium) or infected/DMSO-treated cells ( ∗ P

    Article Snippet: Next, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Scientific, Rockford, IL, United States), blocked with 4% skimmed milk in Tris-buffered saline solution (TBS: 25 mM TRIS and 0.15 M NaCl, pH 7.4) for 1 h, and incubated overnight with goat polyclonal anti-COX-2 (1:100, R & D Systems, Minneapolis, MN, United States) or mouse monoclonal anti-beta-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, United States) in TBS.

    Techniques: Expressing, Infection, Proliferation Assay, Western Blot

    Senescence-associated gene expression in WI-38 and IDH4 fibroblasts. (A) Northern blot analysis of expression of the genes indicated using early-passage (Young; ≈28 pdl) and late-passage (senescent [Sen.]; ≈60 pdl) WI-38 cells, as well as young (dex-treated) and senescent (7 days after removing dex) IDH4 cells. (B) Stabilities of cyclin A, cyclin B1, c-fos, and β-actin mRNAs in IDH4 cells that were either dex-treated [Young (+dex)] or cultured without dex for 7 days [Senescent (−dex)] were assessed after the addition of 2 μg of actinomycin D/ml; preparation of RNA at the times indicated; measurement of cyclin A, cyclin B1, c-fos, and β-actin mRNA Northern blot signals; normalizing them to 18S rRNA; and plotting them on a logarithmic scale (bottom). Dashed horizontal lines, 50% of untreated. The data represent the means ± standard errors of the means of four independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Loss of HuR Is Linked to Reduced Expression of Proliferative Genes during Replicative Senescence

    doi: 10.1128/MCB.21.17.5889-5898.2001

    Figure Lengend Snippet: Senescence-associated gene expression in WI-38 and IDH4 fibroblasts. (A) Northern blot analysis of expression of the genes indicated using early-passage (Young; ≈28 pdl) and late-passage (senescent [Sen.]; ≈60 pdl) WI-38 cells, as well as young (dex-treated) and senescent (7 days after removing dex) IDH4 cells. (B) Stabilities of cyclin A, cyclin B1, c-fos, and β-actin mRNAs in IDH4 cells that were either dex-treated [Young (+dex)] or cultured without dex for 7 days [Senescent (−dex)] were assessed after the addition of 2 μg of actinomycin D/ml; preparation of RNA at the times indicated; measurement of cyclin A, cyclin B1, c-fos, and β-actin mRNA Northern blot signals; normalizing them to 18S rRNA; and plotting them on a logarithmic scale (bottom). Dashed horizontal lines, 50% of untreated. The data represent the means ± standard errors of the means of four independent experiments.

    Article Snippet: Monoclonal anti-β-actin antibody was from Santa Cruz Biotechnologies (Santa Cruz, Calif.).

    Techniques: Expressing, Northern Blot, Cell Culture