monoclonal anti beta actin antibody Millipore Search Results


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  • 99
    Millipore monoclonal anti beta actin antibody
    Parkin truncating variants have reduced protein expression. ( A ) Schematic representation of parkin (NP_004553) functional domains and location of the frameshift variants identified in Portuguese patients. ( B ) Analysis of protein expression of EGFP-tagged parkin clones in HEK293T cells by immunoblotting with anti-EGFP antibody. <t>Beta-actin</t> was used as loading control. Original blots are presented in Supplementary Fig. S2 . Quantification data (graph) are presented as the mean ± SD of three independent experiments; **p
    Monoclonal Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 35215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti beta actin antibody/product/Millipore
    Average 99 stars, based on 35215 article reviews
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    monoclonal anti beta actin antibody - by Bioz Stars, 2020-09
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    99
    Millipore anti beta actin antibody mouse monoclonal
    BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and <t>beta-actin</t> (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.
    Anti Beta Actin Antibody Mouse Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12511 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti beta actin antibody mouse monoclonal/product/Millipore
    Average 99 stars, based on 12511 article reviews
    Price from $9.99 to $1999.99
    anti beta actin antibody mouse monoclonal - by Bioz Stars, 2020-09
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    99
    Millipore monoclonal anti actin alpha smooth muscle
    Immunocytochemical characterization. Confluent HUVEC cell layers after 24 h of NAC supplemented culture. In 15 mM NAC enriched cultivation, endothelial cells changed their morphology from typical cobblestone pattern (a–d) to an elongated, quasi-fusiform configuration (i–l). A slight cellular rearrangement was already detectable at 10 mM (e–h). Cells were positive for common endothelial cell markers CD31 (PECAM-1) and Von-Willebrand-Factor (vWF). Nuclei were counterstained with DAPI. All cells were negative for <t>alpha</t> smooth muscle actin (αSMA), a common myofibroblast marker (scale bar: 100 µ m).
    Monoclonal Anti Actin Alpha Smooth Muscle, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti actin alpha smooth muscle/product/Millipore
    Average 99 stars, based on 4985 article reviews
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    monoclonal anti actin alpha smooth muscle - by Bioz Stars, 2020-09
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    99
    Millipore anti β actin
    MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with <t>β-actin.</t> (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P
    Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti β actin/product/Millipore
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    anti β actin - by Bioz Stars, 2020-09
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    Millipore monoclonal anti beta actin peroxidase antibody
    Knockdown of PTEN and overexpression of AKT reversed the clearance of Aβ caused by alborixin. ( A ) Confocal microscopy and flow cytometric analysis for clearance of amyloid <t>beta</t> (Aβ 1-42 -HiLyte Fluor 555) in siPTEN -transfected and alborixin-treated HMC3 cells. ( B ) Analysis for Aβ clearance in Myr-AKT delta4-129 -transfected HMC3 cells after treatment with alborixin. ( C ) WT PTEN overexpression independent of alborixin caused clearance of Aβ, whereas, the catalytically inactive mutant PTEN C124S did not have any effect on the clearance of Aβ in HMC3 cells. Confocal images of HMC3 cells were taken after treatment with Aβ and alborixin (125 nM) for 12 h. Average RFI of at least 500 cells was used to calculate final average for the same sample from 3 independent experiments (3n). Scale bar used in confocal images: 10 µm. The X axis of dot plots in Figure 8A-C represents Aβ 1-42 -HiLyte Fluor 555 fluorescence. Statistical comparisons for Figure 8A-C were made by using Bonferroni test. p values ***p
    Monoclonal Anti Beta Actin Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2668 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti beta actin peroxidase antibody/product/Millipore
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    99
    Millipore anti actin alpha smooth muscle antibody mouse monoclonal
    Hyperacetylation of microtubules in salivary gland increases K14+ progenitor cells. Representative confocal images of untreated salivary glands (Control–no virus) or glands expressing green fluorescent protein (GFP) (Control-GFP) or GFP-tagged wild-type tubulin (GFP-WT-MT), acetyl-null tubulin K40A (GFP-HypoAcMT-K40A), or acetyl-mimetic tubulin K40Q (GFP-HyperAcMT-K40Q) as indicated and ( A ) immunostained for cytokeratin 14 (K14), cytokeratin 5 (K5), or cytokeratin 19 (K19) or ( C ) immunostained for cytokeratin 14 (K14), <t>alpha</t> smooth muscle myosin (αSMA), or E-cadherin (Ecad). Arrowheads point to basal and suprabasal layers of the epithelium in distal endbuds. Significantly increased K14+ and/or αSMA+ immunostaining of cells in the distal endbuds is observed in HyperAcMT (K40Q) expressing glands. Fluorescence intensity of the cytokeratins (K5, K14, or K19, B ) or αSMA ( D ) in the basal and suprabasal layers of the epithelium as outlined by the dotted white lines in panels A or C, respectively, was quantified and expressed as relative to the GFP control ( N > 3 experiments, n > 12 glands). Data are mean ± SEM. * P
    Anti Actin Alpha Smooth Muscle Antibody Mouse Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2180 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti actin alpha smooth muscle antibody mouse monoclonal/product/Millipore
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    99
    Millipore anti actin alpha smooth muscle cy3 antibody mouse monoclonal
    Microtubules, actin filaments and lipid rafts arrangement during spermiogenesis. Spermatogenic isolated cells were analyzed to test the components of the sperm head elongation complex. The microtubules of the manchette were detected using <t>alpha-tubulin</t> antibody and secondary antibody combined with FITC (tubulin columns). The actin filaments were stained with actin antibody conjugated with <t>Cy3</t> (actin columns). GM1-enriched lipid rafts were detected using cholera toxin conjugated with Alexa flour 594 (GM1 column, red signal) or FITC (GM1 column, green signal). The combination of green and red colors (merge columns) and phase contrast images were also included (CC columns). In NCR, microtubules, actin filaments and GM1 co-localized and were distributed in the manchette zone (mz) opposed to the acrosome zone (az). In HCARDA, it was not possible to detect manchette or acrosome zone. Microtubules, actin filaments and GM1 were equally distributed. Also, an asymmetrical acrosome was observed (asterisk). In ½ HCARDA + ½ OO, cells were polarized and manchette and acrosome zone were detected. Magnification: 620X.
    Anti Actin Alpha Smooth Muscle Cy3 Antibody Mouse Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1806 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore α sma
    IGF-IR and IGF-IIR expression, and VSMCs content in human carotid atherosclerotic plaques. a Representative gels and quantifications of IGF-IR and IGF-IIR protein levels in supernatants from serial immunoprecipitations (IRB and IRA) of non-complicated regions (n = 10) and their respective complicated plaques (n = 10). b Analysis of VSMCs content by Western blot against <t>α-SMA</t> in non-complicated regions (n = 10) and their respective complicated plaques (n = 10). ***p
    α Sma, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3873 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α sma/product/Millipore
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    Image Search Results


    Parkin truncating variants have reduced protein expression. ( A ) Schematic representation of parkin (NP_004553) functional domains and location of the frameshift variants identified in Portuguese patients. ( B ) Analysis of protein expression of EGFP-tagged parkin clones in HEK293T cells by immunoblotting with anti-EGFP antibody. Beta-actin was used as loading control. Original blots are presented in Supplementary Fig. S2 . Quantification data (graph) are presented as the mean ± SD of three independent experiments; **p

    Journal: Scientific Reports

    Article Title: Parkin truncating variants result in a loss-of-function phenotype

    doi: 10.1038/s41598-019-52534-6

    Figure Lengend Snippet: Parkin truncating variants have reduced protein expression. ( A ) Schematic representation of parkin (NP_004553) functional domains and location of the frameshift variants identified in Portuguese patients. ( B ) Analysis of protein expression of EGFP-tagged parkin clones in HEK293T cells by immunoblotting with anti-EGFP antibody. Beta-actin was used as loading control. Original blots are presented in Supplementary Fig. S2 . Quantification data (graph) are presented as the mean ± SD of three independent experiments; **p

    Article Snippet: Antibodies Primary antibodies: mouse monoclonal anti-EGFP antibody (Abnova, MAB1765), mouse monoclonal anti-GFP antibody (Rockland, 600-301-215), mouse monoclonal anti-beta-actin (Sigma-Aldrich, A5441), mouse monoclonal anti-GM130 (BD Biosciences, 610822), mouse monoclonal anti-caspase 3 (Cell Signaling, 9668), mouse monoclonal anti-HDAC2 (Santa Cruz Biotechnology, sc-9959), rabbit polyclonal anti-histone H3 (Abcam, ab1791), mouse monoclonal anti-p62 (Proteintech, 66184-1-Ig), mouse monoclonal anti-TOM20 (BD Biosciences, 612278).

    Techniques: Expressing, Functional Assay, Clone Assay

    Fyn regulates IP 3 -mediated calcium responses. (A) WEHI 7.2 T cells were transfected with Fyn siRNAs (or non-targeting control siRNAs) as described in materials and methods. Fyn levels were measured by western blotting 24 hours post-transfection. β-actin

    Journal: Autophagy

    Article Title: Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

    doi: 10.4161/auto.6.7.13290

    Figure Lengend Snippet: Fyn regulates IP 3 -mediated calcium responses. (A) WEHI 7.2 T cells were transfected with Fyn siRNAs (or non-targeting control siRNAs) as described in materials and methods. Fyn levels were measured by western blotting 24 hours post-transfection. β-actin

    Article Snippet: The following antibodies were used in this study: Fyn (Santa Cruz Biotechnology, sc-16), Lck (Santa Cruz Biotechnology, sc-433), anti-mouse CD3ε (BD Biosciences, 145-2C11), IP3 R3 (BD Biosciences, 610312), β-actin (Sigma-Aldrich, A-5441), p62 (Novus Biologicals, 8878-M03), LC3 (Novus Biologicals, NB100-2220), IP3 R1 (Novus Biologicals, NB120-5908), IP3 R2 (Novus Biologicals, NB100-2466), phospho-S6 kinase (Thr389) (Cell Signaling Technology, 9205), phospho-4EBP1 (Ser65) (Cell Signaling Technology, 9451), Total S6 kinase (Cell Signaling Technology, 9202), Total 4EBP1 (Cell Signaling Technology, 9452).

    Techniques: Transfection, Western Blot

    FL BARD1 functions in DNA damage response. SKNSH and SHSY5Y cell lines were silenced for FL BARD 1 expression upon transfection with lentiviral plasmids (shBARD1#A, shBARD1#B). Unsilenced control cells were transfected with plasmid shCTR. The efficiency of short harpin silencing was verified by western blotting, using an antibody against FL BARD1 isoform. The molecular weight of FL BARD1 isoform is reported. The higher band in the blot is an aspecific staining. β-Actin levels were used as loading control (A). The detection of Υ -H2AX protein was verified in nuclear extract of silenced (shBARD1) and unsilenced control (shCTR) cells, by western blotting. Antibody against histone H3 was used as loading control (B). SKNSH shBARD1 and shCTR cells (C) and SHSY5Y shBARD1 and shCTR cells (D) were treated with 5 Gy IR. The expression of Υ- H2AX was measured by western blotting in a time-course (30 min, 1h, 3h, 6h, 12h, 24h, 36h, 48h) after IR. The integral optical density (IOD) of Υ- H2AX protein bands were measured and normalized respect to loading control protein band H3. The arrows indicate the higher increment of Υ- H2AX in each cell line. The experiments were repeated twice.

    Journal: Journal of Cancer

    Article Title: Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma

    doi: 10.7150/jca.36164

    Figure Lengend Snippet: FL BARD1 functions in DNA damage response. SKNSH and SHSY5Y cell lines were silenced for FL BARD 1 expression upon transfection with lentiviral plasmids (shBARD1#A, shBARD1#B). Unsilenced control cells were transfected with plasmid shCTR. The efficiency of short harpin silencing was verified by western blotting, using an antibody against FL BARD1 isoform. The molecular weight of FL BARD1 isoform is reported. The higher band in the blot is an aspecific staining. β-Actin levels were used as loading control (A). The detection of Υ -H2AX protein was verified in nuclear extract of silenced (shBARD1) and unsilenced control (shCTR) cells, by western blotting. Antibody against histone H3 was used as loading control (B). SKNSH shBARD1 and shCTR cells (C) and SHSY5Y shBARD1 and shCTR cells (D) were treated with 5 Gy IR. The expression of Υ- H2AX was measured by western blotting in a time-course (30 min, 1h, 3h, 6h, 12h, 24h, 36h, 48h) after IR. The integral optical density (IOD) of Υ- H2AX protein bands were measured and normalized respect to loading control protein band H3. The arrows indicate the higher increment of Υ- H2AX in each cell line. The experiments were repeated twice.

    Article Snippet: Mouse monoclonal anti-β-Actin antibody (cod-A5441, Sigma-Aldrich, 1:6000) and anti-H3 (cod-06-755, Millipore, 1:1000), were used as loading control for cytosol and nuclei extracts respectively.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Molecular Weight, Staining

    FL BARD1 functions in regulating apoptosis. Phospho-p53 and p53 and β-Actin protein levels were verified by western blotting in shBARD1 and shCTR cells, in SKNSH (24 hours post-IR) and SHSY5Y (36 hours post-IR) cell lines (A). Caspase-3 activity was evaluated in SKNSH shBARD1 and shCTR IR and V cells (B) and in SHSY5Y shBARD1 and shCTR IR and V cells (C). The asterisk is indicative of p-value ≤ 0.05. The experiments were repeated twice.

    Journal: Journal of Cancer

    Article Title: Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma

    doi: 10.7150/jca.36164

    Figure Lengend Snippet: FL BARD1 functions in regulating apoptosis. Phospho-p53 and p53 and β-Actin protein levels were verified by western blotting in shBARD1 and shCTR cells, in SKNSH (24 hours post-IR) and SHSY5Y (36 hours post-IR) cell lines (A). Caspase-3 activity was evaluated in SKNSH shBARD1 and shCTR IR and V cells (B) and in SHSY5Y shBARD1 and shCTR IR and V cells (C). The asterisk is indicative of p-value ≤ 0.05. The experiments were repeated twice.

    Article Snippet: Mouse monoclonal anti-β-Actin antibody (cod-A5441, Sigma-Aldrich, 1:6000) and anti-H3 (cod-06-755, Millipore, 1:1000), were used as loading control for cytosol and nuclei extracts respectively.

    Techniques: Western Blot, Activity Assay

    BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and beta-actin (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.

    Journal: Current Alzheimer research

    Article Title: BACE1 Levels by APOE Genotype in Non-Demented and Alzheimer\u2019s Post-Mortem Brains

    doi:

    Figure Lengend Snippet: BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and beta-actin (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.

    Article Snippet: The membranes were then stripped and reprobed with mouse anti-β actin (#A1978; Sigma-Aldrich, St. Louis, MO).

    Techniques: Western Blot

    Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

    Journal: Experimental & Molecular Medicine

    Article Title: Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells

    doi: 10.1038/s12276-018-0044-y

    Figure Lengend Snippet: Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

    Article Snippet: After incubation with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature, the membrane was incubated with primary antibodies (mouse anti-β-catenin (Santa Cruz Biotechnology), mouse anti-EN1 (Abcam, Cambridge, UK), and mouse anti-β-actin (Sigma-Aldrich)) for 1 h at room temperature or overnight at 4 °C.

    Techniques: Activation Assay, Derivative Assay, Expressing, ALP Assay, shRNA

    Differential expression of A3A mRNA in progenitor as compared to non-progenitor CBMCs. (A) Six CBMC samples were sorted into progenitor and non-progenitor cells, and A3A mRNA expression was assessed and normalized to GAPDH mRNA level. A3A expression in six pairs of samples was on average 228 (SD = 298) fold higher in non-progenitors as compared to progenitors, corresponding to differential G-to-A editing in these populations. (B) Three pairs of pooled CBMC samples were sorted into progenitor and non-progenitor subpopulations and examined for A3A level, showing higher levels in non-progenitors. (C) Quantitation of the A3A Western blot bands normalized to beta-Actin, confirming higher A3A levels in non-progenitor cells.

    Journal: PLoS ONE

    Article Title: APOBEC3A Is Implicated in a Novel Class of G-to-A mRNA Editing in WT1 Transcripts

    doi: 10.1371/journal.pone.0120089

    Figure Lengend Snippet: Differential expression of A3A mRNA in progenitor as compared to non-progenitor CBMCs. (A) Six CBMC samples were sorted into progenitor and non-progenitor cells, and A3A mRNA expression was assessed and normalized to GAPDH mRNA level. A3A expression in six pairs of samples was on average 228 (SD = 298) fold higher in non-progenitors as compared to progenitors, corresponding to differential G-to-A editing in these populations. (B) Three pairs of pooled CBMC samples were sorted into progenitor and non-progenitor subpopulations and examined for A3A level, showing higher levels in non-progenitors. (C) Quantitation of the A3A Western blot bands normalized to beta-Actin, confirming higher A3A levels in non-progenitor cells.

    Article Snippet: Protein was detected using rabbit anti-APOBEC3A (Santa Cruz Biotechnology) and mouse anti-beta-Actin (Sigma-Aldrich), followed by HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies (Sigma-Aldrich), respectively.

    Techniques: Expressing, Quantitation Assay, Western Blot

    TET1-CD up-regulates TSGs expression. (A). The SMMC 7721 cells were transiently transfected with either TET1-CD plasmids or TET1-mCD plasmids. Expression of TET1-CD and TET1-mCD proteins was analysed by Western blot, and β-actin was used as an internal control. (B) (C). The SMMC 7721 cells were cultured in the 6 mm plate for 24h and then transiently transfected with either pflag-CMV4 vector (control), TET1-CD or TET1-mCD. Expression of TSGs and oncogenes was analyzed by Quantitative RT-PCR 48h after transient transfection, and the results were represented as mean ± SD of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: Effects of a single transient transfection of Ten-eleven translocation 1 catalytic domain on hepatocellular carcinoma

    doi: 10.1371/journal.pone.0207139

    Figure Lengend Snippet: TET1-CD up-regulates TSGs expression. (A). The SMMC 7721 cells were transiently transfected with either TET1-CD plasmids or TET1-mCD plasmids. Expression of TET1-CD and TET1-mCD proteins was analysed by Western blot, and β-actin was used as an internal control. (B) (C). The SMMC 7721 cells were cultured in the 6 mm plate for 24h and then transiently transfected with either pflag-CMV4 vector (control), TET1-CD or TET1-mCD. Expression of TSGs and oncogenes was analyzed by Quantitative RT-PCR 48h after transient transfection, and the results were represented as mean ± SD of three independent experiments. *p

    Article Snippet: Then, the membrane was blocked with 5% non-fat milk for 2h at room temperature, and then incubated at 4°C overnight with anti-TET1 (GeneTex) and anti-FLAG (Stratagene) antibody (1:1000 dilution), or mouse monoclonal anti-β-actin (Sigma) antibody (1:5000 dilution) for the internal control.

    Techniques: Expressing, Transfection, Western Blot, Cell Culture, Plasmid Preparation, Quantitative RT-PCR

    STAT3 expression in the model of chronic hepatitis B. (A) STAT3 levels in the liver tissues at the indicated time points (two representatives of each), the tumor tissues (18M-Tum) and HCC cell lines (HepG2 and PLC/PRF/5) were evaluated by Western blotting, and normalized to β-actin. (B) The liver tissues were stained with hematoxylin and eosin, and analyzed immunohistochemically. #282 and #283 mice (0M: unmanipuladed) were 24-month-old. Original magnifications, x 200 (bar represents 50 μm).

    Journal: PLoS ONE

    Article Title: Gene expression profiling of hepatocarcinogenesis in a mouse model of chronic hepatitis B

    doi: 10.1371/journal.pone.0185442

    Figure Lengend Snippet: STAT3 expression in the model of chronic hepatitis B. (A) STAT3 levels in the liver tissues at the indicated time points (two representatives of each), the tumor tissues (18M-Tum) and HCC cell lines (HepG2 and PLC/PRF/5) were evaluated by Western blotting, and normalized to β-actin. (B) The liver tissues were stained with hematoxylin and eosin, and analyzed immunohistochemically. #282 and #283 mice (0M: unmanipuladed) were 24-month-old. Original magnifications, x 200 (bar represents 50 μm).

    Article Snippet: [ ] Anti-STAT3 monoclonal (79D7; Cell Signaling), anti-mouse p53 polyclonal (Leica, Newcastle, UK), anti-phospho-p53 (Ser6 and Ser15) (Cell Signaling) and anti-β-actin monoclonal (Sigma–Aldrich, St. Louis, MO) antibodies were used for protein detection.

    Techniques: Expressing, Planar Chromatography, Western Blot, Staining, Mouse Assay

    TP53 expression in the model of chronic hepatitis B. Total TP53 levels and phosphorylated TP53 at serines 6 and 15 (p-p53-Ser 6 and -Ser 15) in the liver tissues at the indicated time points (two representatives of each) and the tumor tissues (18M-Tum) were evaluated by Western blotting, and normalized to β -actin.

    Journal: PLoS ONE

    Article Title: Gene expression profiling of hepatocarcinogenesis in a mouse model of chronic hepatitis B

    doi: 10.1371/journal.pone.0185442

    Figure Lengend Snippet: TP53 expression in the model of chronic hepatitis B. Total TP53 levels and phosphorylated TP53 at serines 6 and 15 (p-p53-Ser 6 and -Ser 15) in the liver tissues at the indicated time points (two representatives of each) and the tumor tissues (18M-Tum) were evaluated by Western blotting, and normalized to β -actin.

    Article Snippet: [ ] Anti-STAT3 monoclonal (79D7; Cell Signaling), anti-mouse p53 polyclonal (Leica, Newcastle, UK), anti-phospho-p53 (Ser6 and Ser15) (Cell Signaling) and anti-β-actin monoclonal (Sigma–Aldrich, St. Louis, MO) antibodies were used for protein detection.

    Techniques: Expressing, Western Blot

    ( A ) Western blot analysis showing changes in the protein expression of cleaved caspase-3 in chemo-resistant HCT-116 cells after 48 h incubation of cells in the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin, β-actin ( loading control ) levels are also shown ( upper panel ), and changes in the levels of 19 kDa and 17 kDa fragments of cleaved caspase-3 relative to control normalized to β-actin, as determined by densitometry analysis which, are depicted in the histogram ( lower panel ). ( B ) Changes in the caspase-3 activity in the chemo-resistant HT-29 cells. Values are mean±SD of 4 experiments. p

    Journal: Pharmaceutical research

    Article Title: Difluorinated-Curcumin (CDF): A Novel Curcumin Analog is a Potent Inhibitor of Colon Cancer Stem-Like Cells

    doi: 10.1007/s11095-010-0336-y

    Figure Lengend Snippet: ( A ) Western blot analysis showing changes in the protein expression of cleaved caspase-3 in chemo-resistant HCT-116 cells after 48 h incubation of cells in the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin, β-actin ( loading control ) levels are also shown ( upper panel ), and changes in the levels of 19 kDa and 17 kDa fragments of cleaved caspase-3 relative to control normalized to β-actin, as determined by densitometry analysis which, are depicted in the histogram ( lower panel ). ( B ) Changes in the caspase-3 activity in the chemo-resistant HT-29 cells. Values are mean±SD of 4 experiments. p

    Article Snippet: Rabbit anti-p-EGF-Receptor (Tyr 1173), rabbit anti-c-Myc, rabbit anti-phospho-β-catenin and rabbit anti-cleaved caspase-3 antibodies were the products of Cell Signaling, Danvers, MA, and the mouse anti-β-actin antibodies were purchased from Chemicon International, Billerica, MA.

    Techniques: Western Blot, Expressing, Incubation, Activity Assay

    Quantitative real-time PCR (qRT-PCR) analysis showing mRNA expression of colon CSC markers CD44 and CD166 ( left panel ) and changes in protein levels of ABCG2, pEGFR-Y 1173 and p-IGF-1R in chemo-resistant HCT-116 cells in response to 5-FU + Ox alone or in combination with either curcumin or CDF ( right panel ); β-actin ( loading control ) levels is also shown. The numbers represent percent of corresponding control normalized to β-actin. * p

    Journal: Pharmaceutical research

    Article Title: Difluorinated-Curcumin (CDF): A Novel Curcumin Analog is a Potent Inhibitor of Colon Cancer Stem-Like Cells

    doi: 10.1007/s11095-010-0336-y

    Figure Lengend Snippet: Quantitative real-time PCR (qRT-PCR) analysis showing mRNA expression of colon CSC markers CD44 and CD166 ( left panel ) and changes in protein levels of ABCG2, pEGFR-Y 1173 and p-IGF-1R in chemo-resistant HCT-116 cells in response to 5-FU + Ox alone or in combination with either curcumin or CDF ( right panel ); β-actin ( loading control ) levels is also shown. The numbers represent percent of corresponding control normalized to β-actin. * p

    Article Snippet: Rabbit anti-p-EGF-Receptor (Tyr 1173), rabbit anti-c-Myc, rabbit anti-phospho-β-catenin and rabbit anti-cleaved caspase-3 antibodies were the products of Cell Signaling, Danvers, MA, and the mouse anti-β-actin antibodies were purchased from Chemicon International, Billerica, MA.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    Effect of 5-FU + Ox alone or in combination with either curcumin or CDF on NF-κB DNA binding activity ( left panel ) and changes in the levels β-catenin, p-β-catenin, Cox-2 and c-Myc, in chemo-resistant HCT-116 cells ( right panel ); the levels of β-actin ( loading control ) are also shown. The numbers represent percent of corresponding control normalized to β-actin. * p

    Journal: Pharmaceutical research

    Article Title: Difluorinated-Curcumin (CDF): A Novel Curcumin Analog is a Potent Inhibitor of Colon Cancer Stem-Like Cells

    doi: 10.1007/s11095-010-0336-y

    Figure Lengend Snippet: Effect of 5-FU + Ox alone or in combination with either curcumin or CDF on NF-κB DNA binding activity ( left panel ) and changes in the levels β-catenin, p-β-catenin, Cox-2 and c-Myc, in chemo-resistant HCT-116 cells ( right panel ); the levels of β-actin ( loading control ) are also shown. The numbers represent percent of corresponding control normalized to β-actin. * p

    Article Snippet: Rabbit anti-p-EGF-Receptor (Tyr 1173), rabbit anti-c-Myc, rabbit anti-phospho-β-catenin and rabbit anti-cleaved caspase-3 antibodies were the products of Cell Signaling, Danvers, MA, and the mouse anti-β-actin antibodies were purchased from Chemicon International, Billerica, MA.

    Techniques: Binding Assay, Activity Assay

    ( A ) Western blot showing changes in the levels of pro-apoptotic Bax, and anti-apoptotic Bcl-xL in untreated parental as compared to chemo-resistant HCT-116 cells in response to the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin. The numbers represent percent of corresponding control normalized to β-actin. ( B ) Induction of early apoptosis as determined by acridine-orange/ethidium-bromide staining in chemo-resistant HCT-116 and HT-29 cells after incubation of cells in the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin. Values are mean±SD of 4 experiments. * p

    Journal: Pharmaceutical research

    Article Title: Difluorinated-Curcumin (CDF): A Novel Curcumin Analog is a Potent Inhibitor of Colon Cancer Stem-Like Cells

    doi: 10.1007/s11095-010-0336-y

    Figure Lengend Snippet: ( A ) Western blot showing changes in the levels of pro-apoptotic Bax, and anti-apoptotic Bcl-xL in untreated parental as compared to chemo-resistant HCT-116 cells in response to the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin. The numbers represent percent of corresponding control normalized to β-actin. ( B ) Induction of early apoptosis as determined by acridine-orange/ethidium-bromide staining in chemo-resistant HCT-116 and HT-29 cells after incubation of cells in the absence or presence of 5-Fu + Ox alone or in combination with either CDF or curcumin. Values are mean±SD of 4 experiments. * p

    Article Snippet: Rabbit anti-p-EGF-Receptor (Tyr 1173), rabbit anti-c-Myc, rabbit anti-phospho-β-catenin and rabbit anti-cleaved caspase-3 antibodies were the products of Cell Signaling, Danvers, MA, and the mouse anti-β-actin antibodies were purchased from Chemicon International, Billerica, MA.

    Techniques: Western Blot, Staining, Incubation

    Nuclear import of the fusion protein GST-GFP-NLS in the permeabilized cells. Cells permeabilized with 30 µg/mL digitonin (Dig30) and non-permeabilized cells (Control) were incubated with GST-GFP-NLS in the absence (−Extract) or presence (+Extract) of Xenopus egg extract supplemented with ATP for 1 h at 25 °C. The incorporation of GST-GFP-NLS was assessed by green fluorescence. Cell nuclei were counterstained with Hoechst 33243. Note that without egg extract, the nuclei are not labelled (arrows). Due to light scattering of the fluorescence in the cytoplasm, the nuclei appear smaller than they are. The egg extract restored nuclear import in the permeabilized cells. These pictures are representative of five independent replicates. Scale bar = 20 µm. Detection of importin alpha-1 (55 kDa) ( B ) and Karyopherin beta -1 (KPNB1–97 kDa) ( C ) in Xenopus egg extract by western blot in the presence (+) or in absence (−) of the anti- Xenopus importin alpha-1 antibody (clone 15) and the anti-rat KPNB1 antibody (KPNB1-clone 23). Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gels and were analyzed with the same exposure times (B:1 sec; C:1 min).

    Journal: Scientific Reports

    Article Title: Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

    doi: 10.1038/s41598-019-39500-y

    Figure Lengend Snippet: Nuclear import of the fusion protein GST-GFP-NLS in the permeabilized cells. Cells permeabilized with 30 µg/mL digitonin (Dig30) and non-permeabilized cells (Control) were incubated with GST-GFP-NLS in the absence (−Extract) or presence (+Extract) of Xenopus egg extract supplemented with ATP for 1 h at 25 °C. The incorporation of GST-GFP-NLS was assessed by green fluorescence. Cell nuclei were counterstained with Hoechst 33243. Note that without egg extract, the nuclei are not labelled (arrows). Due to light scattering of the fluorescence in the cytoplasm, the nuclei appear smaller than they are. The egg extract restored nuclear import in the permeabilized cells. These pictures are representative of five independent replicates. Scale bar = 20 µm. Detection of importin alpha-1 (55 kDa) ( B ) and Karyopherin beta -1 (KPNB1–97 kDa) ( C ) in Xenopus egg extract by western blot in the presence (+) or in absence (−) of the anti- Xenopus importin alpha-1 antibody (clone 15) and the anti-rat KPNB1 antibody (KPNB1-clone 23). Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gels and were analyzed with the same exposure times (B:1 sec; C:1 min).

    Article Snippet: After western blotting of the egg extract, the membranes were incubated overnight at 4 °C with rabbit polyclonal Xenopus anti-Lamin B3 (1:10 000, a gift from N. Morin, France), Xenopus anti-importin alpha1 (1:5000, a gift from K. Weiss, Switzerland), mouse monoclonal rat anti-karyopherin β1 (1:1000 KPNB1, Antibodies-online, clone 23), and anti-beta actin (1:5000, Sigma, clone AC15).

    Techniques: Incubation, Fluorescence, Western Blot, Size-exclusion Chromatography

    Incorporation of Lamin B3 from Xenopus eggs into the nuclei of permeabilized cells. ( A ) Detection of Lamin B3 (68 kDa) in Xenopus egg extract (Extract) by western blot in the presence (+) or absence (−) of anti- Xenopus Lamin B3. Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gel and were analyzed with the same exposure times (Lamin B3:2 sec; beta actin: 1 min). ( B ) Nuclear import of Lamin B3 detected by immunofluorescence in permeabilized (Dig30) and non-permeabilized (Control) cells incubated for 60 min in the presence (+Extract) of Xenopus egg extract under energy supplementation at 25 °C. Lamin B3-positive cells presenting a detectable signal (from weak to strong staining) were observed. A high proportion of nuclei were labelled. Inset: magnification of one nucleus showing a strong labelling of the nuclear lamina. These pictures are representative of five independent replicates. Scale bar = 20 µm.

    Journal: Scientific Reports

    Article Title: Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

    doi: 10.1038/s41598-019-39500-y

    Figure Lengend Snippet: Incorporation of Lamin B3 from Xenopus eggs into the nuclei of permeabilized cells. ( A ) Detection of Lamin B3 (68 kDa) in Xenopus egg extract (Extract) by western blot in the presence (+) or absence (−) of anti- Xenopus Lamin B3. Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gel and were analyzed with the same exposure times (Lamin B3:2 sec; beta actin: 1 min). ( B ) Nuclear import of Lamin B3 detected by immunofluorescence in permeabilized (Dig30) and non-permeabilized (Control) cells incubated for 60 min in the presence (+Extract) of Xenopus egg extract under energy supplementation at 25 °C. Lamin B3-positive cells presenting a detectable signal (from weak to strong staining) were observed. A high proportion of nuclei were labelled. Inset: magnification of one nucleus showing a strong labelling of the nuclear lamina. These pictures are representative of five independent replicates. Scale bar = 20 µm.

    Article Snippet: After western blotting of the egg extract, the membranes were incubated overnight at 4 °C with rabbit polyclonal Xenopus anti-Lamin B3 (1:10 000, a gift from N. Morin, France), Xenopus anti-importin alpha1 (1:5000, a gift from K. Weiss, Switzerland), mouse monoclonal rat anti-karyopherin β1 (1:1000 KPNB1, Antibodies-online, clone 23), and anti-beta actin (1:5000, Sigma, clone AC15).

    Techniques: Western Blot, Size-exclusion Chromatography, Immunofluorescence, Incubation, Staining

    Increased expression of TGF-β in retina of mice after Laser-induced CNV formation. ( A ) Flat mounts were fluorescently labeled with TGF-β antibody (green) and the nuclear staining with Hoechst (blue). Low levels of TGF-β expression were detected in ganglion cells of retina and choroid of Nor control mice. However, the strong immunoreactivity could be observed in the ganglion cells, outer nuclear layer, inner nuclear layer of retina and choroid at two weeks after Laser injury (Scale bars = 50 μm). ( B ) Typical result of Western blot showed changes of TGF-β protein levels in RPE/choroid/sclera isolated from the mice with or without Laser-induced CNV formation. ( C ) The quantitative analysis results demonstrated that the TGF-β protein levels were significantly up regulated at one week and reached a peak at three weeks in RPE/choroid/sclera of mice after Laser photocoagulation. The β-actin was used as loading control and reference protein, and the ratio of TGF-β/β-actin in Nor mice was set as 100%. *P

    Journal: Scientific Reports

    Article Title: TGF-β participates choroid neovascularization through Smad2/3-VEGF/TNF-α signaling in mice with Laser-induced wet age-related macular degeneration

    doi: 10.1038/s41598-017-10124-4

    Figure Lengend Snippet: Increased expression of TGF-β in retina of mice after Laser-induced CNV formation. ( A ) Flat mounts were fluorescently labeled with TGF-β antibody (green) and the nuclear staining with Hoechst (blue). Low levels of TGF-β expression were detected in ganglion cells of retina and choroid of Nor control mice. However, the strong immunoreactivity could be observed in the ganglion cells, outer nuclear layer, inner nuclear layer of retina and choroid at two weeks after Laser injury (Scale bars = 50 μm). ( B ) Typical result of Western blot showed changes of TGF-β protein levels in RPE/choroid/sclera isolated from the mice with or without Laser-induced CNV formation. ( C ) The quantitative analysis results demonstrated that the TGF-β protein levels were significantly up regulated at one week and reached a peak at three weeks in RPE/choroid/sclera of mice after Laser photocoagulation. The β-actin was used as loading control and reference protein, and the ratio of TGF-β/β-actin in Nor mice was set as 100%. *P

    Article Snippet: Western Blot Analysis Antibodies used in this study are listed as follows: rabbit anti-TGF-β polyclonal antibody (1:500; catalogue number ab66043), rabbit anti-VEGF polyclonal antibody (1:1,000; catalogue number ab46154) and rabbit anti-TNF-α polyclonal antibody (1:500; catalogue number ab9635, Abcam, Cambridge, UK); rabbit anti-Smad2/3 polyclonal antibody (1:1,000; catalogue number #5678) and anti-Phospho-Smad2/3 monoclonal antibody (1:1,000; catalogue number #8828, Cell Signaling Technology, Boston, MA 02241–3843, USA); mouse anti-β-actin monoclonal antibody (1:3,000; Sigma-Aldrich Corp. St. Louis, MO 63103, USA); and the horseradish peroxidase conjugated goat anti-rabbit or anti-mouse IgG as secondary antibody (1:4,000; Stressgen Biotechnologies Corporation, Victoria BC, Canada).

    Techniques: Expressing, Mouse Assay, Labeling, Staining, Western Blot, Isolation

    Identification of caveolin-1 and -2 in the rat retina. Protein (40 µg) from heart and retina homogenates of adult rats were separated by SDS-PAGE (12%), and then immunoblotted with caveolin-1 (A) and caveolin-2 (B) antibodies, respectively. Beta-actin was used as a control. Molecular weight markers are indicated on the right. Blots are representative of data from a series of four different preparations yielding identical results.

    Journal: Journal of Veterinary Science

    Article Title: Immunohistochemical study of caveolin-1 and -2 in the rat retina

    doi: 10.4142/jvs.2006.7.2.101

    Figure Lengend Snippet: Identification of caveolin-1 and -2 in the rat retina. Protein (40 µg) from heart and retina homogenates of adult rats were separated by SDS-PAGE (12%), and then immunoblotted with caveolin-1 (A) and caveolin-2 (B) antibodies, respectively. Beta-actin was used as a control. Molecular weight markers are indicated on the right. Blots are representative of data from a series of four different preparations yielding identical results.

    Article Snippet: Mouse monoclonal anti-beta-actin was obtained from Sigma (USA).

    Techniques: SDS Page, Molecular Weight

    a Expression levels of TSPO measured in protein extracts from rat and murine glioma cell lines (9L, C6 and GL261). Western blots were normalized using the anti-β-actin antibody. H E staining ( b ) and immunohistochemistry for TSPO ( c ) of coronal brain sections illustrating tumour growth and high level of TSPO expression in a 9L glioma

    Journal: European Journal of Nuclear Medicine and Molecular Imaging

    Article Title: The translocator protein ligand [18F]DPA-714 images glioma and activated microglia in vivo

    doi: 10.1007/s00259-011-2041-4

    Figure Lengend Snippet: a Expression levels of TSPO measured in protein extracts from rat and murine glioma cell lines (9L, C6 and GL261). Western blots were normalized using the anti-β-actin antibody. H E staining ( b ) and immunohistochemistry for TSPO ( c ) of coronal brain sections illustrating tumour growth and high level of TSPO expression in a 9L glioma

    Article Snippet: Incubation with the primary antibodies diluted in PBST with 3% of BSA for the anti-TSPO antibody and 3% non-fat dry milk for the anti-β-actin antibody, respectively (1/10,000 dilution of the anti-rat TSPO antibody NP155 [ ] generously provided by Dr. M. Higuchi, NIRS, Japan, and 1/5,000 dilution of the anti-β-actin antibody purchased from Sigma Aldrich), was performed overnight at 4°C.

    Techniques: Expressing, Western Blot, Staining, Immunohistochemistry

    Western Blot analysis. Sera collected at 28 days post challenge from animals of groups C01 and C03 as well as from one control animal from group C04 were analyzed by Western blot using SBV-infected (+) or non-infected cells (−) as antigens. All sera (dilution 1/100) contained Gc-specific antibodies and enabled staining of SBV-Gc (~120 kDa) in infected cell lysates. In contrast, the SBV-N-protein (~25 kDa) was detected only by sera from the infected control animal as well as by one animal from group C01. Staining of Beta Actin was used as a loading control and is shown (cropped) beneath the corresponding serum-staining. Full-length blots are presented in Supplementary Figure 4 .

    Journal: Scientific Reports

    Article Title: The N-terminal domain of Schmallenberg virus envelope protein Gc is highly immunogenic and can provide protection from infection

    doi: 10.1038/srep42500

    Figure Lengend Snippet: Western Blot analysis. Sera collected at 28 days post challenge from animals of groups C01 and C03 as well as from one control animal from group C04 were analyzed by Western blot using SBV-infected (+) or non-infected cells (−) as antigens. All sera (dilution 1/100) contained Gc-specific antibodies and enabled staining of SBV-Gc (~120 kDa) in infected cell lysates. In contrast, the SBV-N-protein (~25 kDa) was detected only by sera from the infected control animal as well as by one animal from group C01. Staining of Beta Actin was used as a loading control and is shown (cropped) beneath the corresponding serum-staining. Full-length blots are presented in Supplementary Figure 4 .

    Article Snippet: As a loading control, Beta Actin was detected using an anti-Beta Actin monoclonal antibody (1/10 000, Sigma-Aldrich, Darmstadt, Germany) and a peroxidase-conjugated anti-mouse antibody (1/10 000, Dianova, Hamburg, Germany).

    Techniques: Western Blot, Infection, Staining

    CD163 protein induction in THP-1 macrophages challenged with a single LPS stimulation Representative Westernblot images of CD163 and Beta-Actin proteins in LPS-stimulated THP-1 macrophages transfected with Man-PEI complexed with an empty vector (pEmp or pEmpty), a plasmid encoding for CD163 gene (pCD163) or the Man-PEI nanoparticle alone (A). Quantification of the relative expression (band density) of CD163 in LPS-stimulated THP-1 macrophages from 48 hours after transfection (B). The quantification of the relative density was normalized to the respective levels in the pEmpty group, which was assigned a value equal to 1. N = 10 for pCD163 or pEmpty group. *p

    Journal: Immunobiology

    Article Title: Macrophage-specific nanotechnology driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions

    doi: 10.1016/j.imbio.2017.05.011

    Figure Lengend Snippet: CD163 protein induction in THP-1 macrophages challenged with a single LPS stimulation Representative Westernblot images of CD163 and Beta-Actin proteins in LPS-stimulated THP-1 macrophages transfected with Man-PEI complexed with an empty vector (pEmp or pEmpty), a plasmid encoding for CD163 gene (pCD163) or the Man-PEI nanoparticle alone (A). Quantification of the relative expression (band density) of CD163 in LPS-stimulated THP-1 macrophages from 48 hours after transfection (B). The quantification of the relative density was normalized to the respective levels in the pEmpty group, which was assigned a value equal to 1. N = 10 for pCD163 or pEmpty group. *p

    Article Snippet: Blots were stripped (10 min at room temperature) and re-probed with a mouse anti-Beta-Actin antibody incubated overnight at 4°C (1:3000; Sigma Aldrich, St. Louis, MO).

    Techniques: Transfection, Plasmid Preparation, Expressing

    Immunocytochemical characterization. Confluent HUVEC cell layers after 24 h of NAC supplemented culture. In 15 mM NAC enriched cultivation, endothelial cells changed their morphology from typical cobblestone pattern (a–d) to an elongated, quasi-fusiform configuration (i–l). A slight cellular rearrangement was already detectable at 10 mM (e–h). Cells were positive for common endothelial cell markers CD31 (PECAM-1) and Von-Willebrand-Factor (vWF). Nuclei were counterstained with DAPI. All cells were negative for alpha smooth muscle actin (αSMA), a common myofibroblast marker (scale bar: 100 µ m).

    Journal: Cellular and Molecular Bioengineering

    Article Title: Towards a Biohybrid Lung Assist Device: N-Acetylcysteine Reduces Oxygen Toxicity and Changes Endothelial Cells’ Morphology

    doi: 10.1007/s12195-016-0473-4

    Figure Lengend Snippet: Immunocytochemical characterization. Confluent HUVEC cell layers after 24 h of NAC supplemented culture. In 15 mM NAC enriched cultivation, endothelial cells changed their morphology from typical cobblestone pattern (a–d) to an elongated, quasi-fusiform configuration (i–l). A slight cellular rearrangement was already detectable at 10 mM (e–h). Cells were positive for common endothelial cell markers CD31 (PECAM-1) and Von-Willebrand-Factor (vWF). Nuclei were counterstained with DAPI. All cells were negative for alpha smooth muscle actin (αSMA), a common myofibroblast marker (scale bar: 100 µ m).

    Article Snippet: After washing cells, now using permeabilizing washing buffer (0.1% Triton in PBS), staining procedure for intracellular markers was undertaken using primary antibody rabbit-against vWF (Dako, A0082, 1:100) with secondary antibody goat-anti-rabbit (Alexa Fluor 594, Invitrogen A11012, 1:400) and primary antibody mouse against αSMA (Sigma, A2547) with secondary antibody goat-anti-mouse (Invitrogen, Alexa Fluor 594, A11005).

    Techniques: Marker

    MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin. (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P

    Journal: International journal of oncology

    Article Title: MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion

    doi:

    Figure Lengend Snippet: MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin. (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P

    Article Snippet: Anti-β-actin and donkey anti-rabbit IgG HRP-conjugated secondary antibody were purchased from Sigma (St. Louis, MO) and Amersham Biosciences (Piscataway, NJ), respectively.

    Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR

    Effects of MUC1 knockdown or overexpression on multiple cell signals in NSCLC cells. (A) H358, HCC827, and H441 cells were exposed to siRNA for 72 h, and (B) A549 cells were transfected with MUC1-C plasmid for 48 h followed by measurement of phosphorylated tyrosine STAT3, Akt, Src, FAK, and apoptotic-related proteins by Western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin.

    Journal: International journal of oncology

    Article Title: MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion

    doi:

    Figure Lengend Snippet: Effects of MUC1 knockdown or overexpression on multiple cell signals in NSCLC cells. (A) H358, HCC827, and H441 cells were exposed to siRNA for 72 h, and (B) A549 cells were transfected with MUC1-C plasmid for 48 h followed by measurement of phosphorylated tyrosine STAT3, Akt, Src, FAK, and apoptotic-related proteins by Western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin.

    Article Snippet: Anti-β-actin and donkey anti-rabbit IgG HRP-conjugated secondary antibody were purchased from Sigma (St. Louis, MO) and Amersham Biosciences (Piscataway, NJ), respectively.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot

    Increased expression of arterial Ca 2+ transport proteins following a 14 day icv Ang II infusion. Panels A , B and C , representative Western blots and summary data for NCX1, TRPC6 and SERCA2 expression, respectively, in de-endothelialized aorta. Blots were normalized for β-actin as a loading control and the changes shown are relative to the control group. All lanes were loaded with 10–20 µg of membrane protein. The expected molecular weights are: NCX1, 116 KDa; TRPC6, 111 KDa, SERCA2 115 KDA, and β-actin, 42 KDa. C = vehicle control; A = Ang II; A+E = Ang II + eplerenone; A+F = Ang II + FAD-286; *P

    Journal: PLoS ONE

    Article Title: Neuroendocrine Humoral and Vascular Components in the Pressor Pathway for Brain Angiotensin II: A New Axis in Long Term Blood Pressure Control

    doi: 10.1371/journal.pone.0108916

    Figure Lengend Snippet: Increased expression of arterial Ca 2+ transport proteins following a 14 day icv Ang II infusion. Panels A , B and C , representative Western blots and summary data for NCX1, TRPC6 and SERCA2 expression, respectively, in de-endothelialized aorta. Blots were normalized for β-actin as a loading control and the changes shown are relative to the control group. All lanes were loaded with 10–20 µg of membrane protein. The expected molecular weights are: NCX1, 116 KDa; TRPC6, 111 KDa, SERCA2 115 KDA, and β-actin, 42 KDa. C = vehicle control; A = Ang II; A+E = Ang II + eplerenone; A+F = Ang II + FAD-286; *P

    Article Snippet: Gel loading was controlled with monoclonal anti-β-actin antibodies (dilution 1∶10,000; Sigma-Aldrich).

    Techniques: Expressing, Western Blot

    The 3′-UTR of ECE-1 represses ECE-1 protein expression. PC-3, RWPE-1, Huh7 and CHO cells were transiently transfected with empty vector (1), V5-tagged ECE-1c cDNA (2) or V5-tagged ECE-1c cDNA with full-length 3′ UTR (3) and immunoblotted for V5 (A). β-actin was used as a loading control. The PC-3 blot was reprobed for ECE-1 expression. Data represent the ratio of V5:actin expression ± S.E.M. (B; n = 3, * = P

    Journal: PLoS ONE

    Article Title: Endothelin-Converting Enzyme-1 (ECE-1) Is Post-Transcriptionally Regulated by Alternative Polyadenylation

    doi: 10.1371/journal.pone.0083260

    Figure Lengend Snippet: The 3′-UTR of ECE-1 represses ECE-1 protein expression. PC-3, RWPE-1, Huh7 and CHO cells were transiently transfected with empty vector (1), V5-tagged ECE-1c cDNA (2) or V5-tagged ECE-1c cDNA with full-length 3′ UTR (3) and immunoblotted for V5 (A). β-actin was used as a loading control. The PC-3 blot was reprobed for ECE-1 expression. Data represent the ratio of V5:actin expression ± S.E.M. (B; n = 3, * = P

    Article Snippet: The following antibodies were used at the following dilutions: monoclonal anti-V5 antibody 1∶500 (Invitrogen), goat polyclonal anti-ECE-1 1:1000 (R & D Systems), rabbit polyclonal anti-Upf1 1:200 (Santa Cruz) and monoclonal anti-β-actin 1∶10,000 (Sigma).

    Techniques: Expressing, Transfection, Plasmid Preparation

    Effect of TSN on expression of HCV replicon. (A) HCV replicon cells were treated with various concentrations of TSN for 48 h. Replication levels of HCV RNA were analyzed by luciferase assay. Bars indicate luciferase activities relative to that of the drug-negative control. (B) Cell viability was determined by MTS assay. Bars indicate the value relative to that of the drug-negative control. (C) Western blotting analyses. The expression of NS5A and beta-actin was detected using anti-NS5A and anti-beta-actin antibodies. Densitometry of NS5A protein was performed, and the result is indicated as a percentage of the result for the drug-negative control. The assay was repeated three times, and a representative result is shown. (D) A bicistronic reporter gene plasmid, pCIneo-Rluc-IRES-Fluc, was transfected into Huh7 cells. The cells were cultured with TSN at the concentrations indicated, and dual luciferase activities were measured after 24 h of treatment. Values are displayed as ratios of Fluc to Rluc. In panels A, B, and D, the assays were done in triplicate and repeated three times. Error bars indicate means ± SDs.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿ †

    doi: 10.1128/AAC.01780-10

    Figure Lengend Snippet: Effect of TSN on expression of HCV replicon. (A) HCV replicon cells were treated with various concentrations of TSN for 48 h. Replication levels of HCV RNA were analyzed by luciferase assay. Bars indicate luciferase activities relative to that of the drug-negative control. (B) Cell viability was determined by MTS assay. Bars indicate the value relative to that of the drug-negative control. (C) Western blotting analyses. The expression of NS5A and beta-actin was detected using anti-NS5A and anti-beta-actin antibodies. Densitometry of NS5A protein was performed, and the result is indicated as a percentage of the result for the drug-negative control. The assay was repeated three times, and a representative result is shown. (D) A bicistronic reporter gene plasmid, pCIneo-Rluc-IRES-Fluc, was transfected into Huh7 cells. The cells were cultured with TSN at the concentrations indicated, and dual luciferase activities were measured after 24 h of treatment. Values are displayed as ratios of Fluc to Rluc. In panels A, B, and D, the assays were done in triplicate and repeated three times. Error bars indicate means ± SDs.

    Article Snippet: The antibodies used were mouse anti-NS5A (BioDesign, ME), rabbit anti-signal transducer and activator of transcription 1 (anti-STAT1) p84/p91, rabbit anti-phospho-STAT1 (Tyr 701), rabbit anti-STAT2, rabbit anti-phospho-STAT2 (Tyr 690) (Santa Cruz, CA), and anti-beta-actin antibody (Sigma).

    Techniques: Expressing, Luciferase, Negative Control, MTS Assay, Western Blot, Plasmid Preparation, Transfection, Cell Culture

    ISRE reporter screening and aberrant pathway of α-IFN. (A) Pretreatment with TSN. Huh7 cells transfected with a reporter gene (pISRE-Luc and pRL-CMV) were pretreated with TSN (0 or 100 nM) for 0, 24, or 48 h, followed by treatment with α-IFN (0 or 100 IU/ml). Six hours later, the relative ISRE-luciferase activity ( n = 4) was determined as described in Materials and Methods. The data are expressed as means ± SDs and are a representative example of the data from three similar experiments. (B) Pretreatment with TSN at the concentrations indicated for 24 h, followed by treatment with α-IFN (0 to 100 IU/ml). The ISRE reporter assay was performed as described for panel A. (C) Type I IFN-induced antiviral ISG expression in Huh7 cells. Huh7 cells were treated with TSN for 24 h, followed by treatment with α-IFN at 100 IU/ml for 24 h. The total cellular RNA was then isolated for real-time RT-PCR analysis of the mRNAs of 25AS, MxA, p56, viperin, and ISG20. Beta-actin was used as a control. The data are expressed as means ± SDs and are a representative example of data from three similar experiments. *, P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿ †

    doi: 10.1128/AAC.01780-10

    Figure Lengend Snippet: ISRE reporter screening and aberrant pathway of α-IFN. (A) Pretreatment with TSN. Huh7 cells transfected with a reporter gene (pISRE-Luc and pRL-CMV) were pretreated with TSN (0 or 100 nM) for 0, 24, or 48 h, followed by treatment with α-IFN (0 or 100 IU/ml). Six hours later, the relative ISRE-luciferase activity ( n = 4) was determined as described in Materials and Methods. The data are expressed as means ± SDs and are a representative example of the data from three similar experiments. (B) Pretreatment with TSN at the concentrations indicated for 24 h, followed by treatment with α-IFN (0 to 100 IU/ml). The ISRE reporter assay was performed as described for panel A. (C) Type I IFN-induced antiviral ISG expression in Huh7 cells. Huh7 cells were treated with TSN for 24 h, followed by treatment with α-IFN at 100 IU/ml for 24 h. The total cellular RNA was then isolated for real-time RT-PCR analysis of the mRNAs of 25AS, MxA, p56, viperin, and ISG20. Beta-actin was used as a control. The data are expressed as means ± SDs and are a representative example of data from three similar experiments. *, P

    Article Snippet: The antibodies used were mouse anti-NS5A (BioDesign, ME), rabbit anti-signal transducer and activator of transcription 1 (anti-STAT1) p84/p91, rabbit anti-phospho-STAT1 (Tyr 701), rabbit anti-STAT2, rabbit anti-phospho-STAT2 (Tyr 690) (Santa Cruz, CA), and anti-beta-actin antibody (Sigma).

    Techniques: Transfection, Luciferase, Activity Assay, Reporter Assay, Expressing, Isolation, Quantitative RT-PCR

    Suppression of HCV RNA replication by TSN combined with α-IFN. (A and B) Luciferase activity (A, absolute value; B, relative value). Huh7/Rep-Feo cells, which constitutively express an HCV replicon, enable the quantification of replication levels through the measurement of luciferase activity. Absolute and relative dose-response curves in the presence of 24 h of pretreatment of various concentrations of TSN (0, 0.01, 0.03 μg/ml) and α-IFN (0, 100 IU/ml). (A) Bars indicate luciferase activities. (B) Bars indicate luciferase activities relative to the activity of each α-IFN-negative control. Luciferase assays were performed in triplicate. Error bars indicate means ± SDs. (C) MTS assay of Huh7/Rep-Feo cells cultured with the indicated concentrations of TSN and α-IFN. The assays were done in triplicate and repeated three times. Error bars indicate means ± SDs. (D) Western blotting. Ten micrograms of total cellular protein was separated by polyacrylamide gel electrophoresis and transferred onto the membrane. Monoclonal anti-NS5A antibody or an anti-beta-actin antibody was used as the primary antibody. Densitometry of NS5A or beta-actin protein was performed and the result is indicated as a percentage of that for the drug-negative control. The assay was repeated three times, and representative results are shown. (E) Dose-inhibition curves of α-IFN and TSN when they were combined at the indicated ratios, adjusted by the EC 50 of the individual drug. Assays were done in triplicate, and mean values were plotted and indicated as means ± SDs. (F) Graphical representation of the isobologram analysis. For each drug combination in panel E, the EC 50 s of α-IFN and TSN for inhibition of HCV replication were plotted against the fractional concentrations of α-IFN and TSN, which are indicated on the x and y axes, respectively. A theoretical line of additivity is drawn between the EC 50 for each drug alone. All of the fractional EC 50 plots for the TSN and α-IFN combinations fell below the line of additivity, indicating synergy.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿ †

    doi: 10.1128/AAC.01780-10

    Figure Lengend Snippet: Suppression of HCV RNA replication by TSN combined with α-IFN. (A and B) Luciferase activity (A, absolute value; B, relative value). Huh7/Rep-Feo cells, which constitutively express an HCV replicon, enable the quantification of replication levels through the measurement of luciferase activity. Absolute and relative dose-response curves in the presence of 24 h of pretreatment of various concentrations of TSN (0, 0.01, 0.03 μg/ml) and α-IFN (0, 100 IU/ml). (A) Bars indicate luciferase activities. (B) Bars indicate luciferase activities relative to the activity of each α-IFN-negative control. Luciferase assays were performed in triplicate. Error bars indicate means ± SDs. (C) MTS assay of Huh7/Rep-Feo cells cultured with the indicated concentrations of TSN and α-IFN. The assays were done in triplicate and repeated three times. Error bars indicate means ± SDs. (D) Western blotting. Ten micrograms of total cellular protein was separated by polyacrylamide gel electrophoresis and transferred onto the membrane. Monoclonal anti-NS5A antibody or an anti-beta-actin antibody was used as the primary antibody. Densitometry of NS5A or beta-actin protein was performed and the result is indicated as a percentage of that for the drug-negative control. The assay was repeated three times, and representative results are shown. (E) Dose-inhibition curves of α-IFN and TSN when they were combined at the indicated ratios, adjusted by the EC 50 of the individual drug. Assays were done in triplicate, and mean values were plotted and indicated as means ± SDs. (F) Graphical representation of the isobologram analysis. For each drug combination in panel E, the EC 50 s of α-IFN and TSN for inhibition of HCV replication were plotted against the fractional concentrations of α-IFN and TSN, which are indicated on the x and y axes, respectively. A theoretical line of additivity is drawn between the EC 50 for each drug alone. All of the fractional EC 50 plots for the TSN and α-IFN combinations fell below the line of additivity, indicating synergy.

    Article Snippet: The antibodies used were mouse anti-NS5A (BioDesign, ME), rabbit anti-signal transducer and activator of transcription 1 (anti-STAT1) p84/p91, rabbit anti-phospho-STAT1 (Tyr 701), rabbit anti-STAT2, rabbit anti-phospho-STAT2 (Tyr 690) (Santa Cruz, CA), and anti-beta-actin antibody (Sigma).

    Techniques: Luciferase, Activity Assay, Negative Control, MTS Assay, Cell Culture, Western Blot, Polyacrylamide Gel Electrophoresis, Inhibition

    Knockdown of PTEN and overexpression of AKT reversed the clearance of Aβ caused by alborixin. ( A ) Confocal microscopy and flow cytometric analysis for clearance of amyloid beta (Aβ 1-42 -HiLyte Fluor 555) in siPTEN -transfected and alborixin-treated HMC3 cells. ( B ) Analysis for Aβ clearance in Myr-AKT delta4-129 -transfected HMC3 cells after treatment with alborixin. ( C ) WT PTEN overexpression independent of alborixin caused clearance of Aβ, whereas, the catalytically inactive mutant PTEN C124S did not have any effect on the clearance of Aβ in HMC3 cells. Confocal images of HMC3 cells were taken after treatment with Aβ and alborixin (125 nM) for 12 h. Average RFI of at least 500 cells was used to calculate final average for the same sample from 3 independent experiments (3n). Scale bar used in confocal images: 10 µm. The X axis of dot plots in Figure 8A-C represents Aβ 1-42 -HiLyte Fluor 555 fluorescence. Statistical comparisons for Figure 8A-C were made by using Bonferroni test. p values ***p

    Journal: Autophagy

    Article Title: Alborixin clears amyloid-β by inducing autophagy through PTEN-mediated inhibition of the AKT pathway

    doi: 10.1080/15548627.2019.1596476

    Figure Lengend Snippet: Knockdown of PTEN and overexpression of AKT reversed the clearance of Aβ caused by alborixin. ( A ) Confocal microscopy and flow cytometric analysis for clearance of amyloid beta (Aβ 1-42 -HiLyte Fluor 555) in siPTEN -transfected and alborixin-treated HMC3 cells. ( B ) Analysis for Aβ clearance in Myr-AKT delta4-129 -transfected HMC3 cells after treatment with alborixin. ( C ) WT PTEN overexpression independent of alborixin caused clearance of Aβ, whereas, the catalytically inactive mutant PTEN C124S did not have any effect on the clearance of Aβ in HMC3 cells. Confocal images of HMC3 cells were taken after treatment with Aβ and alborixin (125 nM) for 12 h. Average RFI of at least 500 cells was used to calculate final average for the same sample from 3 independent experiments (3n). Scale bar used in confocal images: 10 µm. The X axis of dot plots in Figure 8A-C represents Aβ 1-42 -HiLyte Fluor 555 fluorescence. Statistical comparisons for Figure 8A-C were made by using Bonferroni test. p values ***p

    Article Snippet: Reagents include the following: DMEM (Sigma-Aldrich, D1152), RPM1-1640 (Sigma Aldrich, R6504), MEM (Sigma-Aldrich, M0894) and F12 (Sigma-Aldrich, N3520), non-essential amino acids (Sigma, M7145), streptomycin (Sigma-Aldrich, S6501), penicillin (Sigma-Aldrich, P3032), rapamycin (Sigma-Aldrich, R8781), bafilomycin A1 (Sigma-Aldrich, B1793), sodium bicarbonate (Sigma-Aldrich, S5761), sodium pyruvate (Sigma-Aldrich, P2256), PBS (Sigma-Aldrich, D5652), 4, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, D9542), 2,7-dichlorodihydrofluorescein diacetate (DCFH2ַ -DA) (Sigma-Aldrich, D6883), dimethylsulfoxide (DMSO; Sigma-Aldrich, D2650), thioflavin S (Sigma-Aldrich, T1892), papain (Sigma-Aldrich, 76220), TMRE (Sigma-Aldrich, 87917), poly-L-lysine (Sigma-Aldrich, P4707), RIPA buffer (Sigma-Aldrich, R0278), BSA (Sigma-Aldrich, A2153), skimmed milk (Sigma-Aldrich, 70166), acrylamide (Sigma-Aldrich, A9099), N,N′-Methylenebis acrylamide (Sigma-Aldrich, M7279), sodium orthovanadate (Sigma-Aldrich, 450243), sodium fluoride (Sigma-Aldrich, 215309), glycine (Sigma-Aldrich, G8898), EDTA (Sigma-Aldrich, EDS), triton X-100 (Sigma-Aldrich, T8787), tween 20 (Sigma-Aldrich, P7949), trizma (Sigma-Aldrich, T6066), sodium dodecyl sulphate (Sigma-Aldrich, L3771), sodium chloride (Sigma-Aldrich, S7653), ammonium persulfate (Sigma-Aldrich, A3678), TEMED (Sigma-Aldrich, T7024), glycerol (Sigma-Aldrich, G5516), HEPES (Sigma-Aldrich, H3375), paraformaldehyde (Sigma-Aldrich, P6148), osmium tetroxide solution (Sigma-Aldrich, 75632), glutaraldehyde solution (Sigma-Aldrich, G5882), uranyl acetate (British Drug Houses, 6159-44-0), protease inhibitor cocktail (Sigma-Aldrich, P8340), Aβ1-42 peptide (Sigma-Aldrich, A9810), anti-LC3B-II (Sigma-Aldrich, L7543), anti-SQSTM1 (Sigma-Aldrich, P0067), anti-GFAP (Sigma-Aldrich, G3893), anti-ACTB (Sigma-Aldrich, A3854), sulforhodamine B dye (Sigma-Aldrich, S1402), hibernated media (Gibco, A12476), neurobasal media (Gibco, 21103049), OPTI-MEM media (Gibco, 11058-021), B-27™ Supplement (Gibco, 17504044), glutamax (Gibco, 35050-061), trypsin (Gibco, 25300-054), fetal bovine serum (Gibco, 10270), LysoTracker® Red DND-99 (Thermo Fisher Scientific, L7528), and lipofectamine 3000 (Thermo Fisher Scientific, L3000-001), 3-(4, 5, -dimethylthiazole-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) (Alpha Aesar, ), Aβ1-42 -HiLyte FluorTM 555 (Anaspec, 60480) and Aβ1-42 -HiLyte FluorTM 488 (Anaspec, AS-60479).

    Techniques: Over Expression, Confocal Microscopy, Transfection, Mutagenesis, Fluorescence

    Alborixin enhanced the basal level of autophagy induced by soluble amyloid beta (sAβ) and fAβ. Representative images of transmission electron microscopy (TEM), ( A ) sAβ and ( B ) fibrillar amyloid beta (fAβ). Scale bars: (A) 200 nm, (B) 100 nm. ( C and D ) Level of LC3B-II was increased and that of SQSTM1 was reduced after co-treatment of N9 cells with either soluble or fibrillar forms of Aβ and alborixin 125 nM. Bafilomycin A 1 (20 nM) reversed the effect of alborixin. Rapamycin (200 nM) in this experiment was used as a standard. Autophagic flux was calculated by using LC3B-II:ACTB in the absence and presence of bafilomycin A 1 . Ratio of values obtained through densitometry of western blots were quantified by using ImageJ software. Data presented here are mean±SD of 3 independent experiments and the blots shown in this figure are representative images. Statistical comparisons were made between samples treated with either sAβ or fAβ and all other samples by using Bonferroni test. p values ***p

    Journal: Autophagy

    Article Title: Alborixin clears amyloid-β by inducing autophagy through PTEN-mediated inhibition of the AKT pathway

    doi: 10.1080/15548627.2019.1596476

    Figure Lengend Snippet: Alborixin enhanced the basal level of autophagy induced by soluble amyloid beta (sAβ) and fAβ. Representative images of transmission electron microscopy (TEM), ( A ) sAβ and ( B ) fibrillar amyloid beta (fAβ). Scale bars: (A) 200 nm, (B) 100 nm. ( C and D ) Level of LC3B-II was increased and that of SQSTM1 was reduced after co-treatment of N9 cells with either soluble or fibrillar forms of Aβ and alborixin 125 nM. Bafilomycin A 1 (20 nM) reversed the effect of alborixin. Rapamycin (200 nM) in this experiment was used as a standard. Autophagic flux was calculated by using LC3B-II:ACTB in the absence and presence of bafilomycin A 1 . Ratio of values obtained through densitometry of western blots were quantified by using ImageJ software. Data presented here are mean±SD of 3 independent experiments and the blots shown in this figure are representative images. Statistical comparisons were made between samples treated with either sAβ or fAβ and all other samples by using Bonferroni test. p values ***p

    Article Snippet: Reagents include the following: DMEM (Sigma-Aldrich, D1152), RPM1-1640 (Sigma Aldrich, R6504), MEM (Sigma-Aldrich, M0894) and F12 (Sigma-Aldrich, N3520), non-essential amino acids (Sigma, M7145), streptomycin (Sigma-Aldrich, S6501), penicillin (Sigma-Aldrich, P3032), rapamycin (Sigma-Aldrich, R8781), bafilomycin A1 (Sigma-Aldrich, B1793), sodium bicarbonate (Sigma-Aldrich, S5761), sodium pyruvate (Sigma-Aldrich, P2256), PBS (Sigma-Aldrich, D5652), 4, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, D9542), 2,7-dichlorodihydrofluorescein diacetate (DCFH2ַ -DA) (Sigma-Aldrich, D6883), dimethylsulfoxide (DMSO; Sigma-Aldrich, D2650), thioflavin S (Sigma-Aldrich, T1892), papain (Sigma-Aldrich, 76220), TMRE (Sigma-Aldrich, 87917), poly-L-lysine (Sigma-Aldrich, P4707), RIPA buffer (Sigma-Aldrich, R0278), BSA (Sigma-Aldrich, A2153), skimmed milk (Sigma-Aldrich, 70166), acrylamide (Sigma-Aldrich, A9099), N,N′-Methylenebis acrylamide (Sigma-Aldrich, M7279), sodium orthovanadate (Sigma-Aldrich, 450243), sodium fluoride (Sigma-Aldrich, 215309), glycine (Sigma-Aldrich, G8898), EDTA (Sigma-Aldrich, EDS), triton X-100 (Sigma-Aldrich, T8787), tween 20 (Sigma-Aldrich, P7949), trizma (Sigma-Aldrich, T6066), sodium dodecyl sulphate (Sigma-Aldrich, L3771), sodium chloride (Sigma-Aldrich, S7653), ammonium persulfate (Sigma-Aldrich, A3678), TEMED (Sigma-Aldrich, T7024), glycerol (Sigma-Aldrich, G5516), HEPES (Sigma-Aldrich, H3375), paraformaldehyde (Sigma-Aldrich, P6148), osmium tetroxide solution (Sigma-Aldrich, 75632), glutaraldehyde solution (Sigma-Aldrich, G5882), uranyl acetate (British Drug Houses, 6159-44-0), protease inhibitor cocktail (Sigma-Aldrich, P8340), Aβ1-42 peptide (Sigma-Aldrich, A9810), anti-LC3B-II (Sigma-Aldrich, L7543), anti-SQSTM1 (Sigma-Aldrich, P0067), anti-GFAP (Sigma-Aldrich, G3893), anti-ACTB (Sigma-Aldrich, A3854), sulforhodamine B dye (Sigma-Aldrich, S1402), hibernated media (Gibco, A12476), neurobasal media (Gibco, 21103049), OPTI-MEM media (Gibco, 11058-021), B-27™ Supplement (Gibco, 17504044), glutamax (Gibco, 35050-061), trypsin (Gibco, 25300-054), fetal bovine serum (Gibco, 10270), LysoTracker® Red DND-99 (Thermo Fisher Scientific, L7528), and lipofectamine 3000 (Thermo Fisher Scientific, L3000-001), 3-(4, 5, -dimethylthiazole-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) (Alpha Aesar, ), Aβ1-42 -HiLyte FluorTM 555 (Anaspec, 60480) and Aβ1-42 -HiLyte FluorTM 488 (Anaspec, AS-60479).

    Techniques: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Western Blot, Software

    Hyperacetylation of microtubules in salivary gland increases K14+ progenitor cells. Representative confocal images of untreated salivary glands (Control–no virus) or glands expressing green fluorescent protein (GFP) (Control-GFP) or GFP-tagged wild-type tubulin (GFP-WT-MT), acetyl-null tubulin K40A (GFP-HypoAcMT-K40A), or acetyl-mimetic tubulin K40Q (GFP-HyperAcMT-K40Q) as indicated and ( A ) immunostained for cytokeratin 14 (K14), cytokeratin 5 (K5), or cytokeratin 19 (K19) or ( C ) immunostained for cytokeratin 14 (K14), alpha smooth muscle myosin (αSMA), or E-cadherin (Ecad). Arrowheads point to basal and suprabasal layers of the epithelium in distal endbuds. Significantly increased K14+ and/or αSMA+ immunostaining of cells in the distal endbuds is observed in HyperAcMT (K40Q) expressing glands. Fluorescence intensity of the cytokeratins (K5, K14, or K19, B ) or αSMA ( D ) in the basal and suprabasal layers of the epithelium as outlined by the dotted white lines in panels A or C, respectively, was quantified and expressed as relative to the GFP control ( N > 3 experiments, n > 12 glands). Data are mean ± SEM. * P

    Journal: Journal of Dental Research

    Article Title: Hyperacetylation of Microtubules in Mesenchymal Cells Increases Cytokeratin 14–Positive Epithelial Progenitors in Developing Salivary Glands

    doi: 10.1177/0022034516662450

    Figure Lengend Snippet: Hyperacetylation of microtubules in salivary gland increases K14+ progenitor cells. Representative confocal images of untreated salivary glands (Control–no virus) or glands expressing green fluorescent protein (GFP) (Control-GFP) or GFP-tagged wild-type tubulin (GFP-WT-MT), acetyl-null tubulin K40A (GFP-HypoAcMT-K40A), or acetyl-mimetic tubulin K40Q (GFP-HyperAcMT-K40Q) as indicated and ( A ) immunostained for cytokeratin 14 (K14), cytokeratin 5 (K5), or cytokeratin 19 (K19) or ( C ) immunostained for cytokeratin 14 (K14), alpha smooth muscle myosin (αSMA), or E-cadherin (Ecad). Arrowheads point to basal and suprabasal layers of the epithelium in distal endbuds. Significantly increased K14+ and/or αSMA+ immunostaining of cells in the distal endbuds is observed in HyperAcMT (K40Q) expressing glands. Fluorescence intensity of the cytokeratins (K5, K14, or K19, B ) or αSMA ( D ) in the basal and suprabasal layers of the epithelium as outlined by the dotted white lines in panels A or C, respectively, was quantified and expressed as relative to the GFP control ( N > 3 experiments, n > 12 glands). Data are mean ± SEM. * P

    Article Snippet: Glands were then incubated with blocking buffer (0.05% Tween-20, 5% donkey serum, 1% bovine serum albumin [BSA], and M.O.M. blocking reagent; Vector Laboratories) in phosphate-buffered saline (PBS) before incubating with either primary or secondary antibodies in PBS supplemented with 0.05% Tween-20, 5% donkey serum, and 8% M.O.M. at 4 °C to 23 °C for 2 to 18 h. The primary antibodies used for immunostaining were anti–cytokeratin 14 (1:300; Covance PRB-155P-100), anti–cytokeratin 5 (1:2,000; Covance PRB-160P-100), anti–cytokeratin 19 (1:300; DSHB Troma-III-c), anti–Sox 10 (1:200; Santa Cruz D-20sc-17343), anti-KIT (1:200; mSCF MAB1356), anti–AQP 5 (1:400; Alomone AQP-005), anti–smooth muscle actin (1:200; Sigma A5228), anti-Ki67 (1:300; BD Bioscience 550609), anti-TGFβ (1:200; Abcam ab66043), anti-Hey2 (1:100; Sigma HPA030205), anti–collagen IV (1:400; Millipore AB769), anti–E-cadherin (1:100; Invitrogen 131900), anti–E-cadherin (1:200; BD Biosciences 610182), and anti–E-cadherin (1:100; Cell Signaling 3195S).

    Techniques: Expressing, Immunostaining, Fluorescence

    Microtubules, actin filaments and lipid rafts arrangement during spermiogenesis. Spermatogenic isolated cells were analyzed to test the components of the sperm head elongation complex. The microtubules of the manchette were detected using alpha-tubulin antibody and secondary antibody combined with FITC (tubulin columns). The actin filaments were stained with actin antibody conjugated with Cy3 (actin columns). GM1-enriched lipid rafts were detected using cholera toxin conjugated with Alexa flour 594 (GM1 column, red signal) or FITC (GM1 column, green signal). The combination of green and red colors (merge columns) and phase contrast images were also included (CC columns). In NCR, microtubules, actin filaments and GM1 co-localized and were distributed in the manchette zone (mz) opposed to the acrosome zone (az). In HCARDA, it was not possible to detect manchette or acrosome zone. Microtubules, actin filaments and GM1 were equally distributed. Also, an asymmetrical acrosome was observed (asterisk). In ½ HCARDA + ½ OO, cells were polarized and manchette and acrosome zone were detected. Magnification: 620X.

    Journal: PLoS ONE

    Article Title: Manchette-acrosome disorders and testicular efficiency decline observed in hypercholesterolemic rabbits are recovered with olive oil enriched diet

    doi: 10.1371/journal.pone.0202748

    Figure Lengend Snippet: Microtubules, actin filaments and lipid rafts arrangement during spermiogenesis. Spermatogenic isolated cells were analyzed to test the components of the sperm head elongation complex. The microtubules of the manchette were detected using alpha-tubulin antibody and secondary antibody combined with FITC (tubulin columns). The actin filaments were stained with actin antibody conjugated with Cy3 (actin columns). GM1-enriched lipid rafts were detected using cholera toxin conjugated with Alexa flour 594 (GM1 column, red signal) or FITC (GM1 column, green signal). The combination of green and red colors (merge columns) and phase contrast images were also included (CC columns). In NCR, microtubules, actin filaments and GM1 co-localized and were distributed in the manchette zone (mz) opposed to the acrosome zone (az). In HCARDA, it was not possible to detect manchette or acrosome zone. Microtubules, actin filaments and GM1 were equally distributed. Also, an asymmetrical acrosome was observed (asterisk). In ½ HCARDA + ½ OO, cells were polarized and manchette and acrosome zone were detected. Magnification: 620X.

    Article Snippet: Then, cells were incubated in darkness with different markers alone or combined: antibody against α-tubulin (1:50, MP Biomedicals, 691251) detected with the secondary antibody conjugated with biotin (pan-specific antibody, Vector, PK7800) and avidin-fluorescein complex (Vector, SA5001); antibody against actin conjugated with Cy3 (3 μg/ml, Sigma, C6198, generous gift from Lopez`s lab, IHEM, Mendoza, Argentina); and cholera toxin subunit β conjugated with Alexa fluor 594 (5 μg/ml, MP Biomedicals, ) or fluorescein (Sigma, C1655).

    Techniques: Isolation, Staining

    IGF-IR and IGF-IIR expression, and VSMCs content in human carotid atherosclerotic plaques. a Representative gels and quantifications of IGF-IR and IGF-IIR protein levels in supernatants from serial immunoprecipitations (IRB and IRA) of non-complicated regions (n = 10) and their respective complicated plaques (n = 10). b Analysis of VSMCs content by Western blot against α-SMA in non-complicated regions (n = 10) and their respective complicated plaques (n = 10). ***p

    Journal: Cardiovascular Diabetology

    Article Title: Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis

    doi: 10.1186/s12933-018-0675-2

    Figure Lengend Snippet: IGF-IR and IGF-IIR expression, and VSMCs content in human carotid atherosclerotic plaques. a Representative gels and quantifications of IGF-IR and IGF-IIR protein levels in supernatants from serial immunoprecipitations (IRB and IRA) of non-complicated regions (n = 10) and their respective complicated plaques (n = 10). b Analysis of VSMCs content by Western blot against α-SMA in non-complicated regions (n = 10) and their respective complicated plaques (n = 10). ***p

    Article Snippet: The antibodies used were anti-IRβ, IGF-IRβ and IGF-IIR from Santa Cruz Biotechnology (Dallas, TX, USA); IRS-1 from Millipore (Billerica, MA, USA); p-IRS-1 (Ser307), p-AKT (Thr308), p-p42/44 MAPK (Thr202/Tyr204) and Cleaved Caspase-3 (Asp175) from Cell Signaling Technology (Danvers, MA, USA); anti-β-actin, α-SMA and α-tubulin from Sigma-Aldrich Corp. (St. Louis, MO, USA).

    Techniques: Expressing, Western Blot

    VSMCs content and apoptosis in atherosclerotic plaques from the 24-week-old experimental model. Representative photomicrographs and quantification of immunofluorescence against α-SMA ( a ) and of immunohistochemistry against cleaved PARP ( b ) in aortic roots from 24-week-old Control, ApoE −/− and BATIRKO; ApoE −/− mice. DAPI staining was performed to localize nuclei of cells presented in aortic roots (blue staining). ***p

    Journal: Cardiovascular Diabetology

    Article Title: Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis

    doi: 10.1186/s12933-018-0675-2

    Figure Lengend Snippet: VSMCs content and apoptosis in atherosclerotic plaques from the 24-week-old experimental model. Representative photomicrographs and quantification of immunofluorescence against α-SMA ( a ) and of immunohistochemistry against cleaved PARP ( b ) in aortic roots from 24-week-old Control, ApoE −/− and BATIRKO; ApoE −/− mice. DAPI staining was performed to localize nuclei of cells presented in aortic roots (blue staining). ***p

    Article Snippet: The antibodies used were anti-IRβ, IGF-IRβ and IGF-IIR from Santa Cruz Biotechnology (Dallas, TX, USA); IRS-1 from Millipore (Billerica, MA, USA); p-IRS-1 (Ser307), p-AKT (Thr308), p-p42/44 MAPK (Thr202/Tyr204) and Cleaved Caspase-3 (Asp175) from Cell Signaling Technology (Danvers, MA, USA); anti-β-actin, α-SMA and α-tubulin from Sigma-Aldrich Corp. (St. Louis, MO, USA).

    Techniques: Immunofluorescence, Immunohistochemistry, Mouse Assay, Staining