mouse anti human β actin  (Millipore)


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    Structured Review

    Millipore mouse anti human β actin
    Induction of EMT in PTECs by serum proteins. (A) The potential of the human PTEC cell line HKC8 to undergo EMT was demonstrated by downregulation of E-cadherin and upregulation of α-SMA proteins by Western blotting in response to TGF-β1. Control cells were grown in medium without TGF-β1 (No Rx). Representative blot from two experiments. (B) FACS analysis detected C3d deposited on the surface of PTECs after exposure to NHS but not HIS. (C) Incubation with NHS (5 d) reduced E-cadherin expression and increased α-SMA expression by Western blot. (D and E) <t>β-Actin</t> was used to control for protein loading. The ratio of E-cadherin and α-SMA to β-actin was measured on triplicate cultures. (F) Loss of E-cadherin expression was seen by immunochemistry after exposure of cells to both TGF-β1 and NHS but not exposure to HIS. The reciprocal change in α-SMA expression was seen (representative of two experiments). (G) By real-time PCR (RT-PCR) analysis, incubation for 5 d with TGF-β or NHS increased the level of collagen I mRNA in PTECs ( n = 3 per experiment ± SEM). This did not occur with HIS.
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    Images

    1) Product Images from "C3a Mediates Epithelial-to-Mesenchymal Transition in Proteinuric Nephropathy"

    Article Title: C3a Mediates Epithelial-to-Mesenchymal Transition in Proteinuric Nephropathy

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2008040434

    Induction of EMT in PTECs by serum proteins. (A) The potential of the human PTEC cell line HKC8 to undergo EMT was demonstrated by downregulation of E-cadherin and upregulation of α-SMA proteins by Western blotting in response to TGF-β1. Control cells were grown in medium without TGF-β1 (No Rx). Representative blot from two experiments. (B) FACS analysis detected C3d deposited on the surface of PTECs after exposure to NHS but not HIS. (C) Incubation with NHS (5 d) reduced E-cadherin expression and increased α-SMA expression by Western blot. (D and E) β-Actin was used to control for protein loading. The ratio of E-cadherin and α-SMA to β-actin was measured on triplicate cultures. (F) Loss of E-cadherin expression was seen by immunochemistry after exposure of cells to both TGF-β1 and NHS but not exposure to HIS. The reciprocal change in α-SMA expression was seen (representative of two experiments). (G) By real-time PCR (RT-PCR) analysis, incubation for 5 d with TGF-β or NHS increased the level of collagen I mRNA in PTECs ( n = 3 per experiment ± SEM). This did not occur with HIS.
    Figure Legend Snippet: Induction of EMT in PTECs by serum proteins. (A) The potential of the human PTEC cell line HKC8 to undergo EMT was demonstrated by downregulation of E-cadherin and upregulation of α-SMA proteins by Western blotting in response to TGF-β1. Control cells were grown in medium without TGF-β1 (No Rx). Representative blot from two experiments. (B) FACS analysis detected C3d deposited on the surface of PTECs after exposure to NHS but not HIS. (C) Incubation with NHS (5 d) reduced E-cadherin expression and increased α-SMA expression by Western blot. (D and E) β-Actin was used to control for protein loading. The ratio of E-cadherin and α-SMA to β-actin was measured on triplicate cultures. (F) Loss of E-cadherin expression was seen by immunochemistry after exposure of cells to both TGF-β1 and NHS but not exposure to HIS. The reciprocal change in α-SMA expression was seen (representative of two experiments). (G) By real-time PCR (RT-PCR) analysis, incubation for 5 d with TGF-β or NHS increased the level of collagen I mRNA in PTECs ( n = 3 per experiment ± SEM). This did not occur with HIS.

    Techniques Used: Western Blot, FACS, Incubation, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    2) Product Images from "The Chloroform Fraction of Carpinus tschonoskii Leaves Inhibits the Production of Inflammatory Mediators in HaCaT Keratinocytes and RAW264.7 Macrophages"

    Article Title: The Chloroform Fraction of Carpinus tschonoskii Leaves Inhibits the Production of Inflammatory Mediators in HaCaT Keratinocytes and RAW264.7 Macrophages

    Journal: Toxicological Research

    doi: 10.5487/TR.2012.28.4.255

    Effect of chloroform fraction from Carpinus tschonoskii on the STAT1 signal in IFN-γ-stimulated HaCaT human keratinocytes. (A) Cells were pretreated with the indicated concentration of chloroform fraction of C. tschonoskii for 2 hr, and then the phosphorylation of STAT1 was determined in cells stimulated by IFN-γ (10 ng/ml) for 30 min. The levels of STAT1 and β-actin were identified using Western blotting analysis. (B) Cells were pretreated with the chloroform fraction (50 μg/ml) of C. tschonoskii for 2 hr, and then the translocation of STAT1 protein was determined in cells stimulated by IFN-γ (10 ng/ml) for 60 min. Immunofluorescence stain of STAT1 was stained with DyLight488-conjugated 2 nd antibody and the fluorescence was identified using confocal microscopy (FV500, OLYMPUS) and the images were acquired at constant conditions (PMT, gain, offset, magnification (20 × objective with zoom factor of 2.5) and resolution, etc.). These data are representative of three independent experiments.
    Figure Legend Snippet: Effect of chloroform fraction from Carpinus tschonoskii on the STAT1 signal in IFN-γ-stimulated HaCaT human keratinocytes. (A) Cells were pretreated with the indicated concentration of chloroform fraction of C. tschonoskii for 2 hr, and then the phosphorylation of STAT1 was determined in cells stimulated by IFN-γ (10 ng/ml) for 30 min. The levels of STAT1 and β-actin were identified using Western blotting analysis. (B) Cells were pretreated with the chloroform fraction (50 μg/ml) of C. tschonoskii for 2 hr, and then the translocation of STAT1 protein was determined in cells stimulated by IFN-γ (10 ng/ml) for 60 min. Immunofluorescence stain of STAT1 was stained with DyLight488-conjugated 2 nd antibody and the fluorescence was identified using confocal microscopy (FV500, OLYMPUS) and the images were acquired at constant conditions (PMT, gain, offset, magnification (20 × objective with zoom factor of 2.5) and resolution, etc.). These data are representative of three independent experiments.

    Techniques Used: Concentration Assay, Western Blot, Translocation Assay, Immunofluorescence, Staining, Fluorescence, Confocal Microscopy

    3) Product Images from "Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an in vitro model of CNS tuberculosis"

    Article Title: Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an in vitro model of CNS tuberculosis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16250-3

    CoMtb causes disruption of the blood-brain barrier. Blood-brain barrier (BBB) co-cultures, consisting of brain microvascular endothelial cells and astrocytes in transwells were stimulated for 72 h with control (CoMCont) or conditioned media from Mtb-infected monocytes (CoMtb). ( a ) Trans-endothelial resistance (TEER; Ω × cm 2 ) of BBB cultures with CoMCont or CoMtb stimulation (n = 3). Average background resistance of cell-free coated transwells for each time-point was subtracted. ( b ) Fold-change in BBB permeability to sodium-fluorescein (n = 3). ( c ) Fold-change in BBB permeability to 3KDa FITC-dextran (n = 3). ( d ) Confocal microscopy of the BBB in transwells coated with dye-quenched (DQ) type IV collagen and cells fixed and stained for nucleic acids with DAPI (blue). Green fluorescence depicts areas of collagen degradation. Scale bar: 50 μm. ( e ) Immunoblotting of tight junction proteins, using β-actin as loading control. Membranes were cut according to expected TJP or loading control protein size and each segment incubated with respective antibodies. ( f ) ZO-1, ( g ) Occludin and ( h ) Claudin-5 relative band densities of western blots, normalized to β-actin. Fold-change in ( i ) occludin mRNA (n = 3) and ( j ) claudin-5 mRNA accumulation with CoMtb stimulation (n = 3). β-actin mRNA was used as housekeeping control. Data is represented as mean ± s.d. *p
    Figure Legend Snippet: CoMtb causes disruption of the blood-brain barrier. Blood-brain barrier (BBB) co-cultures, consisting of brain microvascular endothelial cells and astrocytes in transwells were stimulated for 72 h with control (CoMCont) or conditioned media from Mtb-infected monocytes (CoMtb). ( a ) Trans-endothelial resistance (TEER; Ω × cm 2 ) of BBB cultures with CoMCont or CoMtb stimulation (n = 3). Average background resistance of cell-free coated transwells for each time-point was subtracted. ( b ) Fold-change in BBB permeability to sodium-fluorescein (n = 3). ( c ) Fold-change in BBB permeability to 3KDa FITC-dextran (n = 3). ( d ) Confocal microscopy of the BBB in transwells coated with dye-quenched (DQ) type IV collagen and cells fixed and stained for nucleic acids with DAPI (blue). Green fluorescence depicts areas of collagen degradation. Scale bar: 50 μm. ( e ) Immunoblotting of tight junction proteins, using β-actin as loading control. Membranes were cut according to expected TJP or loading control protein size and each segment incubated with respective antibodies. ( f ) ZO-1, ( g ) Occludin and ( h ) Claudin-5 relative band densities of western blots, normalized to β-actin. Fold-change in ( i ) occludin mRNA (n = 3) and ( j ) claudin-5 mRNA accumulation with CoMtb stimulation (n = 3). β-actin mRNA was used as housekeeping control. Data is represented as mean ± s.d. *p

    Techniques Used: Infection, Permeability, Confocal Microscopy, Staining, Fluorescence, Incubation, Western Blot

    4) Product Images from "Influenza infection directly alters innate IL-23 and IL-12p70 and subsequent IL-17A and IFN-γ responses to pneumococcus in vitro in human monocytes"

    Article Title: Influenza infection directly alters innate IL-23 and IL-12p70 and subsequent IL-17A and IFN-γ responses to pneumococcus in vitro in human monocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203521

    Modulation by IAV of innate and adaptive responses to Pneumococcus is not due to cell death or host protein synthesis shutdown. (A) CD14 + cells treated for 24 h with HKSP, live H1N1 or H3N2 alone or in combination with HKSP (or untreated as a control) were dual stained with FITC annexin V and propidium iodide. The percentages of viable, necrotic/late apoptotic, and early apoptotic cells after treatments were ascertained using flow cytometry. Each column represents mean % cell number + SEM of 3 independent donors. (B) Lysates from CD14 + cells treated for 24 h as outlined in (A) above, were prepared and analysed by Western blot for β-actin levels. Western blot is a representative of 2 independent repeats with different donors.
    Figure Legend Snippet: Modulation by IAV of innate and adaptive responses to Pneumococcus is not due to cell death or host protein synthesis shutdown. (A) CD14 + cells treated for 24 h with HKSP, live H1N1 or H3N2 alone or in combination with HKSP (or untreated as a control) were dual stained with FITC annexin V and propidium iodide. The percentages of viable, necrotic/late apoptotic, and early apoptotic cells after treatments were ascertained using flow cytometry. Each column represents mean % cell number + SEM of 3 independent donors. (B) Lysates from CD14 + cells treated for 24 h as outlined in (A) above, were prepared and analysed by Western blot for β-actin levels. Western blot is a representative of 2 independent repeats with different donors.

    Techniques Used: Staining, Flow Cytometry, Cytometry, Western Blot

    5) Product Images from "Novel function for the p38-MK2 signaling pathway in circulating CD1c+ (BDCA-1+) myeloid dendritic cells from healthy donors and advanced cancer patients; inhibition of p38 enhances IL-12 whilst suppressing IL-10"

    Article Title: Novel function for the p38-MK2 signaling pathway in circulating CD1c+ (BDCA-1+) myeloid dendritic cells from healthy donors and advanced cancer patients; inhibition of p38 enhances IL-12 whilst suppressing IL-10

    Journal: International Journal of Cancer. Journal International du Cancer

    doi: 10.1002/ijc.28398

    Comparison of intracellular signaling in myDC and moDC after TLR stimulation and effect of blockade of MAPK pathways in healthy donors. ( a ) MAPK/Rsk/GSK3β signaling. ( i ) Activation of Rsk by both ERK and p38 following TLR ligation in murine splenic and bone-marrow derived DC (arrow = activation), ( ii ) Signaling downstream of ERK and p38 in all other cells after TLR ligation where Rsk is only activated by ERK, ( iii ) Rsk drives GSK3β from the active form (GSK3β*) to the inactive form (GSK3β) with downstream effects on IL-12p70/IL-10 secretion, ( iv ) Sequence of phosphorylation/activation of Rsk by phospho-ERK (p-ERK) and phospho-p38 (p-p38). ( b ) Timecourse of MK2 activation following TLR stimulation in myDC and moDC. Donor-matched myDC and moDC (10 5 ) were stimulated with poly I:C/R848 for the indicated times before Western blotting. To allow cell-for-cell comparison between myDC and moDC, entire samples were used in a single Western blot and probed for phospho-MK2 (p-MK2) with β-actin as a loading control. Infrared secondary antibodies were used to allow for semi-quantitative analysis normalized to loading control. ( i ) Scanned images for one donor of two. ( ii ) Normalized p-MK2 for two donors, open circles = myDC, filled circles = moDC, dotted line = upper p-MK2 band, solid line = lower p-MK2 band. ( c ) Effect of blockade of p38 or MEK on MK2 activation following TLR stimulation in moDC. MoDC were pre-treated with p38 inhibitors SB203580 10 µM (S), BIRB0796 (B) 0.1 or 1.0 µM or MEK inhibitor UO126 10 µM (U) for 1hr followed by poly I:C/R848 stimulation for 30 min then Western blotted for p-MK2 with β-actin loading control as in ( b ). Shown are scanned images for three independent donors and graphs of normalized p-MK2 (gray bars = upper p-MK2 band, black bars = lower p-MK2 band). ( d ) Effect of blockade of p38, MEK or both on ERK, p38 and Rsk activation in moDC. MoDC were treated and analyzed as in ( c ) for phospho-ERK 1/2 (p-ERK 1/2), phospho-p38 (p-p38), phospho-Rsk (p-Rsk, T359/S363, T573 and S380 residues) with total Rsk as loading control. Pre-treatment was with p38 inhibitor BIRB0796 1.0 µM (B), MEK inhibitor UO126 10 µM (U) or both (BU). Shown are scanned images for three independent donors and graphs of normalised p-RskSer380 for each donor. ( e ) Effect of blockade of p38, MEK or both on activation of MK2 and Rsk at Ser380 in myDC. MyDC (three donors) were treated, harvested and analyzed as in ( c ) for p-MK2 and p-RskS380 with β-actin as loading control. Pre-treatment was with p38 inhibitor BIRB0796 1.0µM (B), MEK inhibitor UO126 10µM (U) or both (BU).
    Figure Legend Snippet: Comparison of intracellular signaling in myDC and moDC after TLR stimulation and effect of blockade of MAPK pathways in healthy donors. ( a ) MAPK/Rsk/GSK3β signaling. ( i ) Activation of Rsk by both ERK and p38 following TLR ligation in murine splenic and bone-marrow derived DC (arrow = activation), ( ii ) Signaling downstream of ERK and p38 in all other cells after TLR ligation where Rsk is only activated by ERK, ( iii ) Rsk drives GSK3β from the active form (GSK3β*) to the inactive form (GSK3β) with downstream effects on IL-12p70/IL-10 secretion, ( iv ) Sequence of phosphorylation/activation of Rsk by phospho-ERK (p-ERK) and phospho-p38 (p-p38). ( b ) Timecourse of MK2 activation following TLR stimulation in myDC and moDC. Donor-matched myDC and moDC (10 5 ) were stimulated with poly I:C/R848 for the indicated times before Western blotting. To allow cell-for-cell comparison between myDC and moDC, entire samples were used in a single Western blot and probed for phospho-MK2 (p-MK2) with β-actin as a loading control. Infrared secondary antibodies were used to allow for semi-quantitative analysis normalized to loading control. ( i ) Scanned images for one donor of two. ( ii ) Normalized p-MK2 for two donors, open circles = myDC, filled circles = moDC, dotted line = upper p-MK2 band, solid line = lower p-MK2 band. ( c ) Effect of blockade of p38 or MEK on MK2 activation following TLR stimulation in moDC. MoDC were pre-treated with p38 inhibitors SB203580 10 µM (S), BIRB0796 (B) 0.1 or 1.0 µM or MEK inhibitor UO126 10 µM (U) for 1hr followed by poly I:C/R848 stimulation for 30 min then Western blotted for p-MK2 with β-actin loading control as in ( b ). Shown are scanned images for three independent donors and graphs of normalized p-MK2 (gray bars = upper p-MK2 band, black bars = lower p-MK2 band). ( d ) Effect of blockade of p38, MEK or both on ERK, p38 and Rsk activation in moDC. MoDC were treated and analyzed as in ( c ) for phospho-ERK 1/2 (p-ERK 1/2), phospho-p38 (p-p38), phospho-Rsk (p-Rsk, T359/S363, T573 and S380 residues) with total Rsk as loading control. Pre-treatment was with p38 inhibitor BIRB0796 1.0 µM (B), MEK inhibitor UO126 10 µM (U) or both (BU). Shown are scanned images for three independent donors and graphs of normalised p-RskSer380 for each donor. ( e ) Effect of blockade of p38, MEK or both on activation of MK2 and Rsk at Ser380 in myDC. MyDC (three donors) were treated, harvested and analyzed as in ( c ) for p-MK2 and p-RskS380 with β-actin as loading control. Pre-treatment was with p38 inhibitor BIRB0796 1.0µM (B), MEK inhibitor UO126 10µM (U) or both (BU).

    Techniques Used: Activation Assay, Ligation, Derivative Assay, Sequencing, Western Blot

    6) Product Images from "Role of nucleotide-binding oligomerization domain 1 (NOD1) and its variants in human cytomegalovirus control in vitro and in vivo"

    Article Title: Role of nucleotide-binding oligomerization domain 1 (NOD1) and its variants in human cytomegalovirus control in vitro and in vivo

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

    doi: 10.1073/pnas.1611711113

    NOD1 downstream signaling in WT and mutant NOD1-overexpressing cells. ( A and B ) NF-κB ( A ) and IFN-β ( B ) luciferase reporter assays were performed in 293T cells. pcDNA-NOD1 WT and mutant plasmids were cotransfected with reporter plasmids. After 24 h, cells were lysed, and luciferase activity was determined. ( C and D ) NOD1-overexpressing cells were infected with HCMV (MOI 1) for 24 h, and IL-8 ( C ) and IFN-β ( D ) mRNA expression was measured by qRT-PCR. The depicted mRNA expression experiments represent mean ± SD from triplicate wells of two representative experiments. ( E and F ) The expression levels of NOD1-downstream signaling proteins were determined in total cell lysates ( E ) and cytoplasmic and nuclear fractions ( F ) at 3 hpi. β-actin served as a loading control; histone H3 and lamin B served as loading controls for nuclear proteins. ( G ) WT and NOD1 mutant-overexpressing cells were infected with HCMV Towne, and immunoprecipitation using anti-RIPK2 antibody, followed by immunoblotting for NOD1 using His antibody, were performed at 24 hpi. Data are mean ± SD from triplicate measurements. ** P
    Figure Legend Snippet: NOD1 downstream signaling in WT and mutant NOD1-overexpressing cells. ( A and B ) NF-κB ( A ) and IFN-β ( B ) luciferase reporter assays were performed in 293T cells. pcDNA-NOD1 WT and mutant plasmids were cotransfected with reporter plasmids. After 24 h, cells were lysed, and luciferase activity was determined. ( C and D ) NOD1-overexpressing cells were infected with HCMV (MOI 1) for 24 h, and IL-8 ( C ) and IFN-β ( D ) mRNA expression was measured by qRT-PCR. The depicted mRNA expression experiments represent mean ± SD from triplicate wells of two representative experiments. ( E and F ) The expression levels of NOD1-downstream signaling proteins were determined in total cell lysates ( E ) and cytoplasmic and nuclear fractions ( F ) at 3 hpi. β-actin served as a loading control; histone H3 and lamin B served as loading controls for nuclear proteins. ( G ) WT and NOD1 mutant-overexpressing cells were infected with HCMV Towne, and immunoprecipitation using anti-RIPK2 antibody, followed by immunoblotting for NOD1 using His antibody, were performed at 24 hpi. Data are mean ± SD from triplicate measurements. ** P

    Techniques Used: Mutagenesis, Luciferase, Activity Assay, Infection, Expressing, Quantitative RT-PCR, Immunoprecipitation

    7) Product Images from "Targeting Glutathione and Cystathionine β-Synthase in Ovarian Cancer Treatment by Selenium–Chrysin Polyurea Dendrimer Nanoformulation"

    Article Title: Targeting Glutathione and Cystathionine β-Synthase in Ovarian Cancer Treatment by Selenium–Chrysin Polyurea Dendrimer Nanoformulation

    Journal: Nutrients

    doi: 10.3390/nu11102523

    Putative involvement of cystine/glutamate antiporter system Xc (xCT) and nuclear factor erythroid 2-related factor 2 (Nrf2) in the protective effect of cysteine upon platinum salt exposure. ES2 and OVCAR3 cell lines were incubated with cysteine, carboplatin, and a combination of carboplatin with cysteine for 16 h ( A,B ). Cell death was determined by flow cytometry using annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. The xCT /soluble carrier protein 7A11-encoding gene ( SLC27A11 ) messenger RA (mRNA) expression was analyzed by RT-qPCR ( C ). Hypoxanthine–guanine phosphoribosyltransferase (HPRT) was used as the housekeeping gene. The xCT protein levels were assessed by Western blotting; β-actin was used as the house-keeping protein ( D ). Nrf2 protein levels were measured by immunofluorescence ( E,F ). Results are shown as means ± SD; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 ( A – D , and F ), and represents statistical significance in relation to control conditions, # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001 represents statistical significance between conditions, and p ≤ 0.01 represents statistical significance between conditions among cell lines ( A – D , and F ).
    Figure Legend Snippet: Putative involvement of cystine/glutamate antiporter system Xc (xCT) and nuclear factor erythroid 2-related factor 2 (Nrf2) in the protective effect of cysteine upon platinum salt exposure. ES2 and OVCAR3 cell lines were incubated with cysteine, carboplatin, and a combination of carboplatin with cysteine for 16 h ( A,B ). Cell death was determined by flow cytometry using annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. The xCT /soluble carrier protein 7A11-encoding gene ( SLC27A11 ) messenger RA (mRNA) expression was analyzed by RT-qPCR ( C ). Hypoxanthine–guanine phosphoribosyltransferase (HPRT) was used as the housekeeping gene. The xCT protein levels were assessed by Western blotting; β-actin was used as the house-keeping protein ( D ). Nrf2 protein levels were measured by immunofluorescence ( E,F ). Results are shown as means ± SD; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 ( A – D , and F ), and represents statistical significance in relation to control conditions, # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001 represents statistical significance between conditions, and p ≤ 0.01 represents statistical significance between conditions among cell lines ( A – D , and F ).

    Techniques Used: Incubation, Flow Cytometry, Cytometry, Staining, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

    8) Product Images from "Salmonella polarises peptide-MHC-II presentation towards an unconventional Type B CD4+ T-cell response"

    Article Title: Salmonella polarises peptide-MHC-II presentation towards an unconventional Type B CD4+ T-cell response

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201242983

    MHC-II down-regulation by Salmonella requires clathrin but not invariant chain-directed trafficking. (A) HeLa cells stably expressing HLA-DR WT (DRα,β) and cytoplasmic tail mutants were generated. HLA-DR surface expression was assessed by flow cytometry at 20 h post-infection with invasive GFP- S. Typhimurium and compared with HeLa-CIITA (Ii positive) cells. Refer to Supporting Information Fig. 1 A and B for gating strategy and representative flow cytometry data. Graph shows percent of normal HLA-DR surface expression in uninfected (GFP-negative) cells combined from at least four independent experiments. (B) HeLa cells stably expressing HLA-DR WT (DRα,β)(Ii negative) were transfected with AP-2, clathrin or control siRNAs. Cells were infected with invasive GFP- S. Typhimurium after 5 days of AP-2 or clathrin depletion and surface HLA-DR was assessed as described in (A). Western blot shows AP-2 and clathrin depletion from representative cell lysates after 5 days of siRNA treatment. The loading control is β-actin. Graph shows percent of normal surface HLA-DR expression in uninfected (GFP negative) cells combined from four independent experiments. Comparison of distributions was performed by unpaired (A) or paired (B) two-tailed t -tests.
    Figure Legend Snippet: MHC-II down-regulation by Salmonella requires clathrin but not invariant chain-directed trafficking. (A) HeLa cells stably expressing HLA-DR WT (DRα,β) and cytoplasmic tail mutants were generated. HLA-DR surface expression was assessed by flow cytometry at 20 h post-infection with invasive GFP- S. Typhimurium and compared with HeLa-CIITA (Ii positive) cells. Refer to Supporting Information Fig. 1 A and B for gating strategy and representative flow cytometry data. Graph shows percent of normal HLA-DR surface expression in uninfected (GFP-negative) cells combined from at least four independent experiments. (B) HeLa cells stably expressing HLA-DR WT (DRα,β)(Ii negative) were transfected with AP-2, clathrin or control siRNAs. Cells were infected with invasive GFP- S. Typhimurium after 5 days of AP-2 or clathrin depletion and surface HLA-DR was assessed as described in (A). Western blot shows AP-2 and clathrin depletion from representative cell lysates after 5 days of siRNA treatment. The loading control is β-actin. Graph shows percent of normal surface HLA-DR expression in uninfected (GFP negative) cells combined from four independent experiments. Comparison of distributions was performed by unpaired (A) or paired (B) two-tailed t -tests.

    Techniques Used: Stable Transfection, Expressing, Generated, Flow Cytometry, Cytometry, Infection, Transfection, Western Blot, Two Tailed Test

    9) Product Images from "Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release"

    Article Title: Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-8-14

    Effect of AG490 (AG), PD98059 (PD) and LY294002 (LY) on MMP-2 and MMP-9 activity release and MMP-9 mRNA expression in HDFs. ( A ) HDFs were incubated with AG490 (40 μM), PD98059 (50 μM) and LY294002 (40 μM) in the presence or absence of thrombin (Th, 5 U/ml) at 37°C for 2 h and 6 h, and MMP-2 and MMP-9 activities were detected with zymograph. ( B ) HDFs were incubated with AG490, PD98059 and LY294002 in the presence or absence of Th at 37°C for 6 h, and MMP-9 mRNA was detected by RT-PCR analysis. The relative levels of MMP-9 mRNA were expressed as the ratio to β-actin. The values shown are Mean ± SD for four separate experiments. Cells from each one of the four dermal fibroblast donors were used for one independent experiment. † P
    Figure Legend Snippet: Effect of AG490 (AG), PD98059 (PD) and LY294002 (LY) on MMP-2 and MMP-9 activity release and MMP-9 mRNA expression in HDFs. ( A ) HDFs were incubated with AG490 (40 μM), PD98059 (50 μM) and LY294002 (40 μM) in the presence or absence of thrombin (Th, 5 U/ml) at 37°C for 2 h and 6 h, and MMP-2 and MMP-9 activities were detected with zymograph. ( B ) HDFs were incubated with AG490, PD98059 and LY294002 in the presence or absence of Th at 37°C for 6 h, and MMP-9 mRNA was detected by RT-PCR analysis. The relative levels of MMP-9 mRNA were expressed as the ratio to β-actin. The values shown are Mean ± SD for four separate experiments. Cells from each one of the four dermal fibroblast donors were used for one independent experiment. † P

    Techniques Used: Activity Assay, Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

    Western blot analysis of effect of LY294002 (LY), a PI3K pathway inhibitor on phosphorylation of Akt in HDFs. Cells were incubated with LY (40 μM), thrombin (Th, 5 U/ml) or Th + LY at 37°C for 2 h or 6 h, respectively. Densitometry analysis of immunoblots was carried out using a Scion Image software. The relative levels of phospho-Akt were expressed as the ratio to β-actin. The values shown are Mean ± SD for four separate experiments. Cells from each one of the four dermal fibroblast donors were used for one independent experiment. † P
    Figure Legend Snippet: Western blot analysis of effect of LY294002 (LY), a PI3K pathway inhibitor on phosphorylation of Akt in HDFs. Cells were incubated with LY (40 μM), thrombin (Th, 5 U/ml) or Th + LY at 37°C for 2 h or 6 h, respectively. Densitometry analysis of immunoblots was carried out using a Scion Image software. The relative levels of phospho-Akt were expressed as the ratio to β-actin. The values shown are Mean ± SD for four separate experiments. Cells from each one of the four dermal fibroblast donors were used for one independent experiment. † P

    Techniques Used: Western Blot, Incubation, Software

    Western blot analysis of effect of PD98059 (PD), a MAPK pathway inhibitor on the phosphorylation of ERK1/2 in HDFs. Cells were incubated with PD (50 μM), thrombin (Th, 5 U/ml) or Th + PD at 37°C for 2 h or 6 h, respectively. Densitometry analysis of immunoblots was carried out using a Scion Image software. The relative levels of phospho-ERK1/2 were expressed as the ratio to β-actin. The values shown are Mean ± SD for four separate experiments. Cells from each one of the four dermal fibroblast donors were used for one independent experiment. † P
    Figure Legend Snippet: Western blot analysis of effect of PD98059 (PD), a MAPK pathway inhibitor on the phosphorylation of ERK1/2 in HDFs. Cells were incubated with PD (50 μM), thrombin (Th, 5 U/ml) or Th + PD at 37°C for 2 h or 6 h, respectively. Densitometry analysis of immunoblots was carried out using a Scion Image software. The relative levels of phospho-ERK1/2 were expressed as the ratio to β-actin. The values shown are Mean ± SD for four separate experiments. Cells from each one of the four dermal fibroblast donors were used for one independent experiment. † P

    Techniques Used: Western Blot, Incubation, Software

    Western blot analysis of effect of AG490 (AG), a JAK/STAT pathway inhibitor on the phosphosrylation of STAT3 in HDFs. Cells were incubated with AG (40 μM), thrombin (Th, 5 U/ml) or Th + AG at 37°C for 30 min, 2 h and 6 h, respectively. Densitometry analysis of immunoblots was carried out using a Scion Image software. The relative levels of phospho-STAT3 were expressed as the ratio to β-actin. The values shown are Mean ± SD for four separate experiments. Cells from each one of the four dermal fibroblast donors were used for one independent experiment. † P
    Figure Legend Snippet: Western blot analysis of effect of AG490 (AG), a JAK/STAT pathway inhibitor on the phosphosrylation of STAT3 in HDFs. Cells were incubated with AG (40 μM), thrombin (Th, 5 U/ml) or Th + AG at 37°C for 30 min, 2 h and 6 h, respectively. Densitometry analysis of immunoblots was carried out using a Scion Image software. The relative levels of phospho-STAT3 were expressed as the ratio to β-actin. The values shown are Mean ± SD for four separate experiments. Cells from each one of the four dermal fibroblast donors were used for one independent experiment. † P

    Techniques Used: Western Blot, Incubation, Software

    10) Product Images from "Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo"

    Article Title: Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0613-0

    Expression profile of autophagy-related proteins in MDA-MB-231 cells treated with As III and Tetra, alone or in combination. After treatment with various concentrations of As III and Tetra, alone or in combination, for 48 h, the expression profile of autophagy-related proteins was analyzed using western blot as described in “ Materials and methods ”. Representative image of the expression profile of each protein is shown from three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of control group (untreated cells). As, As III ; Tetra, tetrandrine. Since enough cells cannot be collected in the group treated with 15 µM As III in combination with 4.5 µg/ml Tetra due to its strong cytotoxicity, western blot analysis were not conducted
    Figure Legend Snippet: Expression profile of autophagy-related proteins in MDA-MB-231 cells treated with As III and Tetra, alone or in combination. After treatment with various concentrations of As III and Tetra, alone or in combination, for 48 h, the expression profile of autophagy-related proteins was analyzed using western blot as described in “ Materials and methods ”. Representative image of the expression profile of each protein is shown from three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of control group (untreated cells). As, As III ; Tetra, tetrandrine. Since enough cells cannot be collected in the group treated with 15 µM As III in combination with 4.5 µg/ml Tetra due to its strong cytotoxicity, western blot analysis were not conducted

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot

    Expression profile of autophagy and cell cycle arrest-related proteins in MDA-MB-231 mouse xenografts treated with As III and Tetra, alone or in combination. The experiment was carried out using human breast cancer nude mice implanted subcutaneously with 1 × 10 7 MDA-MB-231 cells. The administration of As III and Tetra, alone or in combination, and the preparation of tumor tissues and their protein samples were carried out as described under “ Materials and methods ”. Results are representatives of three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of vehicle-control group
    Figure Legend Snippet: Expression profile of autophagy and cell cycle arrest-related proteins in MDA-MB-231 mouse xenografts treated with As III and Tetra, alone or in combination. The experiment was carried out using human breast cancer nude mice implanted subcutaneously with 1 × 10 7 MDA-MB-231 cells. The administration of As III and Tetra, alone or in combination, and the preparation of tumor tissues and their protein samples were carried out as described under “ Materials and methods ”. Results are representatives of three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of vehicle-control group

    Techniques Used: Expressing, Multiple Displacement Amplification, Mouse Assay

    11) Product Images from "The Effect of Sortilin Silencing on Ovarian Carcinoma Cells"

    Article Title: The Effect of Sortilin Silencing on Ovarian Carcinoma Cells

    Journal: Avicenna Journal of Medical Biotechnology

    doi:

    Western blot analysis of sortilin expression in ovarian cancer and non-malignant tissues. Seven ovarian carcinoma tissues ( Table 1 ) as well as five ovarian carcinoma cell lines that overexpressed sortilin were compared with five non-malignant ovarian tissues (A and B). The lower band in non-malignant ovarian tissues is likely to be related to the second variant of sortilin with a molecular weight of 80-85 kDa . The level of β-actin as an internal protein loading control was detected in each sample. OC: ovarian carcinoma tissue, N: non-malignant ovarian tissue
    Figure Legend Snippet: Western blot analysis of sortilin expression in ovarian cancer and non-malignant tissues. Seven ovarian carcinoma tissues ( Table 1 ) as well as five ovarian carcinoma cell lines that overexpressed sortilin were compared with five non-malignant ovarian tissues (A and B). The lower band in non-malignant ovarian tissues is likely to be related to the second variant of sortilin with a molecular weight of 80-85 kDa . The level of β-actin as an internal protein loading control was detected in each sample. OC: ovarian carcinoma tissue, N: non-malignant ovarian tissue

    Techniques Used: Western Blot, Expressing, Variant Assay, Molecular Weight

    Western blot analysis of sortilin protein levels in siRNA-transfected cells. Densitometric analysis showed that the level of sortilin was markedly reduced by 72, 69 and 61% in siRNA-treated cells as compared to mock control-treated cells at 48, 72 and 96 hr after transfection, respectively. The level of β-actin as an internal protein loading control was detected in each sample
    Figure Legend Snippet: Western blot analysis of sortilin protein levels in siRNA-transfected cells. Densitometric analysis showed that the level of sortilin was markedly reduced by 72, 69 and 61% in siRNA-treated cells as compared to mock control-treated cells at 48, 72 and 96 hr after transfection, respectively. The level of β-actin as an internal protein loading control was detected in each sample

    Techniques Used: Western Blot, Transfection

    12) Product Images from "A mouse model for distal renal tubular acidosis reveals a previously unrecognized role of the V-ATPase a4 subunit in the proximal tubule"

    Article Title: A mouse model for distal renal tubular acidosis reveals a previously unrecognized role of the V-ATPase a4 subunit in the proximal tubule

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201201527

    Disruption of the a4 subunit in mice results in early mortality Cartoon of the V-ATPase with its transmembrane (V0) and peripheral (V1) sector, each build up by different subunits. The V0 sector including the a subunit is highlighted in orange. Partial genomic structure of the Atp6v0a4 gene (top) and the targeted a4 locus. The dotted line indicates the genomic sequence included in the targeting construct. A neomycin selection cassette flanked by frt sites (black boxes) and single loxP site was inserted into intron 11. A second loxP site and a Bam HI site were introduced into intron 10. Correctly targeted ES cell clones were used for the generation of chimeric mice (below). a4 KO mice were generated by breeding chimeric mice to a cre-deleter mouse strain (bottom). a4 transcript abundance in kidneys of Atp6v0a4 +/+ (WT), Atp6v0a4 +/− (Het) and Atp6v0a4 −/− (KO) mice as revealed by Northern blot. Gapdh mRNA served as a loading control. Detection of the a4 subunit by Western blot analysis of kidney protein lysates of four individual mice per genotype; β-actin served as a loading control. Significant reduction of body weight in Atp6v0a4 −/− mice at 3 weeks of age (** p
    Figure Legend Snippet: Disruption of the a4 subunit in mice results in early mortality Cartoon of the V-ATPase with its transmembrane (V0) and peripheral (V1) sector, each build up by different subunits. The V0 sector including the a subunit is highlighted in orange. Partial genomic structure of the Atp6v0a4 gene (top) and the targeted a4 locus. The dotted line indicates the genomic sequence included in the targeting construct. A neomycin selection cassette flanked by frt sites (black boxes) and single loxP site was inserted into intron 11. A second loxP site and a Bam HI site were introduced into intron 10. Correctly targeted ES cell clones were used for the generation of chimeric mice (below). a4 KO mice were generated by breeding chimeric mice to a cre-deleter mouse strain (bottom). a4 transcript abundance in kidneys of Atp6v0a4 +/+ (WT), Atp6v0a4 +/− (Het) and Atp6v0a4 −/− (KO) mice as revealed by Northern blot. Gapdh mRNA served as a loading control. Detection of the a4 subunit by Western blot analysis of kidney protein lysates of four individual mice per genotype; β-actin served as a loading control. Significant reduction of body weight in Atp6v0a4 −/− mice at 3 weeks of age (** p

    Techniques Used: Mouse Assay, Sequencing, Construct, Selection, Clone Assay, Generated, Northern Blot, Western Blot

    Proximal tubule dysfunction in a4 KO mice For clarity the basolateral and apical borders of the tubular epithelium are indicated by dotted lines and the lumina are marked with an asterisk in immunofluorescence images. A. Western Blot analysis of kidney lysates of five individual mice per genotype shows a significant increase of the a3 subunit and Nhe3 expression levels in kidney lysates of a4 KO mice. Expression of NaPi-IIa is strongly decreased in whole kidney protein lysates of a4 KO mice. β-actin serves as a loading control. B. Visualization of urinary proteins by SDS–polyacrylamide gel electrophoresis and Coomassie staining. Loading volume was adjusted to creatinine levels. C. Albumin ELISA verifies proteinuria in a4 KO spot urine samples (** p
    Figure Legend Snippet: Proximal tubule dysfunction in a4 KO mice For clarity the basolateral and apical borders of the tubular epithelium are indicated by dotted lines and the lumina are marked with an asterisk in immunofluorescence images. A. Western Blot analysis of kidney lysates of five individual mice per genotype shows a significant increase of the a3 subunit and Nhe3 expression levels in kidney lysates of a4 KO mice. Expression of NaPi-IIa is strongly decreased in whole kidney protein lysates of a4 KO mice. β-actin serves as a loading control. B. Visualization of urinary proteins by SDS–polyacrylamide gel electrophoresis and Coomassie staining. Loading volume was adjusted to creatinine levels. C. Albumin ELISA verifies proteinuria in a4 KO spot urine samples (** p

    Techniques Used: Mouse Assay, Immunofluorescence, Western Blot, Expressing, Polyacrylamide Gel Electrophoresis, Staining, Enzyme-linked Immunosorbent Assay

    13) Product Images from "Expression of a2 Vacuolar ATPase in Spermatozoa is Associated with Semen Quality and Chemokine-Cytokine Profiles in Infertile Men"

    Article Title: Expression of a2 Vacuolar ATPase in Spermatozoa is Associated with Semen Quality and Chemokine-Cytokine Profiles in Infertile Men

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070470

    Atp6v0a2 is lower in spermatozoa of infertile men. (A) Atp6v0a2 mRNA expression in spermatozoa was measured by Real-time PCR and gene expression was normalized to β-actin mRNA. Atp6v0a2 mRNA expression in spermatozoa of normal fertile donors was significantly higher than infertile men (P
    Figure Legend Snippet: Atp6v0a2 is lower in spermatozoa of infertile men. (A) Atp6v0a2 mRNA expression in spermatozoa was measured by Real-time PCR and gene expression was normalized to β-actin mRNA. Atp6v0a2 mRNA expression in spermatozoa of normal fertile donors was significantly higher than infertile men (P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Atp6v0a2 is lower in immotile spermatozoa. (A) Western blot analysis shows the levels of Atp6v0a2 in human motile and immotile spermatozoa. (B) The bar diagram shows the quantification of Atp6v0a2 protein expression, as determined by densitometry analysis and normalized to β-actin. Immotile spermatozoa had significantly lower Atp6v0a2 expression than motile spermatozoa (P
    Figure Legend Snippet: Atp6v0a2 is lower in immotile spermatozoa. (A) Western blot analysis shows the levels of Atp6v0a2 in human motile and immotile spermatozoa. (B) The bar diagram shows the quantification of Atp6v0a2 protein expression, as determined by densitometry analysis and normalized to β-actin. Immotile spermatozoa had significantly lower Atp6v0a2 expression than motile spermatozoa (P

    Techniques Used: Western Blot, Expressing

    14) Product Images from "Identification and Characterization of Novel Immunomodulatory Bursal-derived Pentapeptide-II (BPP-II) *"

    Article Title: Identification and Characterization of Novel Immunomodulatory Bursal-derived Pentapeptide-II (BPP-II) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.273854

    Some regulated genes were validated by quantitative RT-PCR. The data are expressed as expression of FGF21 , CD80 , Fas , CBLC , Fyn , Csf1r , CD3D , MS4A2 , CD86 , CDKN2A , and Fgf8 . Gene expressions were normalized to β-actin expression and converted to
    Figure Legend Snippet: Some regulated genes were validated by quantitative RT-PCR. The data are expressed as expression of FGF21 , CD80 , Fas , CBLC , Fyn , Csf1r , CD3D , MS4A2 , CD86 , CDKN2A , and Fgf8 . Gene expressions were normalized to β-actin expression and converted to

    Techniques Used: Quantitative RT-PCR, Expressing

    15) Product Images from "Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo"

    Article Title: Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0613-0

    Expression profile of autophagy-related proteins in MDA-MB-231 cells treated with As III and Tetra, alone or in combination. After treatment with various concentrations of As III ”. Representative image of the expression profile of each protein is shown from three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of control group (untreated cells). As, As III ; Tetra, tetrandrine. Since enough cells cannot be collected in the group treated with 15 µM As III in combination with 4.5 µg/ml Tetra due to its strong cytotoxicity, western blot analysis were not conducted
    Figure Legend Snippet: Expression profile of autophagy-related proteins in MDA-MB-231 cells treated with As III and Tetra, alone or in combination. After treatment with various concentrations of As III ”. Representative image of the expression profile of each protein is shown from three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of control group (untreated cells). As, As III ; Tetra, tetrandrine. Since enough cells cannot be collected in the group treated with 15 µM As III in combination with 4.5 µg/ml Tetra due to its strong cytotoxicity, western blot analysis were not conducted

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot

    Expression profile of autophagy and cell cycle arrest-related proteins in MDA-MB-231 mouse xenografts treated with As III and Tetra, alone or in combination. The experiment was carried out using human breast cancer nude mice implanted subcutaneously with 1 × 10 7 MDA-MB-231 cells. The administration of As III ”. Results are representatives of three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of vehicle-control group
    Figure Legend Snippet: Expression profile of autophagy and cell cycle arrest-related proteins in MDA-MB-231 mouse xenografts treated with As III and Tetra, alone or in combination. The experiment was carried out using human breast cancer nude mice implanted subcutaneously with 1 × 10 7 MDA-MB-231 cells. The administration of As III ”. Results are representatives of three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of vehicle-control group

    Techniques Used: Expressing, Multiple Displacement Amplification, Mouse Assay

    16) Product Images from "Activation of Nucleotide Oligomerization Domain 2 (NOD2) by Human Cytomegalovirus Initiates Innate Immune Responses and Restricts Virus Replication"

    Article Title: Activation of Nucleotide Oligomerization Domain 2 (NOD2) by Human Cytomegalovirus Initiates Innate Immune Responses and Restricts Virus Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092704

    Overexpression of NOD2 restricts HCMV replication and induces antiviral and pro-inflammatory cytokines. A . U373 cells were transiently transfected with pcDNA4/HisMax, pcDNA4/EGFP, pcDNA4/HisMax-hNOD2 or pcDNA4/HisMax-hRIPK2 plasmid. 24 h after transfection cells were infected with pp28-luciferase HCMV. Luciferase activity was measured in cell lysates at 72 hpi. B . Cell lysates from 4 A were used to determine protein expression of HCMV-immediate early (IE1/IE2), early (UL44), and late (pp65) genes. Levels of NOD2 and RIPK2 proteins were measured to confirm NOD2 overexpression; β-actin served as loading control. Western blot data are representative of three independent experiments. Asterisks (*) denote endogenous NOD2 and RIPK2 proteins. C – D. U373 cells were transiently transfected with pcDNA4/HisMax, hNOD2 or hRIPK2 plasmid. 24 h after transfection cells were infected with HCMV Towne and total RNA was isolated at 72 hpi. Levels of IFN-β and IL8 mRNAs were measured by qRT-PCR in non-infected (mock) and HCMV-infected (HCMV) cells. E. HFFs stably expressing empty vector (HFF-control) or HA-tagged NOD2 (HFF-NOD2) were either untreated or treated with 2 μg/ml doxycycline and expression of NOD2 was determined at 48 h using anti-HA antibody. F. HFFs stably expressing empty vector (control) or NOD2 (HFF-NOD2) were induced with doxycycline for 24 h followed by infection with HCMV Towne. Cells were incubated in doxycycline containing media and cell-free supernatants were collected at 96 hpi. Virus progeny released into the supernatants were quantified after infection of fresh HFFs (second cycle) with equal amount of supernatants from control or NOD2-overexpressing HFFs using luciferase assay at 3 dpi (Y-axis on left, in blue) and by real-time PCR (Y-axis on right, in red) in the supernatants of newly-infected HFFs (Fig. 4F). Viral protein expression was determined in newly-infected HFFs at 3 dpi (Fig. 4 G). H, J. Levels of IFN-β and IL8 mRNAs were determined in HFFs-control or HFF-NOD2 cells using qRT-PCR at 96 hpi. I. Levels of IFN-β secreted into the media from HFF-control and HFF-NOD2 cells (first cycle of infection) were measured at 24 hpi using IFN-β ELISA kit. The data shown are the average of three experiments ± SD (*p
    Figure Legend Snippet: Overexpression of NOD2 restricts HCMV replication and induces antiviral and pro-inflammatory cytokines. A . U373 cells were transiently transfected with pcDNA4/HisMax, pcDNA4/EGFP, pcDNA4/HisMax-hNOD2 or pcDNA4/HisMax-hRIPK2 plasmid. 24 h after transfection cells were infected with pp28-luciferase HCMV. Luciferase activity was measured in cell lysates at 72 hpi. B . Cell lysates from 4 A were used to determine protein expression of HCMV-immediate early (IE1/IE2), early (UL44), and late (pp65) genes. Levels of NOD2 and RIPK2 proteins were measured to confirm NOD2 overexpression; β-actin served as loading control. Western blot data are representative of three independent experiments. Asterisks (*) denote endogenous NOD2 and RIPK2 proteins. C – D. U373 cells were transiently transfected with pcDNA4/HisMax, hNOD2 or hRIPK2 plasmid. 24 h after transfection cells were infected with HCMV Towne and total RNA was isolated at 72 hpi. Levels of IFN-β and IL8 mRNAs were measured by qRT-PCR in non-infected (mock) and HCMV-infected (HCMV) cells. E. HFFs stably expressing empty vector (HFF-control) or HA-tagged NOD2 (HFF-NOD2) were either untreated or treated with 2 μg/ml doxycycline and expression of NOD2 was determined at 48 h using anti-HA antibody. F. HFFs stably expressing empty vector (control) or NOD2 (HFF-NOD2) were induced with doxycycline for 24 h followed by infection with HCMV Towne. Cells were incubated in doxycycline containing media and cell-free supernatants were collected at 96 hpi. Virus progeny released into the supernatants were quantified after infection of fresh HFFs (second cycle) with equal amount of supernatants from control or NOD2-overexpressing HFFs using luciferase assay at 3 dpi (Y-axis on left, in blue) and by real-time PCR (Y-axis on right, in red) in the supernatants of newly-infected HFFs (Fig. 4F). Viral protein expression was determined in newly-infected HFFs at 3 dpi (Fig. 4 G). H, J. Levels of IFN-β and IL8 mRNAs were determined in HFFs-control or HFF-NOD2 cells using qRT-PCR at 96 hpi. I. Levels of IFN-β secreted into the media from HFF-control and HFF-NOD2 cells (first cycle of infection) were measured at 24 hpi using IFN-β ELISA kit. The data shown are the average of three experiments ± SD (*p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Infection, Luciferase, Activity Assay, Expressing, Western Blot, Isolation, Quantitative RT-PCR, Stable Transfection, Incubation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    HCMV infection induces NOD2 mRNA and protein in HFFs and U373 cells. A. HFFs were infected with HCMV Towne strain and levels of NOD1, NOD2 and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs were measured by qRT-PCR at indicated time points. B . HFFs were infected with HCMV-TB40 and levels of NOD1, NOD2 and GAPDH mRNAs were measured by qRT-PCR at indicated time points. C . HFFs were infected with a clinical isolate of HCMV and levels of NOD1, NOD2 and GAPDH mRNAs were measured by qRT-PCR at indicated time points. D . HFFs were treated with MDP (10 μg/ml) and levels of NOD1 and NOD2 mRNA were measured as in A, B and C, E . U373 glioma cells were infected with HCMV Towne strain and levels of NOD1, NOD2 and GAPDH mRNAs were measured by qRT-PCR at indicated time points. F. HFFs were infected with HCMV (Towne) at MOI of 1 PFU/cell and levels of NOD2 protein and β-actin were determined 48 and 72 hpi. G. HFFs were infected with HCMV (Towne) strain at MOI of 0.03 or 3 PFU/cell and levels of NOD2 protein and β-actin were determined at 48 hpi. Quantitative data represent mean values (±SD) of triplicate determinations from three independent experiments (*p
    Figure Legend Snippet: HCMV infection induces NOD2 mRNA and protein in HFFs and U373 cells. A. HFFs were infected with HCMV Towne strain and levels of NOD1, NOD2 and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs were measured by qRT-PCR at indicated time points. B . HFFs were infected with HCMV-TB40 and levels of NOD1, NOD2 and GAPDH mRNAs were measured by qRT-PCR at indicated time points. C . HFFs were infected with a clinical isolate of HCMV and levels of NOD1, NOD2 and GAPDH mRNAs were measured by qRT-PCR at indicated time points. D . HFFs were treated with MDP (10 μg/ml) and levels of NOD1 and NOD2 mRNA were measured as in A, B and C, E . U373 glioma cells were infected with HCMV Towne strain and levels of NOD1, NOD2 and GAPDH mRNAs were measured by qRT-PCR at indicated time points. F. HFFs were infected with HCMV (Towne) at MOI of 1 PFU/cell and levels of NOD2 protein and β-actin were determined 48 and 72 hpi. G. HFFs were infected with HCMV (Towne) strain at MOI of 0.03 or 3 PFU/cell and levels of NOD2 protein and β-actin were determined at 48 hpi. Quantitative data represent mean values (±SD) of triplicate determinations from three independent experiments (*p

    Techniques Used: Infection, Quantitative RT-PCR

    17) Product Images from "Constitutive upregulation of the transforming growth factor-? pathway in rheumatoid arthritis synovial fibroblasts"

    Article Title: Constitutive upregulation of the transforming growth factor-? pathway in rheumatoid arthritis synovial fibroblasts

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar2217

    Transforming growth factor (TGF)-β1 protein expression in osteoarthritis (OA) synovial fibroblast (SFBs) ( n = 4) and rheumatoid arthritis (RA) SFBs ( n = 6), as assessed by SDS-PAGE/western blotting. Bars indicate the optical density of the protein bands (median ± 75th and 25th percentiles) relative to the β-actin control.
    Figure Legend Snippet: Transforming growth factor (TGF)-β1 protein expression in osteoarthritis (OA) synovial fibroblast (SFBs) ( n = 4) and rheumatoid arthritis (RA) SFBs ( n = 6), as assessed by SDS-PAGE/western blotting. Bars indicate the optical density of the protein bands (median ± 75th and 25th percentiles) relative to the β-actin control.

    Techniques Used: Expressing, SDS Page, Western Blot

    TGF-β receptor 1 (TGFBR1) and β-actin (control) protein expression in osteoarthritis (OA) synovial fibroblast (SFBs) ( n = 4) and rheumatoid arthritis (RA) SFBs ( n = 5) as assessed by SDS-PAGE/western blotting (upper panel). Bars indicate the median optical density of protein bands ± 75th and 25th percentiles relative to the value of the β-actin bands (lower panel). * p ≤ 0.05 compared to OA.
    Figure Legend Snippet: TGF-β receptor 1 (TGFBR1) and β-actin (control) protein expression in osteoarthritis (OA) synovial fibroblast (SFBs) ( n = 4) and rheumatoid arthritis (RA) SFBs ( n = 5) as assessed by SDS-PAGE/western blotting (upper panel). Bars indicate the median optical density of protein bands ± 75th and 25th percentiles relative to the value of the β-actin bands (lower panel). * p ≤ 0.05 compared to OA.

    Techniques Used: Expressing, SDS Page, Western Blot

    18) Product Images from "Claudin 4 Is Differentially Expressed between Ovarian Cancer Subtypes and Plays a Role in Spheroid Formation"

    Article Title: Claudin 4 Is Differentially Expressed between Ovarian Cancer Subtypes and Plays a Role in Spheroid Formation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms12021334

    Claudin 4 RNA and protein expression in ovarian cancer tissues and cell lines . ( A ) Claudin 4 RNA expression in 13 ovarian cancer cell lines and 7 NOSE cell lines as determined by qRT-PCR. Expression values shown as fold change over the lowest expressing cell line (1816–575), and are the average of two experiments (see Experimental section). ( B ) Claudin 4 protein expression was determined by Western immunoblot analysis of ovarian cancer and NOSE cell lines (50 μg protein/lane). β-actin serves as a loading control. ( C ) Claudin 4 protein expression was determined by Western immunoblot analysis of 7 primary stage III/IV serous ovarian cancer tissues (C1–C7) and 5 normal ovaries (N1–N5) (50 μg protein/lane). β-actin, loading control.
    Figure Legend Snippet: Claudin 4 RNA and protein expression in ovarian cancer tissues and cell lines . ( A ) Claudin 4 RNA expression in 13 ovarian cancer cell lines and 7 NOSE cell lines as determined by qRT-PCR. Expression values shown as fold change over the lowest expressing cell line (1816–575), and are the average of two experiments (see Experimental section). ( B ) Claudin 4 protein expression was determined by Western immunoblot analysis of ovarian cancer and NOSE cell lines (50 μg protein/lane). β-actin serves as a loading control. ( C ) Claudin 4 protein expression was determined by Western immunoblot analysis of 7 primary stage III/IV serous ovarian cancer tissues (C1–C7) and 5 normal ovaries (N1–N5) (50 μg protein/lane). β-actin, loading control.

    Techniques Used: Expressing, RNA Expression, Quantitative RT-PCR, Western Blot

    19) Product Images from "HIV-1 Vpu utilizes both cullin-RING ligase (CRL) dependent and independent mechanisms to downmodulate host proteins"

    Article Title: HIV-1 Vpu utilizes both cullin-RING ligase (CRL) dependent and independent mechanisms to downmodulate host proteins

    Journal: Retrovirology

    doi: 10.1186/s12977-015-0192-2

    siRNA knockdown of cullin 1 hinders surface downmodulation of CD4, but not BST-2, by Vpu. a HeLa-CD4 cells were transfected twice with pooled control or cullin 1 siRNAs. 4 h post the second transfection, the cells were infected with VSV-G pseudotyped DHIV-GFP (Nef−/Vpu−) or DHIV-GFP WARO (Nef−/Vpu+). Cells were subsequently stained to detect surface levels of CD4 and BST-2 between GFP negative ( blue line ) and GFP positive ( red line ) populations 48 h post infection. Gray shaded histogram represents an IgG matched isotype control. b A portion of cells from a were lysed and subjected to Western blot to determine the knockdown efficiency of cullin 1. c Quantification of cullin 1 normalized to β-actin from b .
    Figure Legend Snippet: siRNA knockdown of cullin 1 hinders surface downmodulation of CD4, but not BST-2, by Vpu. a HeLa-CD4 cells were transfected twice with pooled control or cullin 1 siRNAs. 4 h post the second transfection, the cells were infected with VSV-G pseudotyped DHIV-GFP (Nef−/Vpu−) or DHIV-GFP WARO (Nef−/Vpu+). Cells were subsequently stained to detect surface levels of CD4 and BST-2 between GFP negative ( blue line ) and GFP positive ( red line ) populations 48 h post infection. Gray shaded histogram represents an IgG matched isotype control. b A portion of cells from a were lysed and subjected to Western blot to determine the knockdown efficiency of cullin 1. c Quantification of cullin 1 normalized to β-actin from b .

    Techniques Used: Transfection, Infection, Staining, Western Blot

    20) Product Images from "Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine"

    Article Title: Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine

    Journal: American Journal of Translational Research

    doi:

    Upregulation of ICAM-1 and activation of Erk signaling pathway in MDA-MB-231 mouse xenografts treated with As III and Tetra, alone or in combination. MDA-MB-231 tumors in the mice xenograft model treated with As III and Tetra, alone or in combination, were obtained from our previous study, in which long-term (10 weeks) co-administration of As III ]. (A) The expression profiles of ICAM-1, phospho-Erk and total Erk were analyzed using western blot as described in material and methods. Results are representatives of three independent experiments. The relative expression levels of ICAM-1 (B) and phospho-Erk/Erk (C) were expressed as the ratios between each target gene protein and β-actin protein expression levels, and compared with those of untreated control group, respectively. Results are shown as the means ± SD from three independent experiments. p-Erk, phosphorylated Erk; As, As III ; Tetra, tetrandrine. The procedures for establishment of MDA-MB-231 mouse xenografts, and the following administration of As III .
    Figure Legend Snippet: Upregulation of ICAM-1 and activation of Erk signaling pathway in MDA-MB-231 mouse xenografts treated with As III and Tetra, alone or in combination. MDA-MB-231 tumors in the mice xenograft model treated with As III and Tetra, alone or in combination, were obtained from our previous study, in which long-term (10 weeks) co-administration of As III ]. (A) The expression profiles of ICAM-1, phospho-Erk and total Erk were analyzed using western blot as described in material and methods. Results are representatives of three independent experiments. The relative expression levels of ICAM-1 (B) and phospho-Erk/Erk (C) were expressed as the ratios between each target gene protein and β-actin protein expression levels, and compared with those of untreated control group, respectively. Results are shown as the means ± SD from three independent experiments. p-Erk, phosphorylated Erk; As, As III ; Tetra, tetrandrine. The procedures for establishment of MDA-MB-231 mouse xenografts, and the following administration of As III .

    Techniques Used: Activation Assay, Multiple Displacement Amplification, Mouse Assay, Expressing, Western Blot

    Expression profile of differentiation-related proteins in breast cancer cells treated with As III and Tetra, alone or in combination. After treatment with indicated concentrations of As III and Tetra, alone or in combination, for 96 h, the expression profiles of differentiation-related proteins in MDA-MB-231 cells (A) and MCF-7 cells (B) were analyzed using western blot as described in material and methods. Representative image of the expression profile of each protein is shown from three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values above each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of control group (untreated cells). p-Erk, phosphorylated Erk; As, As III ; Tetra, tetrandrine; ATRA, all- trans retinoic acid. ATRA (1 µM) used as a positive control for differentiation.
    Figure Legend Snippet: Expression profile of differentiation-related proteins in breast cancer cells treated with As III and Tetra, alone or in combination. After treatment with indicated concentrations of As III and Tetra, alone or in combination, for 96 h, the expression profiles of differentiation-related proteins in MDA-MB-231 cells (A) and MCF-7 cells (B) were analyzed using western blot as described in material and methods. Representative image of the expression profile of each protein is shown from three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values above each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of control group (untreated cells). p-Erk, phosphorylated Erk; As, As III ; Tetra, tetrandrine; ATRA, all- trans retinoic acid. ATRA (1 µM) used as a positive control for differentiation.

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot, Positive Control

    21) Product Images from "MicroRNA-520b Inhibits Growth of Hepatoma Cells by Targeting MEKK2 and Cyclin D1"

    Article Title: MicroRNA-520b Inhibits Growth of Hepatoma Cells by Targeting MEKK2 and Cyclin D1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031450

    MiR-520b directly inhibits expression of MEKK2 and cyclin D1 via their 3′UTR. (A) Sequence alignment between miR-520b and the 3′UTR of human MEKK2 and cyclin D1 mRNA. Solid line, seed match region; dashed line, seed-mutated region. (B) The effect of miR-520b on the activity of firefly luciferase reporter containing either wild type (WT) or mutant type (Mut) 3′UTR was tested by luciferase reporter gene assays. (C) The effect of miR-520b or Inh-520b on the endogenous expression levels of MEKK2 and cyclin D1 was examined in HepG2 and H7402 cells by western blot analyses. β-actin was used as an internal control. The intensity for each band was quantified densitometrically.
    Figure Legend Snippet: MiR-520b directly inhibits expression of MEKK2 and cyclin D1 via their 3′UTR. (A) Sequence alignment between miR-520b and the 3′UTR of human MEKK2 and cyclin D1 mRNA. Solid line, seed match region; dashed line, seed-mutated region. (B) The effect of miR-520b on the activity of firefly luciferase reporter containing either wild type (WT) or mutant type (Mut) 3′UTR was tested by luciferase reporter gene assays. (C) The effect of miR-520b or Inh-520b on the endogenous expression levels of MEKK2 and cyclin D1 was examined in HepG2 and H7402 cells by western blot analyses. β-actin was used as an internal control. The intensity for each band was quantified densitometrically.

    Techniques Used: Expressing, Sequencing, Activity Assay, Luciferase, Mutagenesis, Western Blot

    22) Product Images from "HER3 targeting sensitizes HNSCC to cetuximab by reducing HER3 activity and HER2/HER3 dimerization - evidence from cell line and patient derived xenograft models"

    Article Title: HER3 targeting sensitizes HNSCC to cetuximab by reducing HER3 activity and HER2/HER3 dimerization - evidence from cell line and patient derived xenograft models

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-16-0558

    HER2/HER3 dimerization is increased upon cetuximab treatment UMSCC1-C cells were exposed to 2µg/ml cetuximab for 24 hr. HER3 was immunoprecipitated from the cell lysate with anti-HER3 antibody. The immunoprecipitate was fractionated on SDS-PAGE followed by immunoblotting with anti-HER2 and HER3 antibodies. As the image shows, cetuximab treatment increased HER3 expression in UMSCC1-C cells, at the same time HER2 and HER3 association was increased as more HER2 was detected by immunoblot in cetuximab treated cells compared with control cells and IgG control. β-actin was used as loading control (figure represents 1 of 3 experiments).
    Figure Legend Snippet: HER2/HER3 dimerization is increased upon cetuximab treatment UMSCC1-C cells were exposed to 2µg/ml cetuximab for 24 hr. HER3 was immunoprecipitated from the cell lysate with anti-HER3 antibody. The immunoprecipitate was fractionated on SDS-PAGE followed by immunoblotting with anti-HER2 and HER3 antibodies. As the image shows, cetuximab treatment increased HER3 expression in UMSCC1-C cells, at the same time HER2 and HER3 association was increased as more HER2 was detected by immunoblot in cetuximab treated cells compared with control cells and IgG control. β-actin was used as loading control (figure represents 1 of 3 experiments).

    Techniques Used: Immunoprecipitation, SDS Page, Expressing

    23) Product Images from "Phosphoinositide 3-kinase inhibition restores neutrophil accuracy in the elderly: toward targeted treatments for immunosenescence"

    Article Title: Phosphoinositide 3-kinase inhibition restores neutrophil accuracy in the elderly: toward targeted treatments for immunosenescence

    Journal: Blood

    doi: 10.1182/blood-2013-08-519520

    PI3K activation in neutrophils from young and old healthy donors. PI3K activity was assessed by western blotting using an antibody to the phosphorylated p85 regulatory subunit of PI3K. β-actin was assessed as the loading control. Cells were unstimulated
    Figure Legend Snippet: PI3K activation in neutrophils from young and old healthy donors. PI3K activity was assessed by western blotting using an antibody to the phosphorylated p85 regulatory subunit of PI3K. β-actin was assessed as the loading control. Cells were unstimulated

    Techniques Used: Activation Assay, Activity Assay, Western Blot

    24) Product Images from "Respiratory Syncytial Virus Impairs Macrophage IFN-?/?- and IFN-?-Stimulated Transcription by Distinct Mechanisms"

    Article Title: Respiratory Syncytial Virus Impairs Macrophage IFN-?/?- and IFN-?-Stimulated Transcription by Distinct Mechanisms

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2008-0229OC

    RSV impairs IFN-mediated transcriptional activation in primary mouse alveolar macrophages. Western immunoblot analysis was performed to determine whether RSV infection (MOI = 1; 24 h) inhibited phosphorylation of STAT1 at Y701 in ( A ) IFN-β– (100 U/ml) or ( C ) IFN-γ–stimulated (10 ng/ml, 30-min treatment) primary mouse alveolar macrophages. RSV infection did not inhibit IFN-γ–stimulated STAT1 (Y701) phosphorylation; however, STAT1 phosphorylation in response to IFN-β was significantly impaired in RSV-infected alveolar macrophages. RSV infection increased the level of STAT1. The levels of β-actin were similar in all samples. Multiplex real-time quantitative PCR analysis was performed to determine the mRNA levels of IFN-responsive genes compared with the expression of GAPDH on mRNA harvested from alveolar macrophages stimulated with either IFN-β (100 U/ml) or IFN-γ (10 ng/ml) for 6 hours. RSV infection significantly inhibited the IFN-β–induced expression of Nod1 ( B ), and the IFN-γ–induced expression of C2ta ( D ). The data represent three independent experiments run in triplicate, and are presented as means (±SEM); * P
    Figure Legend Snippet: RSV impairs IFN-mediated transcriptional activation in primary mouse alveolar macrophages. Western immunoblot analysis was performed to determine whether RSV infection (MOI = 1; 24 h) inhibited phosphorylation of STAT1 at Y701 in ( A ) IFN-β– (100 U/ml) or ( C ) IFN-γ–stimulated (10 ng/ml, 30-min treatment) primary mouse alveolar macrophages. RSV infection did not inhibit IFN-γ–stimulated STAT1 (Y701) phosphorylation; however, STAT1 phosphorylation in response to IFN-β was significantly impaired in RSV-infected alveolar macrophages. RSV infection increased the level of STAT1. The levels of β-actin were similar in all samples. Multiplex real-time quantitative PCR analysis was performed to determine the mRNA levels of IFN-responsive genes compared with the expression of GAPDH on mRNA harvested from alveolar macrophages stimulated with either IFN-β (100 U/ml) or IFN-γ (10 ng/ml) for 6 hours. RSV infection significantly inhibited the IFN-β–induced expression of Nod1 ( B ), and the IFN-γ–induced expression of C2ta ( D ). The data represent three independent experiments run in triplicate, and are presented as means (±SEM); * P

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

    RSV impairs IFN-α/β– but not IFN-γ–mediated signal transducer and activator of transcription (STAT)-1 phosphorylation. Western immunoblot analysis was performed to determine whether RSV infection (MOI = 1; 24 h) inhibited phosphorylation of STAT1 at Y701 in ( A ) IFN-α– (100 U/ml), IFN-β– (100 U/ml), or ( B ) IFN-γ–stimulated (10 ng/ml, 30-min treatment) RAW macrophages. RSV infection did not inhibit IFN-γ–stimulated STAT1 (Y701) phosphorylation; however, STAT1 phosphorylation in response to either IFN-α or IFN-β was significantly impaired in RSV-infected macrophages. RSV infection increased the level of STAT1. The levels of β-actin were similar in all samples. Each lane of the immunoblot was loaded with 20 μg of protein, and the immunoblots shown are representative of three independent experiments.
    Figure Legend Snippet: RSV impairs IFN-α/β– but not IFN-γ–mediated signal transducer and activator of transcription (STAT)-1 phosphorylation. Western immunoblot analysis was performed to determine whether RSV infection (MOI = 1; 24 h) inhibited phosphorylation of STAT1 at Y701 in ( A ) IFN-α– (100 U/ml), IFN-β– (100 U/ml), or ( B ) IFN-γ–stimulated (10 ng/ml, 30-min treatment) RAW macrophages. RSV infection did not inhibit IFN-γ–stimulated STAT1 (Y701) phosphorylation; however, STAT1 phosphorylation in response to either IFN-α or IFN-β was significantly impaired in RSV-infected macrophages. RSV infection increased the level of STAT1. The levels of β-actin were similar in all samples. Each lane of the immunoblot was loaded with 20 μg of protein, and the immunoblots shown are representative of three independent experiments.

    Techniques Used: Western Blot, Infection

    RSV impairs IFN-α/β–mediated STAT2 phosphorylation. ( A ) Western immunoblot analysis was performed to determine whether RSV infection (MOI = 1; 24 h) inhibited phosphorylation of STAT2 in IFN-α– (100 U/ml) or IFN-β–stimulated (100 U/ml, 30-min treatment) RAW macrophages. STAT2 phosphorylation in response to either IFN-α or IFN-β was significantly impaired in RSV-infected macrophages. RSV infection increased the level of STAT2, whereas β-actin levels were similar in all samples. Each lane of the immunoblot was loaded with 20 μg of protein, and the immunoblots shown are representative of three independent experiments. ( B ) Flow cytometric analysis was performed on macrophages to determine the effect of RSV infection on STAT2 protein expression on a per-cell basis. RSV-infected macrophages ( closed bar ) had increased levels of STAT2 when compared with mock-infected cells ( open bar ). After infection, both RSV-negative and RSV-positive macrophages ( hatched bars ) had elevated levels of STAT2 when compared with mock-infected cells. Data are means (±SEM; n = 6); * P
    Figure Legend Snippet: RSV impairs IFN-α/β–mediated STAT2 phosphorylation. ( A ) Western immunoblot analysis was performed to determine whether RSV infection (MOI = 1; 24 h) inhibited phosphorylation of STAT2 in IFN-α– (100 U/ml) or IFN-β–stimulated (100 U/ml, 30-min treatment) RAW macrophages. STAT2 phosphorylation in response to either IFN-α or IFN-β was significantly impaired in RSV-infected macrophages. RSV infection increased the level of STAT2, whereas β-actin levels were similar in all samples. Each lane of the immunoblot was loaded with 20 μg of protein, and the immunoblots shown are representative of three independent experiments. ( B ) Flow cytometric analysis was performed on macrophages to determine the effect of RSV infection on STAT2 protein expression on a per-cell basis. RSV-infected macrophages ( closed bar ) had increased levels of STAT2 when compared with mock-infected cells ( open bar ). After infection, both RSV-negative and RSV-positive macrophages ( hatched bars ) had elevated levels of STAT2 when compared with mock-infected cells. Data are means (±SEM; n = 6); * P

    Techniques Used: Western Blot, Infection, Flow Cytometry, Expressing

    25) Product Images from "EphB and Ephrin-B Interactions Mediate Human Mesenchymal Stem Cell Suppression of Activated T-Cells"

    Article Title: EphB and Ephrin-B Interactions Mediate Human Mesenchymal Stem Cell Suppression of Activated T-Cells

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2012.0676

    Gene expression of soluble factors in MSC and T-cells. (A–C) The expression of immunosuppressive factors: indoleamine 2,3-dioxygenase (IDO) (A) in MSC were induced after 24 h stimulation with INF-γ (25 ng/mL) and EphB4-Fc (5 μg/mL) or ephrin-B1-Fc (5 μg/mL) compared with human-Fc control; while transforming growth factor (TGF-β1) (B) and inducible nitric oxide synthase (iNOS) (C) expression was up-regulated after 48 h stimulation with interferon (IFN)-γ (25 ng/mL) and EphB4-Fc (5 μg/mL) compared with human-Fc control. (D–G) The expression of key molecules involved in T-cell activation and proliferation: IFN-γ (D) , tumor necrosis factor-α (TNF-α) (E) and interleukin (IL)-17 (F) are down-regulated after 24 h stimulation with EphB2-Fc (1 μg/mL) or ephrin-B2-Fc (1 μg/mL) compared with human-Fc control, whereas IL-2 (G) expression was reduced after 48 h stimulation with EphB2-Fc (1 μg/mL) or ephrin-B2-Fc (1 μg/mL) compared with human-Fc control. Gene expression analysis was performed by RT-PCR. Representative data of three independent experiments, expression is relative to β-actin control. One-way ANOVA, dunnett post-test, * P
    Figure Legend Snippet: Gene expression of soluble factors in MSC and T-cells. (A–C) The expression of immunosuppressive factors: indoleamine 2,3-dioxygenase (IDO) (A) in MSC were induced after 24 h stimulation with INF-γ (25 ng/mL) and EphB4-Fc (5 μg/mL) or ephrin-B1-Fc (5 μg/mL) compared with human-Fc control; while transforming growth factor (TGF-β1) (B) and inducible nitric oxide synthase (iNOS) (C) expression was up-regulated after 48 h stimulation with interferon (IFN)-γ (25 ng/mL) and EphB4-Fc (5 μg/mL) compared with human-Fc control. (D–G) The expression of key molecules involved in T-cell activation and proliferation: IFN-γ (D) , tumor necrosis factor-α (TNF-α) (E) and interleukin (IL)-17 (F) are down-regulated after 24 h stimulation with EphB2-Fc (1 μg/mL) or ephrin-B2-Fc (1 μg/mL) compared with human-Fc control, whereas IL-2 (G) expression was reduced after 48 h stimulation with EphB2-Fc (1 μg/mL) or ephrin-B2-Fc (1 μg/mL) compared with human-Fc control. Gene expression analysis was performed by RT-PCR. Representative data of three independent experiments, expression is relative to β-actin control. One-way ANOVA, dunnett post-test, * P

    Techniques Used: Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction

    EphB/ephrin-B expression by human mesenchymal stromal/stem cells (MSC). Human ex vivo expanded MSC were purified by STRO-1 + magnetic-activated cell sorting from bone marrow mononuclear cells of healthy donors. (A) Gene expression analysis obtained using RT-PCR and normalized to β-actin. Data represent the mean±SEM of three independent donors. (B) Supportive western blot data of EphB2 and ephrin-B2 protein expressed by human MSC from four donors and the corresponding β-actin controls. RT-PCR and western blot data are consistent with our previously published data. 14
    Figure Legend Snippet: EphB/ephrin-B expression by human mesenchymal stromal/stem cells (MSC). Human ex vivo expanded MSC were purified by STRO-1 + magnetic-activated cell sorting from bone marrow mononuclear cells of healthy donors. (A) Gene expression analysis obtained using RT-PCR and normalized to β-actin. Data represent the mean±SEM of three independent donors. (B) Supportive western blot data of EphB2 and ephrin-B2 protein expressed by human MSC from four donors and the corresponding β-actin controls. RT-PCR and western blot data are consistent with our previously published data. 14

    Techniques Used: Expressing, Ex Vivo, Purification, FACS, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Erythropoietin-producing hepatocellular B (EphB)/ephrin-B expression by human T-cells. Human primary T-cells were purified by CD3 + magnetic-activated cell sorting from peripheral blood mononuclear cells of healthy donors. (A) Gene expression data was obtained using real-time polymerase chain reaction (RT-PCR) and normalized to β-actin. Data represent the mean±SEM of three independent donors. (B) The data represent western blot analyses of EphB4 and ephrin-B1 protein expressed by human primary T-cells from three donors and the corresponding β-actin controls.
    Figure Legend Snippet: Erythropoietin-producing hepatocellular B (EphB)/ephrin-B expression by human T-cells. Human primary T-cells were purified by CD3 + magnetic-activated cell sorting from peripheral blood mononuclear cells of healthy donors. (A) Gene expression data was obtained using real-time polymerase chain reaction (RT-PCR) and normalized to β-actin. Data represent the mean±SEM of three independent donors. (B) The data represent western blot analyses of EphB4 and ephrin-B1 protein expressed by human primary T-cells from three donors and the corresponding β-actin controls.

    Techniques Used: Expressing, Purification, FACS, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

    shRNA knockdown of EphB2 or ephrin-B2 expression in MSC reduces their inhibitory function on T-cell proliferation. (A) EphB2 gene expression is reduced in EphB2 knockdown MSC lines compared with nonsilencing scramble MSC control. (B) ephrin-B2 gene expression is decreased in ephrin-B2 knockdown MSC lines compared with nonsilencing scramble MSC control. Gene expression analysis obtained using RT-PCR and normalized to β-actin. (C) T-cell proliferation was significantly suppressed in a dose-dependent manner ( open bars ) in the presence of MSC compared with a culture without MSC ( gray bar ). However, MSC-mediated T-cell suppression was significantly reversed in the presence of shRNA knockdown EphB2 MSC compared with the nonsilencing scramble MSC control. (D) MSC-induced T-cell suppression was significantly reduced in the presence of shRNA ephrin-B2 MSC compared with the nonsilencing scramble MSC control. Data represent the mean±SEM of four independent experiments; * P
    Figure Legend Snippet: shRNA knockdown of EphB2 or ephrin-B2 expression in MSC reduces their inhibitory function on T-cell proliferation. (A) EphB2 gene expression is reduced in EphB2 knockdown MSC lines compared with nonsilencing scramble MSC control. (B) ephrin-B2 gene expression is decreased in ephrin-B2 knockdown MSC lines compared with nonsilencing scramble MSC control. Gene expression analysis obtained using RT-PCR and normalized to β-actin. (C) T-cell proliferation was significantly suppressed in a dose-dependent manner ( open bars ) in the presence of MSC compared with a culture without MSC ( gray bar ). However, MSC-mediated T-cell suppression was significantly reversed in the presence of shRNA knockdown EphB2 MSC compared with the nonsilencing scramble MSC control. (D) MSC-induced T-cell suppression was significantly reduced in the presence of shRNA ephrin-B2 MSC compared with the nonsilencing scramble MSC control. Data represent the mean±SEM of four independent experiments; * P

    Techniques Used: shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction

    26) Product Images from "Particled Mica, STB-HO has chemopreventive potential via G1 arrest, and inhibition of proliferation and vascular endothelial growth factor receptor 2 in HCT colorectal cancer cells"

    Article Title: Particled Mica, STB-HO has chemopreventive potential via G1 arrest, and inhibition of proliferation and vascular endothelial growth factor receptor 2 in HCT colorectal cancer cells

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-13-189

    Effect of STB-HO on pVEGFR2, PI3K and Akt in colon cancer cells. (A) Basal expression of pVEGFR2 was confirmed in various colon cancer cells by Western blotting. (B) STB-HO (0, 250 or 500 μg/ml) was treated in HCT15, SW620 and HCT116 colon cancer cells for 24 h. Western blotting was performed to determine the expression of pVEGFR2, VEGFR2, PI3K, pAKT, AKT and β-actin in STB-HO treated colon cancer cells.
    Figure Legend Snippet: Effect of STB-HO on pVEGFR2, PI3K and Akt in colon cancer cells. (A) Basal expression of pVEGFR2 was confirmed in various colon cancer cells by Western blotting. (B) STB-HO (0, 250 or 500 μg/ml) was treated in HCT15, SW620 and HCT116 colon cancer cells for 24 h. Western blotting was performed to determine the expression of pVEGFR2, VEGFR2, PI3K, pAKT, AKT and β-actin in STB-HO treated colon cancer cells.

    Techniques Used: Expressing, Western Blot

    Effect of STB-HO on pVEGFR2, PI3K and Akt in HUVECs. HUVECs were starved for 24 h in M199 containing 1% heat-inactivated FBS and then treated with various concentrations of STB-HO (0, 250 or 500 μg/ml) in M199 containing 1% heat-inactivated FBS, 10 ng/ml VEGF and 5 units/ml heparin for 10 min, 30 min, 1 h, 2 h, and 24 h. The cells were harvested and western blotting was performed to determine the expression of pVEGFR2, VEGFR2, pAKT, AKT and β-actin.
    Figure Legend Snippet: Effect of STB-HO on pVEGFR2, PI3K and Akt in HUVECs. HUVECs were starved for 24 h in M199 containing 1% heat-inactivated FBS and then treated with various concentrations of STB-HO (0, 250 or 500 μg/ml) in M199 containing 1% heat-inactivated FBS, 10 ng/ml VEGF and 5 units/ml heparin for 10 min, 30 min, 1 h, 2 h, and 24 h. The cells were harvested and western blotting was performed to determine the expression of pVEGFR2, VEGFR2, pAKT, AKT and β-actin.

    Techniques Used: Western Blot, Expressing

    27) Product Images from "Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue"

    Article Title: Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006385

    Platelets sequester circulating histone H2A in plasma from dengue-infected patients. ( A-B ) Western blot analysis for histone H2A and β-actin in ( A ) freshly isolated platelets from three healthy volunteers (Control) and three patients with dengue; and in ( B ) platelets from three healthy volunteers that were kept unstimulated (Unst) or stimulated with thrombin (Thr), DENV or Mock for 6h. Human peripheral blood mononuclear cells (PBMC) were used as positive control for histone H2A expression. ( C ) Histone H2A concentration in plasma from control subjects or patients with mild dengue or dengue with warning signs and severe dengue (WS+Sev). Boxes indicate the median and interquartile ranges and whiskers indicate minimal and maximal values in each group. ( D-E ) Platelets were isolated from a healthy volunteer and incubated with 20% plasma from five dengue-infected patients (dengue plasma) or five healthy volunteers (control plasma) for 4 hours in the presence or absence of cyclohexamide (CHX), cytochalasin B (CTB) or DMSO (vehicle). ( F ) Histone H2A concentration in plasma from control subjects or patients with dengue, zika or chikungunya fever. Each dot represents the level of histone H2A in plasma from one patient or control. Lines represent median and interquartile range. ( G ) Western blot analysis for histone H2A and β-actin in platelets incubated with 20% plasma from three control subjects or three patients with dengue, zika or chikungunya. * means p
    Figure Legend Snippet: Platelets sequester circulating histone H2A in plasma from dengue-infected patients. ( A-B ) Western blot analysis for histone H2A and β-actin in ( A ) freshly isolated platelets from three healthy volunteers (Control) and three patients with dengue; and in ( B ) platelets from three healthy volunteers that were kept unstimulated (Unst) or stimulated with thrombin (Thr), DENV or Mock for 6h. Human peripheral blood mononuclear cells (PBMC) were used as positive control for histone H2A expression. ( C ) Histone H2A concentration in plasma from control subjects or patients with mild dengue or dengue with warning signs and severe dengue (WS+Sev). Boxes indicate the median and interquartile ranges and whiskers indicate minimal and maximal values in each group. ( D-E ) Platelets were isolated from a healthy volunteer and incubated with 20% plasma from five dengue-infected patients (dengue plasma) or five healthy volunteers (control plasma) for 4 hours in the presence or absence of cyclohexamide (CHX), cytochalasin B (CTB) or DMSO (vehicle). ( F ) Histone H2A concentration in plasma from control subjects or patients with dengue, zika or chikungunya fever. Each dot represents the level of histone H2A in plasma from one patient or control. Lines represent median and interquartile range. ( G ) Western blot analysis for histone H2A and β-actin in platelets incubated with 20% plasma from three control subjects or three patients with dengue, zika or chikungunya. * means p

    Techniques Used: Infection, Western Blot, Isolation, Positive Control, Expressing, Concentration Assay, Incubation, CtB Assay

    Increased HLA class I on DENV-infected platelets depends on protein translation and proteasome activity. (A) Western blot analysis for HLA class I and β-actin in freshly isolated platelets from two healthy control volunteers and two dengue-infected patients. ( B-H ) Platelets isolated from healthy volunteers were kept unstimulated (Unst) or stimulated with thrombin (Thr), DENV or Mock for the indicated times. Panel B shows the overall HLA class I expression in platelets from two independent donors at 6 hours post stimulation; and panel C shows the percent of platelets with high surface expression of HLA class I (HLA class I High ) in each condition. ( D-H ) Platelets were exposed to DENV or Mock in the presence of DMSO (vehicle), bortezomib (1 μM) or cyclohexamide (10 μM). Panel D show the HLA class I expression at 6 hours post infection, panels E - F show the percent of HLA class I High expression and panels G-H depicts the P-selectin (CD62P) surface expression in platelets incubated in each condition. Bars represent mean ± standard error of the mean of 3 to 7 independent experiments from individual platelet donors. * indicates p
    Figure Legend Snippet: Increased HLA class I on DENV-infected platelets depends on protein translation and proteasome activity. (A) Western blot analysis for HLA class I and β-actin in freshly isolated platelets from two healthy control volunteers and two dengue-infected patients. ( B-H ) Platelets isolated from healthy volunteers were kept unstimulated (Unst) or stimulated with thrombin (Thr), DENV or Mock for the indicated times. Panel B shows the overall HLA class I expression in platelets from two independent donors at 6 hours post stimulation; and panel C shows the percent of platelets with high surface expression of HLA class I (HLA class I High ) in each condition. ( D-H ) Platelets were exposed to DENV or Mock in the presence of DMSO (vehicle), bortezomib (1 μM) or cyclohexamide (10 μM). Panel D show the HLA class I expression at 6 hours post infection, panels E - F show the percent of HLA class I High expression and panels G-H depicts the P-selectin (CD62P) surface expression in platelets incubated in each condition. Bars represent mean ± standard error of the mean of 3 to 7 independent experiments from individual platelet donors. * indicates p

    Techniques Used: Infection, Activity Assay, Western Blot, Isolation, Expressing, Incubation

    Dengue triggers platelet activation and chemokine secretion. ( A ) P-selectin (CD62P) surface expression by platelets isolated from healthy volunteers (Control) and patients with mild dengue, dengue with warning signs (WS) and severe dengue (Sev). (percentage of positive cells by flow cytometry). Boxes indicate the median and interquartile ranges and whiskers indicate minimal and maximal values in each group. Representative dot plots are shown. ( B ) Western blot analysis of platelet factor 4 (PF4/CXCL4) and β-actin in platelets isolated from three control subjects and three patients with dengue. ( C and E ) PF4/CXCL4 and RANTES/CCL5 concentration in supernatants of platelets (1 x 10 9 per mL) from 8 control subjects and 8 dengue patients that were cultured for 4h ex vivo . Platelets obtained from the same patients in the recovery phase (Recov) were also evaluated. Each dot represents the level of chemokines secreted by platelets from one dengue patient or control. Horizontal lines represent median. ( D and F ) Concentration of PF4/CXCL4 and RANTES/CCL5 in plasma from control subjects and patients with dengue. Boxes indicate the median and interquartile ranges and whiskers indicate minimal and maximal values in each group. ( G-I ) Platelets isolated from healthy volunteers were kept unstimulated (Unst) or stimulated with thrombin (Thr), DENV or Mock for the indicated times. Panel G shows the percentage of CD62P-expressing platelets, and panels H to I show the concentration of PF4/CXCL4 and RANTES/CCL5 in the supernatant of platelets incubated in each condition for 6h. Bars represent mean ± standard error of the mean of 4 to 8 independent experiments performed with platelets from individual donors.* means p
    Figure Legend Snippet: Dengue triggers platelet activation and chemokine secretion. ( A ) P-selectin (CD62P) surface expression by platelets isolated from healthy volunteers (Control) and patients with mild dengue, dengue with warning signs (WS) and severe dengue (Sev). (percentage of positive cells by flow cytometry). Boxes indicate the median and interquartile ranges and whiskers indicate minimal and maximal values in each group. Representative dot plots are shown. ( B ) Western blot analysis of platelet factor 4 (PF4/CXCL4) and β-actin in platelets isolated from three control subjects and three patients with dengue. ( C and E ) PF4/CXCL4 and RANTES/CCL5 concentration in supernatants of platelets (1 x 10 9 per mL) from 8 control subjects and 8 dengue patients that were cultured for 4h ex vivo . Platelets obtained from the same patients in the recovery phase (Recov) were also evaluated. Each dot represents the level of chemokines secreted by platelets from one dengue patient or control. Horizontal lines represent median. ( D and F ) Concentration of PF4/CXCL4 and RANTES/CCL5 in plasma from control subjects and patients with dengue. Boxes indicate the median and interquartile ranges and whiskers indicate minimal and maximal values in each group. ( G-I ) Platelets isolated from healthy volunteers were kept unstimulated (Unst) or stimulated with thrombin (Thr), DENV or Mock for the indicated times. Panel G shows the percentage of CD62P-expressing platelets, and panels H to I show the concentration of PF4/CXCL4 and RANTES/CCL5 in the supernatant of platelets incubated in each condition for 6h. Bars represent mean ± standard error of the mean of 4 to 8 independent experiments performed with platelets from individual donors.* means p

    Techniques Used: Activation Assay, Expressing, Isolation, Flow Cytometry, Cytometry, Western Blot, Concentration Assay, Cell Culture, Ex Vivo, Incubation

    28) Product Images from "ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †"

    Article Title: ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00974-07

    Expression of mAPE1 in response to sodium arsenite in 10T½ cells. (A) Dose-response experiments. Confluent cells were treated for 30 min in medium containing the indicated concentration of arsenite and then washed and incubated in fresh medium for 2 h before harvesting for Northern blotting analysis. (B) Kinetics. Confluent cells were treated with 50 μM arsenite for the indicated times, then washed, and incubated in fresh medium for 2 h before harvesting for Northern blotting analysis. (C and D) Cellular viability under conditions corresponding to the experiments shown in panels A and B, respectively. (E) Four hours after arsenite treatment, Ape1 protein levels were detected by immunoblotting. (F) Six hours after arsenite treatment, the AP endonuclease assay of Ape1 activity was performed. The 35-mer band is the substrate, and the 12-mer band is the AP endonuclease cleavage product. AP, purified hApe1 as a positive control. Three independent sets of assays were performed and normalized to GAPDH mRNA for Northern blotting or to β-actin for immunoblotting. Standard deviations are indicated by error bars. Values that were significantly different ( P
    Figure Legend Snippet: Expression of mAPE1 in response to sodium arsenite in 10T½ cells. (A) Dose-response experiments. Confluent cells were treated for 30 min in medium containing the indicated concentration of arsenite and then washed and incubated in fresh medium for 2 h before harvesting for Northern blotting analysis. (B) Kinetics. Confluent cells were treated with 50 μM arsenite for the indicated times, then washed, and incubated in fresh medium for 2 h before harvesting for Northern blotting analysis. (C and D) Cellular viability under conditions corresponding to the experiments shown in panels A and B, respectively. (E) Four hours after arsenite treatment, Ape1 protein levels were detected by immunoblotting. (F) Six hours after arsenite treatment, the AP endonuclease assay of Ape1 activity was performed. The 35-mer band is the substrate, and the 12-mer band is the AP endonuclease cleavage product. AP, purified hApe1 as a positive control. Three independent sets of assays were performed and normalized to GAPDH mRNA for Northern blotting or to β-actin for immunoblotting. Standard deviations are indicated by error bars. Values that were significantly different ( P

    Techniques Used: Expressing, Concentration Assay, Incubation, Northern Blot, Activity Assay, Purification, Positive Control

    Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P
    Figure Legend Snippet: Changes of Ape1 levels in ATF4-deficient cells. The cells infected with a retrovirus expressing siRNA against ATF4 (S-ATF4) or with an mApe1 retroviral expression vector were cultured to confluence under the appropriate selection conditions (puromycin for the siRNA vectors and hygromycin for the mApe1 expression vector). The cells were then treated with 20 to 50 μM sodium arsenite for 30 min. The cells were washed twice, and the incubation in fresh medium continued for 4 h. The Ape1 protein levels were determined by immunoblotting and phosphorimaging and normalized to the β-actin signal. (A) 10T½ cells. (B) TK6 cells. The top panel in each frame shows a representative immunoblot, and the graph shows the quantification from three independent experiments. Standard deviations are indicated by error bars. Values that were significantly different ( P

    Techniques Used: Infection, Expressing, Plasmid Preparation, Cell Culture, Selection, Incubation

    29) Product Images from "Overexpression and oncogenic function of aldo-keto reductase family 1B10 (AKR1B10) in pancreatic carcinoma"

    Article Title: Overexpression and oncogenic function of aldo-keto reductase family 1B10 (AKR1B10) in pancreatic carcinoma

    Journal: Modern Pathology

    doi: 10.1038/modpathol.2011.191

    Western blot analysis of prenylated proteins including HDJ2 and KRAS as well its downstream signals. Increased non-farnesylated HDJ2 protein ( upper black arrow ) and decreased membrane-bound KRAS protein were observed in the siRNA AKR1B10 pancreatic cancer cell line when compared to the scrambled siRNA control. Increased levels of membrane-bound E-cadherin ( lower black arrow ) and decreased levels of KRAS downstream effectors, phosphor-ERK and phosphor-MEK1/2, were also found. Flottin-2 was used as a loading control for membrane-bound proteins, while β-actin is used for cytosolic proteins.
    Figure Legend Snippet: Western blot analysis of prenylated proteins including HDJ2 and KRAS as well its downstream signals. Increased non-farnesylated HDJ2 protein ( upper black arrow ) and decreased membrane-bound KRAS protein were observed in the siRNA AKR1B10 pancreatic cancer cell line when compared to the scrambled siRNA control. Increased levels of membrane-bound E-cadherin ( lower black arrow ) and decreased levels of KRAS downstream effectors, phosphor-ERK and phosphor-MEK1/2, were also found. Flottin-2 was used as a loading control for membrane-bound proteins, while β-actin is used for cytosolic proteins.

    Techniques Used: Western Blot

    30) Product Images from "Aptamer-mediated survivin RNAi enables 5-fluorouracil to eliminate colorectal cancer stem cells"

    Article Title: Aptamer-mediated survivin RNAi enables 5-fluorouracil to eliminate colorectal cancer stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05859-z

    EpCAM aptamer-guided RNAi effectively silenced survivin. ( a ) Specificity and efficacy of EpCAM-aptamer guided RNAi in knocking down survivin mRNA. Chimera or negative control chimera were incubated with HT-29 or HEK-2913T cells for 24 hours and the total RNA was extracted for qRT-PCR analysis of survivin mRNA levels. GAPDH was used as an internal control. ( b , c ) HT-29 Tumour-bearing mice were treated with 2 nmol/mouse of PEG-labelled chimera for 48 hours. The tumours were collected for RNA extraction followed by qRT-PCR analysis of survivin mRNA expression ( b ) and 5′RACE assay ( c ). ( d ) Effective downregulation of survivin protein via EpCAM aptamer-guided RNAi. Chimera or negative control chimera were incubated with HT-29 or HEK-2913T cells for 48 hours and the survivin protein levels were analyzed using Western blot analysis. β-actin was used as a loading control. ( e ) The bar graph shows the survivin protein levels in various treatment groups. Data shown are means ± SEM, n = 3. * p
    Figure Legend Snippet: EpCAM aptamer-guided RNAi effectively silenced survivin. ( a ) Specificity and efficacy of EpCAM-aptamer guided RNAi in knocking down survivin mRNA. Chimera or negative control chimera were incubated with HT-29 or HEK-2913T cells for 24 hours and the total RNA was extracted for qRT-PCR analysis of survivin mRNA levels. GAPDH was used as an internal control. ( b , c ) HT-29 Tumour-bearing mice were treated with 2 nmol/mouse of PEG-labelled chimera for 48 hours. The tumours were collected for RNA extraction followed by qRT-PCR analysis of survivin mRNA expression ( b ) and 5′RACE assay ( c ). ( d ) Effective downregulation of survivin protein via EpCAM aptamer-guided RNAi. Chimera or negative control chimera were incubated with HT-29 or HEK-2913T cells for 48 hours and the survivin protein levels were analyzed using Western blot analysis. β-actin was used as a loading control. ( e ) The bar graph shows the survivin protein levels in various treatment groups. Data shown are means ± SEM, n = 3. * p

    Techniques Used: Negative Control, Incubation, Quantitative RT-PCR, Mouse Assay, RNA Extraction, Expressing, Western Blot

    31) Product Images from "MicroRNA-211 Expression Promotes Colorectal Cancer Cell Growth In Vitro and In Vivo by Targeting Tumor Suppressor CHD5"

    Article Title: MicroRNA-211 Expression Promotes Colorectal Cancer Cell Growth In Vitro and In Vivo by Targeting Tumor Suppressor CHD5

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029750

    miR-211 inhibits CHD5 expression in colorectal cancer cells stably expressing exogenous miR-211. (A) Schematic representation of the miR-211 vector, which contains an expression cassette of the P CMV promoter, EGFP, and miR-211 precursor and a selective cassette of the P PGK promoter and Puro r . (B) CHD5 protein levels in cell lines HCT-116 vec and HCT-116 miR-211 were evaluated by Western blot and (C) semi-quantified based on CHD5/β-actin relative intensities. (D) Luciferase reporter assay using HEK 293T cells that were transfected with either the luciferase/3′-UTR miR-211 reporter vector, the miR-211 vector, or both. Schematic representation of the luciferase-CHD5 reporter vector and luciferase reporter control vector were also inserted. * indicated as P
    Figure Legend Snippet: miR-211 inhibits CHD5 expression in colorectal cancer cells stably expressing exogenous miR-211. (A) Schematic representation of the miR-211 vector, which contains an expression cassette of the P CMV promoter, EGFP, and miR-211 precursor and a selective cassette of the P PGK promoter and Puro r . (B) CHD5 protein levels in cell lines HCT-116 vec and HCT-116 miR-211 were evaluated by Western blot and (C) semi-quantified based on CHD5/β-actin relative intensities. (D) Luciferase reporter assay using HEK 293T cells that were transfected with either the luciferase/3′-UTR miR-211 reporter vector, the miR-211 vector, or both. Schematic representation of the luciferase-CHD5 reporter vector and luciferase reporter control vector were also inserted. * indicated as P

    Techniques Used: Expressing, Stable Transfection, Plasmid Preparation, Western Blot, Luciferase, Reporter Assay, Transfection

    Comparison of CHD5 and miR-211 expression in various colorectal cancer cell lines. (A) RNA sequence map of the 3′-UTR of CHD5 (Gene ID 26038) mRNA with the complementary site (3′-UTR 130–136) for the seed region of miRNA-211. (B) miR-211 levels in RKO, HCT-116, RKO-S, and RKO-AS cell lines by quantitative RT-PCR based on miR-211/U6 expression fold values. (C) CHD5 protein levels in two colon cancer cell lines (RKO and HCT-116) and two established cell lines (RKO-S and RKO-AS) with enforced CHD5-S expression were analyzed by Western blot and semi-quantified based on CHD5/β-actin relative intensities. Bio-Rad Quantity One software was used for densitometric analysis of the Western blots. (D) Comparison of CHD5 and miR-211 expression levels in HCT-116 and RKO cell lines. * indicated as P
    Figure Legend Snippet: Comparison of CHD5 and miR-211 expression in various colorectal cancer cell lines. (A) RNA sequence map of the 3′-UTR of CHD5 (Gene ID 26038) mRNA with the complementary site (3′-UTR 130–136) for the seed region of miRNA-211. (B) miR-211 levels in RKO, HCT-116, RKO-S, and RKO-AS cell lines by quantitative RT-PCR based on miR-211/U6 expression fold values. (C) CHD5 protein levels in two colon cancer cell lines (RKO and HCT-116) and two established cell lines (RKO-S and RKO-AS) with enforced CHD5-S expression were analyzed by Western blot and semi-quantified based on CHD5/β-actin relative intensities. Bio-Rad Quantity One software was used for densitometric analysis of the Western blots. (D) Comparison of CHD5 and miR-211 expression levels in HCT-116 and RKO cell lines. * indicated as P

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR, Western Blot, Software

    Effects of exogenous miR-211 on cell growth-associated proteins and p53-related pathway proteins in colorectal cancer cell lines. (A) The levels of cell growth-associated proteins in HCT-116 vec and HCT-116 miR-211 cell lines were analyzed by Western blot and (B) semi-quantified based on targeted protein/β-actin relative intensities. The levels of p53-related pathway proteins in HCT-116 vec and HCT-116 miR-211 cell lines were analyzed by Western blot (C) and semi-quantified based on targeted protein/β-actin relative intensities (D).
    Figure Legend Snippet: Effects of exogenous miR-211 on cell growth-associated proteins and p53-related pathway proteins in colorectal cancer cell lines. (A) The levels of cell growth-associated proteins in HCT-116 vec and HCT-116 miR-211 cell lines were analyzed by Western blot and (B) semi-quantified based on targeted protein/β-actin relative intensities. The levels of p53-related pathway proteins in HCT-116 vec and HCT-116 miR-211 cell lines were analyzed by Western blot (C) and semi-quantified based on targeted protein/β-actin relative intensities (D).

    Techniques Used: Western Blot

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    Article Title: Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray
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    Purification:

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    Produced:

    Article Title: R9AP Stabilizes RGS11-G?5 and Accelerates the Early Light Response of ON-Bipolar Cells
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    Incubation:

    Article Title: Effects of MS-153 on chronic ethanol consumption and GLT1 modulation of glutamate levels in male alcohol-preferring rats
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    Millipore mouse anti human β actin monoclonal antibody
    Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with <t>anti-β-actin</t> antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.
    Mouse Anti Human β Actin Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human β actin monoclonal antibody/product/Millipore
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    Millipore mouse β actin antibody
    Amyloid plaque load and soluble Aβ levels in the brain of APP swe /PS1 in a context of partial MyD88 deficiency . Deposition of Aβ plaques is significantly more abundant in 6 and 9 month-old APP swe /PS1 compared to APP swe /PS1-Myd88 +/- mice (a) . Aβ immunoreactivity in cortex and hippocampus is shown in brain sections of 9 month-old APP swe /PS1 and APP swe /PS1-Myd88 +/- mice. Percentage of area covered by plaques was quantified for mice of 3, 6 and 9 month-old, respectively. n = 9-10. (Two-way ANOVA was performed revealing a significant interaction between factors age and genotype. The comparison of genotype for each age was performed by Student's t -test). To detect soluble Aβ, western blot analysis on 10-20% Tris-Tricine denaturing polyacrylamide gels of extracellular (b) , intracellular (c) and membrane-associated (d) enriched proteins of 6 month-old mice were assessed using monoclonal 6E10 antibody to reveal the different species. Most of Aβ oligomers were significantly higher in the brains of APP swe /PS1-MyD88 +/- (AM) mice than that of APP swe /PS1 (A) in all protein fractions. Bands depicted here were cut from the same membrane for each protein fraction. Values are expressed as optical densities (OD) in arbitrary units (a.u.) of Aβ normalized with <t>β-actin.</t> n = 4-7; Student's t -test; * P
    Mouse β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with anti-β-actin antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.

    Journal: PLoS ONE

    Article Title: Ser276 Phosphorylation of NF-kB p65 by MSK1 Controls SCF Expression in Inflammation

    doi: 10.1371/journal.pone.0004393

    Figure Lengend Snippet: Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with anti-β-actin antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.

    Article Snippet: Immunoblotting used the following antibodies: rabbit anti-human IκB-α polyclonal antibody (1/1000, Calbiochem, La Jolla, CA), mouse anti-human phospho- IκB-α monoclonal antibody, (1/1000, Ab-1, Oncogene Research Product, Boston, MA), rabbit anti-human phospho-Ser276 p65 antibody (1/1000, 3037, Cell Signaling Technology, Danvers MA), rabbit anti-human phospho-Ser536 p65 antibody (1/1000, 3031, Cell Signaling Technology), rabbit anti-human p65 polyclonal antibody (1/200, sc-109, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human CBP polyclonal antibody (1/200, sc-369, Santa Cruz Biotechnology), mouse anti-human β-actin monoclonal antibody (1/5000, Ab-1, Oncogene Research Product), goat anti-human MSK1 (1/200, sc-9392, Santa Cruz Biotechnology.

    Techniques: Expressing, Transfection, Luciferase, Construct, Incubation, Activity Assay, Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

    Amyloid plaque load and soluble Aβ levels in the brain of APP swe /PS1 in a context of partial MyD88 deficiency . Deposition of Aβ plaques is significantly more abundant in 6 and 9 month-old APP swe /PS1 compared to APP swe /PS1-Myd88 +/- mice (a) . Aβ immunoreactivity in cortex and hippocampus is shown in brain sections of 9 month-old APP swe /PS1 and APP swe /PS1-Myd88 +/- mice. Percentage of area covered by plaques was quantified for mice of 3, 6 and 9 month-old, respectively. n = 9-10. (Two-way ANOVA was performed revealing a significant interaction between factors age and genotype. The comparison of genotype for each age was performed by Student's t -test). To detect soluble Aβ, western blot analysis on 10-20% Tris-Tricine denaturing polyacrylamide gels of extracellular (b) , intracellular (c) and membrane-associated (d) enriched proteins of 6 month-old mice were assessed using monoclonal 6E10 antibody to reveal the different species. Most of Aβ oligomers were significantly higher in the brains of APP swe /PS1-MyD88 +/- (AM) mice than that of APP swe /PS1 (A) in all protein fractions. Bands depicted here were cut from the same membrane for each protein fraction. Values are expressed as optical densities (OD) in arbitrary units (a.u.) of Aβ normalized with β-actin. n = 4-7; Student's t -test; * P

    Journal: Molecular Neurodegeneration

    Article Title: MyD88-adaptor protein acts as a preventive mechanism for memory deficits in a mouse model of Alzheimer's disease

    doi: 10.1186/1750-1326-6-5

    Figure Lengend Snippet: Amyloid plaque load and soluble Aβ levels in the brain of APP swe /PS1 in a context of partial MyD88 deficiency . Deposition of Aβ plaques is significantly more abundant in 6 and 9 month-old APP swe /PS1 compared to APP swe /PS1-Myd88 +/- mice (a) . Aβ immunoreactivity in cortex and hippocampus is shown in brain sections of 9 month-old APP swe /PS1 and APP swe /PS1-Myd88 +/- mice. Percentage of area covered by plaques was quantified for mice of 3, 6 and 9 month-old, respectively. n = 9-10. (Two-way ANOVA was performed revealing a significant interaction between factors age and genotype. The comparison of genotype for each age was performed by Student's t -test). To detect soluble Aβ, western blot analysis on 10-20% Tris-Tricine denaturing polyacrylamide gels of extracellular (b) , intracellular (c) and membrane-associated (d) enriched proteins of 6 month-old mice were assessed using monoclonal 6E10 antibody to reveal the different species. Most of Aβ oligomers were significantly higher in the brains of APP swe /PS1-MyD88 +/- (AM) mice than that of APP swe /PS1 (A) in all protein fractions. Bands depicted here were cut from the same membrane for each protein fraction. Values are expressed as optical densities (OD) in arbitrary units (a.u.) of Aβ normalized with β-actin. n = 4-7; Student's t -test; * P

    Article Snippet: Membranes were stripped in 25 mM glycine-HCl, pH 2.0, containing 1% SDS to allow β-actin revelation using first a mouse β-actin antibody (MAB1501, 1:10 000; Millipore Bioscience Research Reagents) and then a goat anti-mouse peroxidase conjugated secondary antibody (1:10 000; Jackson ImmunoResearch).

    Techniques: Mouse Assay, Western Blot

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep26992

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

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

    Techniques: Staining, Western Blot, Fluorescence, Significance Assay

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep26992

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

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

    Techniques: Expressing, Staining, Western Blot, Fluorescence

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep26992

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

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

    Techniques: Staining, Western Blot, Fluorescence, Significance Assay

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep26992

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

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

    Techniques: Staining, Western Blot, Fluorescence, Significance Assay

    Digitoxin induces autophagic and proteasomal degradation of the α1 subunit. (A) HFFs were infected and treated with digitoxin (30 nM), AICAR (0.8 mM), digitoxin plus AICAR, or GCV (5 μM). Cells were treated with MG132 (10 μM) 10 h before harvest. Lysates were used to detect p-AMPK and α1. Na/K α1 subunit expression was normalized against β-actin expression. The ratios of normalized α1 levels in MG132-treated and untreated samples were quantitated by densitometry. (B) Infected and treated HFFs were immunoprecipitated with anti-ubiquitin antibody and probed for the Na + ,K + /ATPase α1 subunit in the presence of MG132 (10 μM). (C) HCMV-infected cells were treated or not treated with digitoxin. At 24 hpi, cycloheximide (CHX; 100 μg/ml) was added. Cells were harvested at the indicated time intervals following CHX addition for SDS-PAGE analysis to measure α1 subunit degradation. (D) Infected and digitoxin-treated cells were treated with bafilomycin A1 (50 nM) for 4 h before harvest. Lysates were prepared at 48 hpi, and levels of the Na + ,K + /ATPase α1 subunit and p62 were detected by WB. (E) Model depicting the mechanism of digitoxin-mediated autophagy induction and HCMV inhibition. (i and ii) HCMV activates mTOR (i), which suppresses ULK1 by phosphorylating it at Ser757 to inhibit autophagy (ii), creating an intracellular microenvironment favorable for HCMV replication. (iii) Digitoxin degrades the α1 subunit by ubiquitination and autophagy. (iv) AMPK activation through the α1 subunit is independent of α1 degradation. (v) pAMPK phosphorylates ULK1 at Ser317 and interacts with it to induce autophagy, which is detrimental for HCMV.

    Journal: Journal of Virology

    Article Title: Digitoxin Suppresses Human Cytomegalovirus Replication via Na+, K+/ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

    doi: 10.1128/JVI.01861-17

    Figure Lengend Snippet: Digitoxin induces autophagic and proteasomal degradation of the α1 subunit. (A) HFFs were infected and treated with digitoxin (30 nM), AICAR (0.8 mM), digitoxin plus AICAR, or GCV (5 μM). Cells were treated with MG132 (10 μM) 10 h before harvest. Lysates were used to detect p-AMPK and α1. Na/K α1 subunit expression was normalized against β-actin expression. The ratios of normalized α1 levels in MG132-treated and untreated samples were quantitated by densitometry. (B) Infected and treated HFFs were immunoprecipitated with anti-ubiquitin antibody and probed for the Na + ,K + /ATPase α1 subunit in the presence of MG132 (10 μM). (C) HCMV-infected cells were treated or not treated with digitoxin. At 24 hpi, cycloheximide (CHX; 100 μg/ml) was added. Cells were harvested at the indicated time intervals following CHX addition for SDS-PAGE analysis to measure α1 subunit degradation. (D) Infected and digitoxin-treated cells were treated with bafilomycin A1 (50 nM) for 4 h before harvest. Lysates were prepared at 48 hpi, and levels of the Na + ,K + /ATPase α1 subunit and p62 were detected by WB. (E) Model depicting the mechanism of digitoxin-mediated autophagy induction and HCMV inhibition. (i and ii) HCMV activates mTOR (i), which suppresses ULK1 by phosphorylating it at Ser757 to inhibit autophagy (ii), creating an intracellular microenvironment favorable for HCMV replication. (iii) Digitoxin degrades the α1 subunit by ubiquitination and autophagy. (iv) AMPK activation through the α1 subunit is independent of α1 degradation. (v) pAMPK phosphorylates ULK1 at Ser317 and interacts with it to induce autophagy, which is detrimental for HCMV.

    Article Snippet: Other antibodies and related reagents were as follows: mouse monoclonal anti-β-actin (Millipore, Billerica, MA); rabbit polyclonal anti-LC3-II (Novus Biologicals, LLC, Littleton, CO, USA); anti-p62, -Beclin1, and -PI3K class III antibodies (Cell Signaling Technology, Beverly, MA); Na+ ,K+ /ATPase α1 subunit and ubiquitin antibodies (Santa Cruz Biotechnology); pAMPK (T172), AMPK, pULK1(Ser317/Ser757), and ULK1-1 antibodies, an mTOR substrate kit, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling); and HRP-conjugated sheep anti-mouse IgG (GE Healthcare, Waukesha, WI).

    Techniques: Infection, Expressing, Immunoprecipitation, SDS Page, Western Blot, Inhibition, Activation Assay

    Digitoxin-mediated inhibition of mTOR activity in noninfected ATG5 KD cells is lost in HCMV-infected ATG5 KD cells. Infected (A and B) or uninfected (C) control and ATG5 KD cells were treated with digitoxin (30 nM) or GCV (5 μM) for the indicated time points, and cell lysates were used to detect viral proteins IE1/2 and UL44 and cellular proteins ATG5, p-mTOR, mTOR, p-Ser S6K, p-Thr S6K, S6K, and p4E-BP-1. β-Actin was used as a loading control.

    Journal: Journal of Virology

    Article Title: Digitoxin Suppresses Human Cytomegalovirus Replication via Na+, K+/ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

    doi: 10.1128/JVI.01861-17

    Figure Lengend Snippet: Digitoxin-mediated inhibition of mTOR activity in noninfected ATG5 KD cells is lost in HCMV-infected ATG5 KD cells. Infected (A and B) or uninfected (C) control and ATG5 KD cells were treated with digitoxin (30 nM) or GCV (5 μM) for the indicated time points, and cell lysates were used to detect viral proteins IE1/2 and UL44 and cellular proteins ATG5, p-mTOR, mTOR, p-Ser S6K, p-Thr S6K, S6K, and p4E-BP-1. β-Actin was used as a loading control.

    Article Snippet: Other antibodies and related reagents were as follows: mouse monoclonal anti-β-actin (Millipore, Billerica, MA); rabbit polyclonal anti-LC3-II (Novus Biologicals, LLC, Littleton, CO, USA); anti-p62, -Beclin1, and -PI3K class III antibodies (Cell Signaling Technology, Beverly, MA); Na+ ,K+ /ATPase α1 subunit and ubiquitin antibodies (Santa Cruz Biotechnology); pAMPK (T172), AMPK, pULK1(Ser317/Ser757), and ULK1-1 antibodies, an mTOR substrate kit, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling); and HRP-conjugated sheep anti-mouse IgG (GE Healthcare, Waukesha, WI).

    Techniques: Inhibition, Activity Assay, Infection

    Digitoxin induces AMPK phosphorylation and autophagy flux in infected and noninfected HFFs. (A) Cells were infected with HCMV (MOI of 1 PFU/cell) and treated with digitoxin (30 nM) or GCV (5 μM) (I) or noninfected and treated with digitoxin or GCV (II). Lysates were prepared at the indicated time points and used for detection of pAMPK, AMPK, and IE1/2 by WB. (B) Model showing the function of bafilomycin A1 in inhibiting autophagosome-lysosome fusion, resulting in accumulation of LC3-II and p62 instead of their degradation. (C) Cells were treated as described for panel A for the indicated times. Bafilomycin A1 (50 nM) was added 4 h before harvest, and the levels of LC3-II and p62 were detected by WB. β-Actin was used as a loading control. (D) Cells were infected or treated with digitoxin (30 nM). LC3 puncta were detected by confocal microscopy at 24 and 72 hpi. The total number of LC3-II puncta/field was estimated by use of ImageJ software. The mean ± SD for three fields is provided for each condition. (E) HFFs were treated with digitoxin or GCV. Cell lysates were collected at the indicated time points for detection of pAMPK and p62 by WB. β-Actin was used as a loading control. Blots are the best representatives of three independent experiments.

    Journal: Journal of Virology

    Article Title: Digitoxin Suppresses Human Cytomegalovirus Replication via Na+, K+/ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

    doi: 10.1128/JVI.01861-17

    Figure Lengend Snippet: Digitoxin induces AMPK phosphorylation and autophagy flux in infected and noninfected HFFs. (A) Cells were infected with HCMV (MOI of 1 PFU/cell) and treated with digitoxin (30 nM) or GCV (5 μM) (I) or noninfected and treated with digitoxin or GCV (II). Lysates were prepared at the indicated time points and used for detection of pAMPK, AMPK, and IE1/2 by WB. (B) Model showing the function of bafilomycin A1 in inhibiting autophagosome-lysosome fusion, resulting in accumulation of LC3-II and p62 instead of their degradation. (C) Cells were treated as described for panel A for the indicated times. Bafilomycin A1 (50 nM) was added 4 h before harvest, and the levels of LC3-II and p62 were detected by WB. β-Actin was used as a loading control. (D) Cells were infected or treated with digitoxin (30 nM). LC3 puncta were detected by confocal microscopy at 24 and 72 hpi. The total number of LC3-II puncta/field was estimated by use of ImageJ software. The mean ± SD for three fields is provided for each condition. (E) HFFs were treated with digitoxin or GCV. Cell lysates were collected at the indicated time points for detection of pAMPK and p62 by WB. β-Actin was used as a loading control. Blots are the best representatives of three independent experiments.

    Article Snippet: Other antibodies and related reagents were as follows: mouse monoclonal anti-β-actin (Millipore, Billerica, MA); rabbit polyclonal anti-LC3-II (Novus Biologicals, LLC, Littleton, CO, USA); anti-p62, -Beclin1, and -PI3K class III antibodies (Cell Signaling Technology, Beverly, MA); Na+ ,K+ /ATPase α1 subunit and ubiquitin antibodies (Santa Cruz Biotechnology); pAMPK (T172), AMPK, pULK1(Ser317/Ser757), and ULK1-1 antibodies, an mTOR substrate kit, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling); and HRP-conjugated sheep anti-mouse IgG (GE Healthcare, Waukesha, WI).

    Techniques: Infection, Western Blot, Confocal Microscopy, Software

    AICAR antagonizes HCMV inhibition by digitoxin in conjunction with reduced AMPK phosphorylation and autophagy. (A) HFFs were infected with HCMV (100 PFU/well), followed by treatment with compounds at the indicated doses. (Left) Digitoxin plus AICAR; (center) digitoxin plus CC; (right) digitoxin plus GCV. The effects of drug combinations were analyzed by WB at 24 hpi (B) and 72 hpi (C) for LC3-II, pAMPK, AMPK, UL44, and pp65. (D) Control and ATG5 KD cells were infected with HCMV (MOI = 1) and treated with AICAR (0.8 mM), digitoxin (30 nM), or GCV (5 μM). Lysates were prepared to detect p62 and viral pp65. β-Actin was used as a loading control. Blots are representative of three independent experiments.

    Journal: Journal of Virology

    Article Title: Digitoxin Suppresses Human Cytomegalovirus Replication via Na+, K+/ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

    doi: 10.1128/JVI.01861-17

    Figure Lengend Snippet: AICAR antagonizes HCMV inhibition by digitoxin in conjunction with reduced AMPK phosphorylation and autophagy. (A) HFFs were infected with HCMV (100 PFU/well), followed by treatment with compounds at the indicated doses. (Left) Digitoxin plus AICAR; (center) digitoxin plus CC; (right) digitoxin plus GCV. The effects of drug combinations were analyzed by WB at 24 hpi (B) and 72 hpi (C) for LC3-II, pAMPK, AMPK, UL44, and pp65. (D) Control and ATG5 KD cells were infected with HCMV (MOI = 1) and treated with AICAR (0.8 mM), digitoxin (30 nM), or GCV (5 μM). Lysates were prepared to detect p62 and viral pp65. β-Actin was used as a loading control. Blots are representative of three independent experiments.

    Article Snippet: Other antibodies and related reagents were as follows: mouse monoclonal anti-β-actin (Millipore, Billerica, MA); rabbit polyclonal anti-LC3-II (Novus Biologicals, LLC, Littleton, CO, USA); anti-p62, -Beclin1, and -PI3K class III antibodies (Cell Signaling Technology, Beverly, MA); Na+ ,K+ /ATPase α1 subunit and ubiquitin antibodies (Santa Cruz Biotechnology); pAMPK (T172), AMPK, pULK1(Ser317/Ser757), and ULK1-1 antibodies, an mTOR substrate kit, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling); and HRP-conjugated sheep anti-mouse IgG (GE Healthcare, Waukesha, WI).

    Techniques: Inhibition, Infection, Western Blot