mouse anti human β actin  (Millipore)


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    Structured Review

    Millipore mouse anti human β actin
    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 <t>β-actin</t> 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.
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    Images

    1) 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

    2) Product Images from "Interferon-? Upregulates Expression of IFP35 Gene in HeLa Cells via Interferon Regulatory Factor-1"

    Article Title: Interferon-? Upregulates Expression of IFP35 Gene in HeLa Cells via Interferon Regulatory Factor-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050932

    ISRE is responsible for IFN-γ induced IFP35 promoter activation. ( A ) HeLa cells were transfected with pGL-210 or pGL-359 and stimulated with IFN-γ (10 ng/ml) for different time periods. The response to IFN-γ is presented as fold induction relative to pGL3-Basic. ( B ) HeLa cells were stimulated with IFN-γ (10 ng/ml) for different time periods. The expression of IFP35 and α-tubulin was monitored by Western blot analysis. ( C ) HeLa cells were transfected with the pGL3-Basic, pGL-210 or pGL-210mISRE constructs. At 36 h after transfecion, cells were incubated with medium alone or with IFN-γ (10 ng/ml) for 12 h before luciferase assays were performed.
    Figure Legend Snippet: ISRE is responsible for IFN-γ induced IFP35 promoter activation. ( A ) HeLa cells were transfected with pGL-210 or pGL-359 and stimulated with IFN-γ (10 ng/ml) for different time periods. The response to IFN-γ is presented as fold induction relative to pGL3-Basic. ( B ) HeLa cells were stimulated with IFN-γ (10 ng/ml) for different time periods. The expression of IFP35 and α-tubulin was monitored by Western blot analysis. ( C ) HeLa cells were transfected with the pGL3-Basic, pGL-210 or pGL-210mISRE constructs. At 36 h after transfecion, cells were incubated with medium alone or with IFN-γ (10 ng/ml) for 12 h before luciferase assays were performed.

    Techniques Used: Activation Assay, Transfection, Expressing, Western Blot, Construct, Incubation, Luciferase

    IRF-1 and IRF-2 differentially upregulate IFP35 expression in HeLa cell. ( A ) HeLa cells were transfected with shIRF-1 or shCON. At 36 h after transfection, cells were incubated for 12 h in the presence or absence of IFN-γ (10 ng/ml). Immunoblotting was then performed to examine the protein levels of IFP35, IRF-1 and α-tubulin. ( B ) The experiments were similarly performed as in (A) except that HeLa cells were transfected with shIRF-2 and shCONr. ( C and D ) HeLa cells were co-transfected with pGL-210 and the indicated shRNA expression plasmids. At 36 h after transfection, cells were cultured in the presence or absence of IFN-γ (10 ng/ml) for 12 h before luciferase assays were performed. The response to IFN-γ is presented as fold induction relative to unstimulated cells. Data are the mean and standard error from three experiments. * P
    Figure Legend Snippet: IRF-1 and IRF-2 differentially upregulate IFP35 expression in HeLa cell. ( A ) HeLa cells were transfected with shIRF-1 or shCON. At 36 h after transfection, cells were incubated for 12 h in the presence or absence of IFN-γ (10 ng/ml). Immunoblotting was then performed to examine the protein levels of IFP35, IRF-1 and α-tubulin. ( B ) The experiments were similarly performed as in (A) except that HeLa cells were transfected with shIRF-2 and shCONr. ( C and D ) HeLa cells were co-transfected with pGL-210 and the indicated shRNA expression plasmids. At 36 h after transfection, cells were cultured in the presence or absence of IFN-γ (10 ng/ml) for 12 h before luciferase assays were performed. The response to IFN-γ is presented as fold induction relative to unstimulated cells. Data are the mean and standard error from three experiments. * P

    Techniques Used: Expressing, Transfection, Incubation, shRNA, Cell Culture, Luciferase

    IRF-1 and IRF-2 upregulate IFP35 expression in HeLa and 293T cells. ( A and B ) HeLa cells were co-transfected with pGL-210 and the indicated expression plasmids. Luciferase assay was performed 48 h after transfection. ( C and D ) HeLa cells were transfected with the indicated expression plasmids. At 48 h after transfection, whole cell extracts were prepared and analyzed by Western blot. α-tubulin serves as an internal standard for normalization. ( E and F ) 293T cells were transfected with the indicated expression plasmids. Luciferase assay was performed 48 h after transfection. ( G and H ) 293T cells were transfected with the indicated expression plasmids. Western blot analysis was performed 48 h after transfection.
    Figure Legend Snippet: IRF-1 and IRF-2 upregulate IFP35 expression in HeLa and 293T cells. ( A and B ) HeLa cells were co-transfected with pGL-210 and the indicated expression plasmids. Luciferase assay was performed 48 h after transfection. ( C and D ) HeLa cells were transfected with the indicated expression plasmids. At 48 h after transfection, whole cell extracts were prepared and analyzed by Western blot. α-tubulin serves as an internal standard for normalization. ( E and F ) 293T cells were transfected with the indicated expression plasmids. Luciferase assay was performed 48 h after transfection. ( G and H ) 293T cells were transfected with the indicated expression plasmids. Western blot analysis was performed 48 h after transfection.

    Techniques Used: Expressing, Transfection, Luciferase, Western Blot

    3) Product Images from "Silencing Primary Dystonia: Lentiviral-Mediated RNA Interference Therapy for DYT1 Dystonia"

    Article Title: Silencing Primary Dystonia: Lentiviral-Mediated RNA Interference Therapy for DYT1 Dystonia

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3016-05.2005

    Allele-specific silencing of torsinA(ΔE) in a simulated heterozygous state. Cos7 cells were transiently transfected with torsinA(wt)-GFP, HA-torsinA(ΔE), and different pVETL-(GFP)shRNAs. A , Schematic diagram of pVETL constructs used for expression of shRNAs and the GFP reporter. B , Western blot analysis showing allele-specific suppression of HA-torsinA(ΔE) by shTAmut5, and potent suppression of both forms of torsinA by shTAcom. C , Quantification of three experiments as in B . Error bars indicate SE. mis, U6shTAmis; com, U6shTAcom; mut5, U6shTAmut5; torA, torsinA; tub, α-tubulin; WB, Western blot.
    Figure Legend Snippet: Allele-specific silencing of torsinA(ΔE) in a simulated heterozygous state. Cos7 cells were transiently transfected with torsinA(wt)-GFP, HA-torsinA(ΔE), and different pVETL-(GFP)shRNAs. A , Schematic diagram of pVETL constructs used for expression of shRNAs and the GFP reporter. B , Western blot analysis showing allele-specific suppression of HA-torsinA(ΔE) by shTAmut5, and potent suppression of both forms of torsinA by shTAcom. C , Quantification of three experiments as in B . Error bars indicate SE. mis, U6shTAmis; com, U6shTAcom; mut5, U6shTAmut5; torA, torsinA; tub, α-tubulin; WB, Western blot.

    Techniques Used: Transfection, Construct, Expressing, Western Blot

    Silencing endogenous torsinA in wild-type primary neuronal cultures. Murine primary neuronal cultures were transduced with different FIVeGFP constructs and assayed for silencing of torsinA. A , Western blot analysis showing endogenous torsinA suppression by FIVeGFP.shTAcom. The arrowhead indicates torsinA at the expected molecular weight of 37.8kDa. Three additional nonspecific bands are recognized by the DM2A8 antibody. Tubulin is shown as a loading control. B , Quantification of Western blot signal as in A . Error bars indicate SE. C , Indirect immunofluorescence showing decreased torsinA signal in neurons transduced by FIVeGFP.shTAcom (arrowheads), but not FIVeGFP or FIVeGFP.shTAmut5, when compared with surrounding nontransduced cells. tub, α-Tubulin; WB, Western blot.
    Figure Legend Snippet: Silencing endogenous torsinA in wild-type primary neuronal cultures. Murine primary neuronal cultures were transduced with different FIVeGFP constructs and assayed for silencing of torsinA. A , Western blot analysis showing endogenous torsinA suppression by FIVeGFP.shTAcom. The arrowhead indicates torsinA at the expected molecular weight of 37.8kDa. Three additional nonspecific bands are recognized by the DM2A8 antibody. Tubulin is shown as a loading control. B , Quantification of Western blot signal as in A . Error bars indicate SE. C , Indirect immunofluorescence showing decreased torsinA signal in neurons transduced by FIVeGFP.shTAcom (arrowheads), but not FIVeGFP or FIVeGFP.shTAmut5, when compared with surrounding nontransduced cells. tub, α-Tubulin; WB, Western blot.

    Techniques Used: Transduction, Construct, Western Blot, Molecular Weight, Immunofluorescence

    FIVeGFP.shRNA-mediated silencing of torsinA(ΔE) prevents formation of spheroid bodies. A , Western blot analysis of PC6-3 torsinA(ΔE)-expressing cell lines, showing potent suppression of torsinA(ΔE) expression by FIVeGFP.shTAcom and shTAmut5 but not a FIVeGFP control. The formation of a higher molecular weight, torsinA-immunoreactive band of ∼85 kDa (arrow), was also suppressed. GFP, FIVeGFP; com, FIVeGFP.shTAcom; mut5, FIVeGFP.shTAmut5. B , Quantification of results from three experiments as in A . C , Immunofluorescence image, with GFP as a reporter for transduced cells, showing prevention of spheroid body formation by FIVeGFP.shTAmut5 but not by FIVeGFP. Blue, DAPI nuclear staining. D , Quantification of the presence of spheroid bodies in three independent experiments in nontransduced control cells or cells transduced by different viral vectors. Error bars indicate SE. DOX, Doxycycline; com, U6shTAcom; mut5, U6shTAmut5; tub, α-tubulin; WB, Western blot; torA, torsinA.
    Figure Legend Snippet: FIVeGFP.shRNA-mediated silencing of torsinA(ΔE) prevents formation of spheroid bodies. A , Western blot analysis of PC6-3 torsinA(ΔE)-expressing cell lines, showing potent suppression of torsinA(ΔE) expression by FIVeGFP.shTAcom and shTAmut5 but not a FIVeGFP control. The formation of a higher molecular weight, torsinA-immunoreactive band of ∼85 kDa (arrow), was also suppressed. GFP, FIVeGFP; com, FIVeGFP.shTAcom; mut5, FIVeGFP.shTAmut5. B , Quantification of results from three experiments as in A . C , Immunofluorescence image, with GFP as a reporter for transduced cells, showing prevention of spheroid body formation by FIVeGFP.shTAmut5 but not by FIVeGFP. Blue, DAPI nuclear staining. D , Quantification of the presence of spheroid bodies in three independent experiments in nontransduced control cells or cells transduced by different viral vectors. Error bars indicate SE. DOX, Doxycycline; com, U6shTAcom; mut5, U6shTAmut5; tub, α-tubulin; WB, Western blot; torA, torsinA.

    Techniques Used: shRNA, Western Blot, Expressing, Molecular Weight, Immunofluorescence, Staining

    Allele-specific silencing of transiently expressed torsinA(ΔE) in Cos7 cells. A , Target cDNA region of torsinA(ΔE) and different short hairpin RNAs used in this study. B , Western blot results showing the effect of different shRNAs on torsinA(wt)-GFP or HA-torsinA(ΔE) expression. α-Tubulin is shown as a loading control. C , Similar experiment performed with shmis, shTAcom, and shTAmut2-5. D , Quantification of results from three experiments as in B and C . Error bars indicate SE. mis, U6shTAmis; mut, U6shTAmut5; com, U6shTAcom; torA, torsinA; tub, α-tubulin.
    Figure Legend Snippet: Allele-specific silencing of transiently expressed torsinA(ΔE) in Cos7 cells. A , Target cDNA region of torsinA(ΔE) and different short hairpin RNAs used in this study. B , Western blot results showing the effect of different shRNAs on torsinA(wt)-GFP or HA-torsinA(ΔE) expression. α-Tubulin is shown as a loading control. C , Similar experiment performed with shmis, shTAcom, and shTAmut2-5. D , Quantification of results from three experiments as in B and C . Error bars indicate SE. mis, U6shTAmis; mut, U6shTAmut5; com, U6shTAcom; torA, torsinA; tub, α-tubulin.

    Techniques Used: Western Blot, Expressing

    Assessment of the interferon response in neuronal cells transduced by FIVeGFP.shRNA. A , Western blot analysis of PC6-3 torsinA(ΔE)-inducible cell lines showing increased PKR and STAT1 signal and STAT1 phosphorylation when treated with interferon-α, but not with the different FIVeGFP constructs, despite effective torsinA silencing. B , Western blot analysis of the same experiment performed in primary neuronal cultures, showing similar results. GFP, FIVeGFP; com, FIVeGFP.shTAcom; mut5, FIVeGFP.shTAmut5; DOX, doxycycline; IFN, interferon; torA, torsinA; tub, α-tubulin.
    Figure Legend Snippet: Assessment of the interferon response in neuronal cells transduced by FIVeGFP.shRNA. A , Western blot analysis of PC6-3 torsinA(ΔE)-inducible cell lines showing increased PKR and STAT1 signal and STAT1 phosphorylation when treated with interferon-α, but not with the different FIVeGFP constructs, despite effective torsinA silencing. B , Western blot analysis of the same experiment performed in primary neuronal cultures, showing similar results. GFP, FIVeGFP; com, FIVeGFP.shTAcom; mut5, FIVeGFP.shTAmut5; DOX, doxycycline; IFN, interferon; torA, torsinA; tub, α-tubulin.

    Techniques Used: shRNA, Western Blot, Construct

    Preferential shRNA strand participation in the RNAi pathway. A , Shown is the sequence of the targeted torsinA(ΔE) cDNA region used to generate the GFP-guide and GFP-passenger reporter constructs, underlining the specific sequence used for each, with a schematic representation of the resulting coding region. B , Western blot analysis of transiently transfected Cos7 cells showing the effect of different shRNAs on GFP-guide and GFP-passenger. α-Tubulin is shown as a loading control. C , Quantification of results from three experiments as in B . Error bars indicate SE. com, U6shTAcom; mut, U6shTAmut; tub, α-tubulin; WB, Western blot.
    Figure Legend Snippet: Preferential shRNA strand participation in the RNAi pathway. A , Shown is the sequence of the targeted torsinA(ΔE) cDNA region used to generate the GFP-guide and GFP-passenger reporter constructs, underlining the specific sequence used for each, with a schematic representation of the resulting coding region. B , Western blot analysis of transiently transfected Cos7 cells showing the effect of different shRNAs on GFP-guide and GFP-passenger. α-Tubulin is shown as a loading control. C , Quantification of results from three experiments as in B . Error bars indicate SE. com, U6shTAcom; mut, U6shTAmut; tub, α-tubulin; WB, Western blot.

    Techniques Used: shRNA, Sequencing, Construct, Western Blot, Transfection

    4) Product Images from "VSL#3 Probiotic Stimulates T-Cell Protein Tyrosine Phosphatase-Mediated Recovery of IFN-γ Induced Intestinal Epithelial Barrier Defects"

    Article Title: VSL#3 Probiotic Stimulates T-Cell Protein Tyrosine Phosphatase-Mediated Recovery of IFN-γ Induced Intestinal Epithelial Barrier Defects

    Journal: Inflammatory bowel diseases

    doi: 10.1097/MIB.0000000000000954

    VSL#3 attenuated STAT1 signaling in a TCPTP-dependent manner (A) T 84 monolayers were basolaterally treated with IFN-γ (1000 U/ml) for 24 hrs or VSL#3 (10 2 , 10 4 , 10 6 , 10 8 CFU/mL) apically for 9 hrs and STAT1 phosphorylation and total STAT1 levels were determined by subsequent Western blotting of cell lysates. While IFN- γ as expected increased phospho-STAT1, VSL#3 alone was without effect (representative blot of 3 experiments. (B,C) Densitometric analysis of Western blots from T 84 monolayers treated basolaterally with IFN-γ (1000 U/ml) for 24 hrs showed a significant increase in STAT1 phosphorylation relative to total STAT1 and β-actin (p
    Figure Legend Snippet: VSL#3 attenuated STAT1 signaling in a TCPTP-dependent manner (A) T 84 monolayers were basolaterally treated with IFN-γ (1000 U/ml) for 24 hrs or VSL#3 (10 2 , 10 4 , 10 6 , 10 8 CFU/mL) apically for 9 hrs and STAT1 phosphorylation and total STAT1 levels were determined by subsequent Western blotting of cell lysates. While IFN- γ as expected increased phospho-STAT1, VSL#3 alone was without effect (representative blot of 3 experiments. (B,C) Densitometric analysis of Western blots from T 84 monolayers treated basolaterally with IFN-γ (1000 U/ml) for 24 hrs showed a significant increase in STAT1 phosphorylation relative to total STAT1 and β-actin (p

    Techniques Used: Western Blot

    5) Product Images from "Overexpressed tryptophanyl-tRNA synthetase, an angiostatic protein, enhances oral cancer cell invasiveness"

    Article Title: Overexpressed tryptophanyl-tRNA synthetase, an angiostatic protein, enhances oral cancer cell invasiveness

    Journal: Oncotarget

    doi:

    The detection of surface-bound TrpRS on INF-γ-treated OSCC cells A. The detection of intracellular and extracellular TrpRS expressions in IFN-γ-treated OSCC cells. OEC-M1 cells were treated with IFN-γ (200 U/ml) for 24 h. Cell extracts (CE) and CM were obtained from control and IFN-γ treated cells, and the proteins were detected via Western blot using an anti-TrpRS antibody. β-actin was used as the loading control. B. TrpRS was detected in the plasma membrane (PM) fraction of IFN-γ-treated OEC-M1 cells. OEC-M1 cells were treated with IFN-γ (200 U/ml) for 24 h. The whole-cell extract (CE), cytosolic (Cytosol) and PM fractions were prepared as described in the Materials and Methods section. The proteins were subjected to Western blot using anti-EGFR, anti-TrpRS and anti-GAPDH antibodies as indicated. C. Cy3-labeled T2-TrpRS was detected on the cell surface via immunofluorescence staining as described in the Materials and Methods section. Cells pre-incubated without a–c. or with 50 μg/ml unlabeled GST d–f. full-length GST-TrpRS g–i. GST-mini-TrpRS j–l. or GST-T2-TrpRS m–o. are presented. The Cy3-labeled T2-TrpRS (red), WGA (green) and merged images are presented as indicated. DNA was stained with Hoechst 33258 (blue).
    Figure Legend Snippet: The detection of surface-bound TrpRS on INF-γ-treated OSCC cells A. The detection of intracellular and extracellular TrpRS expressions in IFN-γ-treated OSCC cells. OEC-M1 cells were treated with IFN-γ (200 U/ml) for 24 h. Cell extracts (CE) and CM were obtained from control and IFN-γ treated cells, and the proteins were detected via Western blot using an anti-TrpRS antibody. β-actin was used as the loading control. B. TrpRS was detected in the plasma membrane (PM) fraction of IFN-γ-treated OEC-M1 cells. OEC-M1 cells were treated with IFN-γ (200 U/ml) for 24 h. The whole-cell extract (CE), cytosolic (Cytosol) and PM fractions were prepared as described in the Materials and Methods section. The proteins were subjected to Western blot using anti-EGFR, anti-TrpRS and anti-GAPDH antibodies as indicated. C. Cy3-labeled T2-TrpRS was detected on the cell surface via immunofluorescence staining as described in the Materials and Methods section. Cells pre-incubated without a–c. or with 50 μg/ml unlabeled GST d–f. full-length GST-TrpRS g–i. GST-mini-TrpRS j–l. or GST-T2-TrpRS m–o. are presented. The Cy3-labeled T2-TrpRS (red), WGA (green) and merged images are presented as indicated. DNA was stained with Hoechst 33258 (blue).

    Techniques Used: Western Blot, Labeling, Immunofluorescence, Staining, Incubation, Whole Genome Amplification

    TrpRS is overexpressed in OSCC tissues A. Total cell lysates (30 μg per lane) prepared from OSCC tumor tissue (T) and adjacent normal tissue (N) were subjected to SDS-PAGE, transferred to PVDF membrane and then stained with Fast Green FCF dye (lower panel). The proteins were detected by probing with anti-TrpRS, anti-STAT1, anti-MX1, anti-ANXA2 and anti-β-actin antibodies as indicated. β-actin was used as the loading control. The TrpRS signal detected via Western blot was acquired, quantified and normalized to the corresponding β-actin signal. The value represents the normalized fold-change in the tumor tissue relative to the corresponding normal adjacent tissue. B. IHC analysis of TrpRS expression in the tumor tissue (T), adjacent normal tissue (N), and the metastatic lymph node tissue (LN) from one representative case. The brown signal indicates the cytosolic distribution of TrpRS in the tumor tissue. C. Statistical analysis of the IHC staining scores for TrpRS from 146 paired tumor (T) and adjacent normal tissues (N, 16 normal tissues were missing from the surgically resected OSCC tissue sections). The red line indicates the median with the interquartile range of the IHC staining score. D. Statistical analysis of the IHC staining scores for TrpRS from 28 paired tumor tissue (T) and metastatic lymph node (LN) sections. *, a p value of less than 0.05 indicates significance based on the paired t test.
    Figure Legend Snippet: TrpRS is overexpressed in OSCC tissues A. Total cell lysates (30 μg per lane) prepared from OSCC tumor tissue (T) and adjacent normal tissue (N) were subjected to SDS-PAGE, transferred to PVDF membrane and then stained with Fast Green FCF dye (lower panel). The proteins were detected by probing with anti-TrpRS, anti-STAT1, anti-MX1, anti-ANXA2 and anti-β-actin antibodies as indicated. β-actin was used as the loading control. The TrpRS signal detected via Western blot was acquired, quantified and normalized to the corresponding β-actin signal. The value represents the normalized fold-change in the tumor tissue relative to the corresponding normal adjacent tissue. B. IHC analysis of TrpRS expression in the tumor tissue (T), adjacent normal tissue (N), and the metastatic lymph node tissue (LN) from one representative case. The brown signal indicates the cytosolic distribution of TrpRS in the tumor tissue. C. Statistical analysis of the IHC staining scores for TrpRS from 146 paired tumor (T) and adjacent normal tissues (N, 16 normal tissues were missing from the surgically resected OSCC tissue sections). The red line indicates the median with the interquartile range of the IHC staining score. D. Statistical analysis of the IHC staining scores for TrpRS from 28 paired tumor tissue (T) and metastatic lymph node (LN) sections. *, a p value of less than 0.05 indicates significance based on the paired t test.

    Techniques Used: SDS Page, Staining, Western Blot, Immunohistochemistry, Expressing

    TrpRS overexpression promotes cell migration and invasion A. OEC-M1 cells were transfected with the pcDNA 3.1/Myc-His empty vector (V) or a pcDNA 3.1/Myc-His plasmid carrying one of three isoforms of TrpRS (FL: full-length TrpRS; Mi: mini-TrpRS; or T2: T2-TrpRS) as indicated. At 48 h after transfection, cell lysates were prepared, and the proteins were detected via Western blot using an anti-myc antibody. β-actin was used as the loading control. Simultaneously, transfected cells were subjected to cell counting, migration and invasion assays as described in the Materials and Methods section. B. Quantitative data show the relative percentage of cell viability obtained from three independent cell counting assays. The error bars indicate the standard error of the mean. Quantitative analysis of the migration C. and invasion assays D. Photographs obtained from the migration and invasion assays (left panel). The data are presented as values with standard deviations obtained from three independent experiments (right panel). *, a p value of less than 0.05 indicates significance based on the Mann-Whitney U test.
    Figure Legend Snippet: TrpRS overexpression promotes cell migration and invasion A. OEC-M1 cells were transfected with the pcDNA 3.1/Myc-His empty vector (V) or a pcDNA 3.1/Myc-His plasmid carrying one of three isoforms of TrpRS (FL: full-length TrpRS; Mi: mini-TrpRS; or T2: T2-TrpRS) as indicated. At 48 h after transfection, cell lysates were prepared, and the proteins were detected via Western blot using an anti-myc antibody. β-actin was used as the loading control. Simultaneously, transfected cells were subjected to cell counting, migration and invasion assays as described in the Materials and Methods section. B. Quantitative data show the relative percentage of cell viability obtained from three independent cell counting assays. The error bars indicate the standard error of the mean. Quantitative analysis of the migration C. and invasion assays D. Photographs obtained from the migration and invasion assays (left panel). The data are presented as values with standard deviations obtained from three independent experiments (right panel). *, a p value of less than 0.05 indicates significance based on the Mann-Whitney U test.

    Techniques Used: Over Expression, Migration, Transfection, Plasmid Preparation, Western Blot, Cell Counting, MANN-WHITNEY

    Secreted TrpRS promotes OSCC cell invasion A. The detection of secreted TrpRS in CM via Western blot. Cell extracts (CE; 50 μg) and CM (10 μg) obtained from transfected OEC-M1 cells (V: empty vector; FL: full-length TrpRS; Mi: mini-TrpRS; T2: T2-TrpRS) were prepared for Western blot using an anti-myc antibody. β-actin was used as the loading control. B. The treatment of recipient cells with CM harvested from TrpRS-expressing cells promoted cell invasion. The recipient OEC-M1 cells were treated with CM harvested from TrpRS-expressing cells throughout the invasion assay. C. The treatment of recipient cells with CM harvested from TrpRS-knockdown cells reduced cell invasion. CE from TrpRS-knockdown OEC-M1 cells were prepared for Western blot using an anti-TrpRS antibody. β-actin was used as the loading control. The recipient OEC-M1 cells were treated with CM harvested from TrpRS-knockdown cells throughout the invasion assay. D. The extracellular addition of recombinant TrpRS protein rescued the invasion ability of TrpRS-knockdown oral cancer cells. Purified GST-TrpRS fusion proteins (2 μg per lane) were separated via SDS-PAGE and stained with Coomassie Brilliant Blue (left panel). The TrpRS-knockdown OSCC cells were treated with a GST-TrpRS fusion protein (1 μg/ml) throughout the invasion assay. The quantitative analysis of the invasion assays (C–D) is presented as the mean values with standard deviations obtained from three independent experiments. *, a p value of less than 0.05 indicates significance based on the Mann-Whitney U test.
    Figure Legend Snippet: Secreted TrpRS promotes OSCC cell invasion A. The detection of secreted TrpRS in CM via Western blot. Cell extracts (CE; 50 μg) and CM (10 μg) obtained from transfected OEC-M1 cells (V: empty vector; FL: full-length TrpRS; Mi: mini-TrpRS; T2: T2-TrpRS) were prepared for Western blot using an anti-myc antibody. β-actin was used as the loading control. B. The treatment of recipient cells with CM harvested from TrpRS-expressing cells promoted cell invasion. The recipient OEC-M1 cells were treated with CM harvested from TrpRS-expressing cells throughout the invasion assay. C. The treatment of recipient cells with CM harvested from TrpRS-knockdown cells reduced cell invasion. CE from TrpRS-knockdown OEC-M1 cells were prepared for Western blot using an anti-TrpRS antibody. β-actin was used as the loading control. The recipient OEC-M1 cells were treated with CM harvested from TrpRS-knockdown cells throughout the invasion assay. D. The extracellular addition of recombinant TrpRS protein rescued the invasion ability of TrpRS-knockdown oral cancer cells. Purified GST-TrpRS fusion proteins (2 μg per lane) were separated via SDS-PAGE and stained with Coomassie Brilliant Blue (left panel). The TrpRS-knockdown OSCC cells were treated with a GST-TrpRS fusion protein (1 μg/ml) throughout the invasion assay. The quantitative analysis of the invasion assays (C–D) is presented as the mean values with standard deviations obtained from three independent experiments. *, a p value of less than 0.05 indicates significance based on the Mann-Whitney U test.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing, Invasion Assay, Recombinant, Purification, SDS Page, Staining, MANN-WHITNEY

    6) Product Images from "ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response"

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05559-w

    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
    Figure Legend Snippet: ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

    Techniques Used: Transfection, Incubation, In Vitro, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction

    7) Product Images from "STAT1 regulates marginal zone B cell differentiation in response to inflammation and infection with blood-borne bacteria"

    Article Title: STAT1 regulates marginal zone B cell differentiation in response to inflammation and infection with blood-borne bacteria

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151620

    Development and TLR-induced activation, proliferation, and apoptosis of Stat1 −/− MZ B cells are normal. (A) Splenocytes of WT and Stat1 −/− mice were stained with antibodies to B220, CD21, and CD23, gated on B220, and analyzed for MZ B cells (B220 + CD21 hi CD23 lo ) by flow cytometry. One experiment out of three is shown. (B) Mean numbers of WT and Stat1 −/− MZ B cells are shown ( n = 4–9). Results represent three experiments. (C–F) MZ B cells of WT and Stat1 −/− mice were stimulated with 1 µg/ml CpG (C and D) or 2 µg/ml LPS (E and F) for 24 h, stained with antibodies to CD69 (C and E) and MHC class II (D and F), respectively, and analyzed by flow cytometry. One experiment out of two is shown. (G and H) Same as in C–F, except the culture supernatant of the stimulated cells were subjected to ELISA for measuring the production of IL-6 (G) and IL-10 (H) before and after the treatments ( n = 2). Results represent two experiments. (I) Same as in A and B except cells were stimulated for the indicated times, pulsed with BrdU for the last 2 h, and stained with anti–BrdU-FITC, and then BrdU incorporation measured by flow cytometry. One experiment out of two is shown. (J) Same as in I except the cells were treated for the indicated times, stained with Annexin V, and analyzed with flow cytometry ( n = 3). Results represent three experiments. All values are shown as the means ± SD. *, P
    Figure Legend Snippet: Development and TLR-induced activation, proliferation, and apoptosis of Stat1 −/− MZ B cells are normal. (A) Splenocytes of WT and Stat1 −/− mice were stained with antibodies to B220, CD21, and CD23, gated on B220, and analyzed for MZ B cells (B220 + CD21 hi CD23 lo ) by flow cytometry. One experiment out of three is shown. (B) Mean numbers of WT and Stat1 −/− MZ B cells are shown ( n = 4–9). Results represent three experiments. (C–F) MZ B cells of WT and Stat1 −/− mice were stimulated with 1 µg/ml CpG (C and D) or 2 µg/ml LPS (E and F) for 24 h, stained with antibodies to CD69 (C and E) and MHC class II (D and F), respectively, and analyzed by flow cytometry. One experiment out of two is shown. (G and H) Same as in C–F, except the culture supernatant of the stimulated cells were subjected to ELISA for measuring the production of IL-6 (G) and IL-10 (H) before and after the treatments ( n = 2). Results represent two experiments. (I) Same as in A and B except cells were stimulated for the indicated times, pulsed with BrdU for the last 2 h, and stained with anti–BrdU-FITC, and then BrdU incorporation measured by flow cytometry. One experiment out of two is shown. (J) Same as in I except the cells were treated for the indicated times, stained with Annexin V, and analyzed with flow cytometry ( n = 3). Results represent three experiments. All values are shown as the means ± SD. *, P

    Techniques Used: Activation Assay, Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

    Impaired plasma cell differentiation in Stat1 −/− MZ B cells in response to TLR stimulation. MZ B cells of WT and Stat1 −/− mice were stimulated with 1 µg/ml CpG (A) and 2 µg/ml LPS (B), respectively, for 2 d, stained with antibodies to B220 and CD138, and analyzed by flow cytometry. One experiment out of three is shown. (C) The mean percentages of plasma cell (B220 − CD138 + ) after stimulation are shown ( n = 3). Results represent three experiments. MZ B cells were stimulated with 1 µg/ml CpG for 3 d, and then subjected to ELISPOT assay. (D) The spots of ASCs are shown. One experiment out of three is shown. The frequencies of ASCs per 10 3 MZ B cells (E) and mean area of each spot (F) in the ELISPOT assay is shown ( n = 3). Results represent three experiments. Same as in B, except mRNA was prepared from the cells before and after stimulation with LPS and subjected to RT-QPCR using primers to Pax5 (G), Bcl6 (H), Prdm1 (I), and Xbp1s (J). All genes were normalized to Rpl7 ( n = 4). Results represent two experiments. All values are shown as the means ± SD. *, P
    Figure Legend Snippet: Impaired plasma cell differentiation in Stat1 −/− MZ B cells in response to TLR stimulation. MZ B cells of WT and Stat1 −/− mice were stimulated with 1 µg/ml CpG (A) and 2 µg/ml LPS (B), respectively, for 2 d, stained with antibodies to B220 and CD138, and analyzed by flow cytometry. One experiment out of three is shown. (C) The mean percentages of plasma cell (B220 − CD138 + ) after stimulation are shown ( n = 3). Results represent three experiments. MZ B cells were stimulated with 1 µg/ml CpG for 3 d, and then subjected to ELISPOT assay. (D) The spots of ASCs are shown. One experiment out of three is shown. The frequencies of ASCs per 10 3 MZ B cells (E) and mean area of each spot (F) in the ELISPOT assay is shown ( n = 3). Results represent three experiments. Same as in B, except mRNA was prepared from the cells before and after stimulation with LPS and subjected to RT-QPCR using primers to Pax5 (G), Bcl6 (H), Prdm1 (I), and Xbp1s (J). All genes were normalized to Rpl7 ( n = 4). Results represent two experiments. All values are shown as the means ± SD. *, P

    Techniques Used: Cell Differentiation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Quantitative RT-PCR

    STAT1 binds to Prdm1 promoter and regulates Prdm1 expression and IgM production in MZ B cells in response to TLR stimulation. (A) CH12F3 cells were subjected to lentivirus-based knockdown of luciferase or Stat1 , and then stimulated with or without LPS for 4 d. IgM in the culture supernatant was measured by ELISA ( n = 3). Results represent three experiments. (B) Same as in A, except total RNA from the cells treated for 2 d were subjected to RT-QPCR using primers to Prdm1 ( n = 6). Results represent three experiments. (C) ChIP-seq data ( Robertson et al., 2007 ) were analyzed for STAT1 binding regions in human Prdm1 loci. The corresponding exons and introns of Prdm1 are shown. Comparison of conserved GAS-like 1 (GASL1) and GAS-like 2 (GASL2) in human and mouse Prdm1 promoters are shown. (D) CH12F3 cells were stimulated with 5 µg/ml LPS for 6 h, and ChIP was performed using anti-STAT1 antibody. Relative abundance of GASL1 (A), GASL2 (B), and exon 7 are shown ( n = 6). Results represent three experiments. (E) Reporter plasmid containing the promoter region of Prdm1 (–2,052 to +207 bp) was cotransfected with pCMV-DsRed, an internal control plasmid, into sh LacZ - or sh Stat1 -treated CH12F3 cells for 24 h, followed by stimulation with 5 µg/ml LPS for the indicated times before being harvested and subjected to reporter activity assay. Reporter activity was normalized to the percentage of DsRed-positive cells ( n = 3). Results represent three experiments. (F) MZ B cells from WT or Stat1 −/− mice were stimulated with 2 µg/ml LPS for 24 h and transduced with recombinant retrovirus pGC-YFP or pGC-Blimp-1-YFP for 2 d. The YFP + cells were sorted out and incubated for another 24 h before measuring IgM production by ELISA ( n = 3). Results represent three experiments. All values are shown as the means ± SD *, P
    Figure Legend Snippet: STAT1 binds to Prdm1 promoter and regulates Prdm1 expression and IgM production in MZ B cells in response to TLR stimulation. (A) CH12F3 cells were subjected to lentivirus-based knockdown of luciferase or Stat1 , and then stimulated with or without LPS for 4 d. IgM in the culture supernatant was measured by ELISA ( n = 3). Results represent three experiments. (B) Same as in A, except total RNA from the cells treated for 2 d were subjected to RT-QPCR using primers to Prdm1 ( n = 6). Results represent three experiments. (C) ChIP-seq data ( Robertson et al., 2007 ) were analyzed for STAT1 binding regions in human Prdm1 loci. The corresponding exons and introns of Prdm1 are shown. Comparison of conserved GAS-like 1 (GASL1) and GAS-like 2 (GASL2) in human and mouse Prdm1 promoters are shown. (D) CH12F3 cells were stimulated with 5 µg/ml LPS for 6 h, and ChIP was performed using anti-STAT1 antibody. Relative abundance of GASL1 (A), GASL2 (B), and exon 7 are shown ( n = 6). Results represent three experiments. (E) Reporter plasmid containing the promoter region of Prdm1 (–2,052 to +207 bp) was cotransfected with pCMV-DsRed, an internal control plasmid, into sh LacZ - or sh Stat1 -treated CH12F3 cells for 24 h, followed by stimulation with 5 µg/ml LPS for the indicated times before being harvested and subjected to reporter activity assay. Reporter activity was normalized to the percentage of DsRed-positive cells ( n = 3). Results represent three experiments. (F) MZ B cells from WT or Stat1 −/− mice were stimulated with 2 µg/ml LPS for 24 h and transduced with recombinant retrovirus pGC-YFP or pGC-Blimp-1-YFP for 2 d. The YFP + cells were sorted out and incubated for another 24 h before measuring IgM production by ELISA ( n = 3). Results represent three experiments. All values are shown as the means ± SD *, P

    Techniques Used: Expressing, Luciferase, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Plasmid Preparation, Activity Assay, Mouse Assay, Transduction, Recombinant, Pyrolysis Gas Chromatography, Incubation

    Impaired IgM response in Stat1 −/− MZ B cells in response to TLR stimulation in vitro and in vivo . MZ B (A) or FO B (B) cells of WT and Stat1 −/− mice were treated with or without 1 µg/ml Pam3CSK4 (Pam), 2 µg/ml LPS, 10 µg/ml R848, or 1 µg/ml CpG for 4 d ( n = 3–5). Results represent three experiments. Same as in A, except MZ B cells of WT and (C) Ifnar1 −/− or (D) Ifngr1 −/− mice were stimulated with the indicated TLR agonists for 4 d. Secreted IgM in the culture supernatant was measured by ELISA ( n = 2–5). Results represent two to three experiments. (E) WT and Stat1 −/− mice were intravenously injected with 25 µg LPS for 0, 3, and 5 d ( n = 5). Results represent three experiments. (F) MZ B cells of WT or Stat1 −/− mice (2 × 10 6 each) were adoptively transferred into RBP-J CKO mice for 1 d, and then 25 µg LPS was intravenously injected for the indicated times. Total serum IgM of the treated mice was measured by ELISA ( n = 4). Results represent two experiments. All values are shown as the means ± SD. *, P
    Figure Legend Snippet: Impaired IgM response in Stat1 −/− MZ B cells in response to TLR stimulation in vitro and in vivo . MZ B (A) or FO B (B) cells of WT and Stat1 −/− mice were treated with or without 1 µg/ml Pam3CSK4 (Pam), 2 µg/ml LPS, 10 µg/ml R848, or 1 µg/ml CpG for 4 d ( n = 3–5). Results represent three experiments. Same as in A, except MZ B cells of WT and (C) Ifnar1 −/− or (D) Ifngr1 −/− mice were stimulated with the indicated TLR agonists for 4 d. Secreted IgM in the culture supernatant was measured by ELISA ( n = 2–5). Results represent two to three experiments. (E) WT and Stat1 −/− mice were intravenously injected with 25 µg LPS for 0, 3, and 5 d ( n = 5). Results represent three experiments. (F) MZ B cells of WT or Stat1 −/− mice (2 × 10 6 each) were adoptively transferred into RBP-J CKO mice for 1 d, and then 25 µg LPS was intravenously injected for the indicated times. Total serum IgM of the treated mice was measured by ELISA ( n = 4). Results represent two experiments. All values are shown as the means ± SD. *, P

    Techniques Used: In Vitro, In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

    8) Product Images from "PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation"

    Article Title: PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation

    Journal: Nature Communications

    doi: 10.1038/ncomms12849

    PARP9 and PARP14 expression in vitro and in vivo . ( a ) TMT-derived 0-h-normalized protein abundance profiles for PARP9 and PARP14 from mouse RAW264.7 and human THP-1 M(IFNγ) and M(IL-4) data sets. ( b ) PARP9 and PARP14 gene expression at 24 h after stimulation ( n =3). ( c ) PARP9 and PARP14 protein expression visualized by western blot. The time course in the relative protein abundances of PARP9 and PARP14 normalized to β-actin were quantified (graph, n =3). * P
    Figure Legend Snippet: PARP9 and PARP14 expression in vitro and in vivo . ( a ) TMT-derived 0-h-normalized protein abundance profiles for PARP9 and PARP14 from mouse RAW264.7 and human THP-1 M(IFNγ) and M(IL-4) data sets. ( b ) PARP9 and PARP14 gene expression at 24 h after stimulation ( n =3). ( c ) PARP9 and PARP14 protein expression visualized by western blot. The time course in the relative protein abundances of PARP9 and PARP14 normalized to β-actin were quantified (graph, n =3). * P

    Techniques Used: Expressing, In Vitro, In Vivo, Derivative Assay, Western Blot

    Haematopoietic PARP14 in mouse atheromata and PARP9–PARP14 expression in human plaques. ( a ) Representative image and quantification of aortic root lesion formation and CD68+ macrophage accumulation (green, Alexa 488) in high-fat and high-cholesterol diet-fed LDLR −/− mice whose bone marrow was reconstituted by PARP14 −/− mice (BMT PARP14 −/−→ LDLR −/− mice, n =5), compared with LDLR −/− mice with PARP14 +/+ bone marrow (BMT PARP14 +/+→ LDLR −/− mice, n =6–7). Scale bars, 100 μm. ( b ) mRNA expression of the aorta from a . n =6–8. ( c ) Immunofluorescence staining of PARP14 and PARP9 proteins (green, Alexa 488) in human carotid plaques. CD68 (red, Alexa 594). Nuclei (blue, 4,6-diamidino-2-phenylindole, DAPI). Scale bars, 100 μm; insets, 10 μm ( n =5). Prevalence of PARP14+ or PARP9+ macrophages in macrophage-poor versus macrophage-rich plaques. * P
    Figure Legend Snippet: Haematopoietic PARP14 in mouse atheromata and PARP9–PARP14 expression in human plaques. ( a ) Representative image and quantification of aortic root lesion formation and CD68+ macrophage accumulation (green, Alexa 488) in high-fat and high-cholesterol diet-fed LDLR −/− mice whose bone marrow was reconstituted by PARP14 −/− mice (BMT PARP14 −/−→ LDLR −/− mice, n =5), compared with LDLR −/− mice with PARP14 +/+ bone marrow (BMT PARP14 +/+→ LDLR −/− mice, n =6–7). Scale bars, 100 μm. ( b ) mRNA expression of the aorta from a . n =6–8. ( c ) Immunofluorescence staining of PARP14 and PARP9 proteins (green, Alexa 488) in human carotid plaques. CD68 (red, Alexa 594). Nuclei (blue, 4,6-diamidino-2-phenylindole, DAPI). Scale bars, 100 μm; insets, 10 μm ( n =5). Prevalence of PARP14+ or PARP9+ macrophages in macrophage-poor versus macrophage-rich plaques. * P

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Staining

    Network analysis links PARP9–PARP14 with coronary artery disease. The PARP14 (blue)–PARP9 (purple) module consists of the first neighbours of each protein (light blue and orange nodes, respectively). The significance of closeness of the PARP9–PARP14 first neighbours in the interactome (PARP9–PARP14 module) and disease modules compared with random expectation is indicated by P values. The random expectation was same size-connected components of PARP9–PARP14 module and a disease module drawn randomly from the network. Closeness between PARP9–PARP14 modules and other diseases such as cardiovascular, metabolic and IFNγ-related diseases was evaluated in the network. The inner circle contains significantly close disease modules.
    Figure Legend Snippet: Network analysis links PARP9–PARP14 with coronary artery disease. The PARP14 (blue)–PARP9 (purple) module consists of the first neighbours of each protein (light blue and orange nodes, respectively). The significance of closeness of the PARP9–PARP14 first neighbours in the interactome (PARP9–PARP14 module) and disease modules compared with random expectation is indicated by P values. The random expectation was same size-connected components of PARP9–PARP14 module and a disease module drawn randomly from the network. Closeness between PARP9–PARP14 modules and other diseases such as cardiovascular, metabolic and IFNγ-related diseases was evaluated in the network. The inner circle contains significantly close disease modules.

    Techniques Used:

    Potential interaction of PARP9 and PARP14. ( a ) PARP14 silencing and enforced expression significantly affected PARP9 gene expression in IFNγ-stimulated THP-1 cells ( n =3). PARP9 silencing increased PARP14 gene expression ( n =3). ( b ) Co-IP assay revealed a complex between PARP9 and PARP14. ( c ) Intracellular colocalization of PARP9 and PARP14 in the cytosol in M(-) and M(IFNγ). ( d ) PARP9 inhibits ADP-ribosylation of STAT1α and STAT6 by PARP14 (protein ribosylation assay). PARP14 auto-ribosylation is also indicated. * P
    Figure Legend Snippet: Potential interaction of PARP9 and PARP14. ( a ) PARP14 silencing and enforced expression significantly affected PARP9 gene expression in IFNγ-stimulated THP-1 cells ( n =3). PARP9 silencing increased PARP14 gene expression ( n =3). ( b ) Co-IP assay revealed a complex between PARP9 and PARP14. ( c ) Intracellular colocalization of PARP9 and PARP14 in the cytosol in M(-) and M(IFNγ). ( d ) PARP9 inhibits ADP-ribosylation of STAT1α and STAT6 by PARP14 (protein ribosylation assay). PARP14 auto-ribosylation is also indicated. * P

    Techniques Used: Expressing, Co-Immunoprecipitation Assay

    The molecular functions of PARP9 and PARP14 in macrophages in vitro . ( a ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulated (TNFα, IL-1β and CCL2/MCP-1) and IL-4 stimulated (MRC1) gene expression in human primary macrophages ( n =8). ( b ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulation (TNFα and iNOS) and IL-4 stimulation (Arg1 and MRC1) gene expression in mouse bone marrow-derived macrophages ( n =3). ( c ) The ratio of phosphorylated STAT1 and STAT6 protein levels to total STAT1 and STAT6 (p STAT1/tSTAT1 ratio and pSTAT6/tSTAT6 ratio) in human primary macrophages ( n =6 and n =5, respectively) of the PARP9 and PARP14 silencing experiments. * P
    Figure Legend Snippet: The molecular functions of PARP9 and PARP14 in macrophages in vitro . ( a ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulated (TNFα, IL-1β and CCL2/MCP-1) and IL-4 stimulated (MRC1) gene expression in human primary macrophages ( n =8). ( b ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulation (TNFα and iNOS) and IL-4 stimulation (Arg1 and MRC1) gene expression in mouse bone marrow-derived macrophages ( n =3). ( c ) The ratio of phosphorylated STAT1 and STAT6 protein levels to total STAT1 and STAT6 (p STAT1/tSTAT1 ratio and pSTAT6/tSTAT6 ratio) in human primary macrophages ( n =6 and n =5, respectively) of the PARP9 and PARP14 silencing experiments. * P

    Techniques Used: In Vitro, Expressing, Derivative Assay

    Identification of PARP14-induced ribosylation sites in STAT1. ( a ) The amino-acid sequence of human STAT1α C terminus. Green amino acids indicate ribosylated peptides; confirmed ribosylation sites are underlined. STAT1 is phosphorylated at indicated tyrosine (red). ( b ; Left panels) MS/MS spectra for the mono-ADP-ribosylated peptides and corresponding unmodified forms. ADP-ribose fragments are annotated in green. *, ribosylation site; m, oxidized methionine. The grey circles indicate background or undetermined ions. (Right panels) MS1-based quantification of PARP9 inhibition of PARP14-mediated STAT1α ribosylation at E657 (upper panel) and E705 (lower panel), respectively. ( c ) Effects of mutated amino acids at E657 and E705 in STAT1 (ribosylation sites for PARP14) on its Tyr701 phosphorylation and pro-inflammatory gene expression in mouse bone marrow-derived macrophages ( n =4). * P
    Figure Legend Snippet: Identification of PARP14-induced ribosylation sites in STAT1. ( a ) The amino-acid sequence of human STAT1α C terminus. Green amino acids indicate ribosylated peptides; confirmed ribosylation sites are underlined. STAT1 is phosphorylated at indicated tyrosine (red). ( b ; Left panels) MS/MS spectra for the mono-ADP-ribosylated peptides and corresponding unmodified forms. ADP-ribose fragments are annotated in green. *, ribosylation site; m, oxidized methionine. The grey circles indicate background or undetermined ions. (Right panels) MS1-based quantification of PARP9 inhibition of PARP14-mediated STAT1α ribosylation at E657 (upper panel) and E705 (lower panel), respectively. ( c ) Effects of mutated amino acids at E657 and E705 in STAT1 (ribosylation sites for PARP14) on its Tyr701 phosphorylation and pro-inflammatory gene expression in mouse bone marrow-derived macrophages ( n =4). * P

    Techniques Used: Sequencing, Mass Spectrometry, Inhibition, Expressing, Derivative Assay

    Role of haematopoietic PARP14 in acute arterial lesion formation in mice. ( a–c ) Cultured peritoneal macrophages derived from PARP14 −/− and PARP14 +/+ mice. ( a ) IFNγ and IL-4 pathway gene expression profiles ( n =3). ( b ) Secretion of inflammatory factors into culture media ( n =3). ( c ) Western blot and corresponding densitometry quantification of phosphorylated STAT1 and STAT6. Each data point is the average of triplicate samples per donor ( n =3). ( d ) Left: representative images of haematoxylin and eosin (H E; top) and Mac3 (bottom) staining. Scale bars, 100 μm. Right: quantification of lesion formation in mechanically injured femoral arteries of PARP14 −/− and PARP +/+ mice. Mac3 staining represents macrophage accumulation ( n =4–5). ( e ) LCM of the neointima followed by gene expression analysis ( n =4). ( f ) Flow cytometry analysis of splenic CD11b+Ly6G− monocytes after induction of mechanically injured femoral arteries of PARP14 +/+ and PARP14 −/− mice ( n =3). ( g ) Representative H E staining images and quantification of neointima formation in mechanically injured femoral arteries after bone marrow transplantation (BMT) PARP14 +/+→+/+ and PARP14 −/−→+/+ mice ( n =6). Scale bars, 100 μm. * P
    Figure Legend Snippet: Role of haematopoietic PARP14 in acute arterial lesion formation in mice. ( a–c ) Cultured peritoneal macrophages derived from PARP14 −/− and PARP14 +/+ mice. ( a ) IFNγ and IL-4 pathway gene expression profiles ( n =3). ( b ) Secretion of inflammatory factors into culture media ( n =3). ( c ) Western blot and corresponding densitometry quantification of phosphorylated STAT1 and STAT6. Each data point is the average of triplicate samples per donor ( n =3). ( d ) Left: representative images of haematoxylin and eosin (H E; top) and Mac3 (bottom) staining. Scale bars, 100 μm. Right: quantification of lesion formation in mechanically injured femoral arteries of PARP14 −/− and PARP +/+ mice. Mac3 staining represents macrophage accumulation ( n =4–5). ( e ) LCM of the neointima followed by gene expression analysis ( n =4). ( f ) Flow cytometry analysis of splenic CD11b+Ly6G− monocytes after induction of mechanically injured femoral arteries of PARP14 +/+ and PARP14 −/− mice ( n =3). ( g ) Representative H E staining images and quantification of neointima formation in mechanically injured femoral arteries after bone marrow transplantation (BMT) PARP14 +/+→+/+ and PARP14 −/−→+/+ mice ( n =6). Scale bars, 100 μm. * P

    Techniques Used: Mouse Assay, Cell Culture, Derivative Assay, Expressing, Western Blot, Staining, Laser Capture Microdissection, Flow Cytometry, Cytometry, Transplantation Assay

    9) Product Images from "STAT1β Is Not Dominant Negative and Is Capable of Contributing to Gamma Interferon-Dependent Innate Immunity"

    Article Title: STAT1β Is Not Dominant Negative and Is Capable of Contributing to Gamma Interferon-Dependent Innate Immunity

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00295-14

    STAT1β shows prolonged tyrosine 701 phosphorylation. (A to D) BMMϕ derived from WT (+/+), Stat1 β/β (β/β), and Stat1 α/α (α/α) mice were stimulated with IFN-β (A) or
    Figure Legend Snippet: STAT1β shows prolonged tyrosine 701 phosphorylation. (A to D) BMMϕ derived from WT (+/+), Stat1 β/β (β/β), and Stat1 α/α (α/α) mice were stimulated with IFN-β (A) or

    Techniques Used: Derivative Assay, Mouse Assay

    STAT1β shows prolonged nuclear localization and prolonged promoter binding after IFN-γ treatment compared to STAT1α. (A) BMMϕ derived from WT (+/+), Stat1 β/β (β/β), and Stat1 α/α
    Figure Legend Snippet: STAT1β shows prolonged nuclear localization and prolonged promoter binding after IFN-γ treatment compared to STAT1α. (A) BMMϕ derived from WT (+/+), Stat1 β/β (β/β), and Stat1 α/α

    Techniques Used: Binding Assay, Derivative Assay

    STAT1α and STAT1β can mediate type I and type III IFN-dependent antiviral immunity in vivo . (A) EMCV (50 PFU/mouse) was administered i.p. to WT ( Stat1 +/+ ), Stat1 β/β , Stat1 α/α , and Stat1 −/−
    Figure Legend Snippet: STAT1α and STAT1β can mediate type I and type III IFN-dependent antiviral immunity in vivo . (A) EMCV (50 PFU/mouse) was administered i.p. to WT ( Stat1 +/+ ), Stat1 β/β , Stat1 α/α , and Stat1 −/−

    Techniques Used: In Vivo

    STAT1β is transcriptionally active in response to IFN-β and IFN-γ. BMMϕ isolated from WT (+/+), Stat1 β/β (β/β), Stat1 α/α (α/α), and Stat1 −/−
    Figure Legend Snippet: STAT1β is transcriptionally active in response to IFN-β and IFN-γ. BMMϕ isolated from WT (+/+), Stat1 β/β (β/β), Stat1 α/α (α/α), and Stat1 −/−

    Techniques Used: Isolation

    STAT1α and STAT1β show differential efficiencies in immune defense against MCMV and L. monocytogenes infections. (A) WT ( Stat1 +/+ ), Stat1 β/β , Stat1 α/α , and Stat1 −/− mice were infected i.p.
    Figure Legend Snippet: STAT1α and STAT1β show differential efficiencies in immune defense against MCMV and L. monocytogenes infections. (A) WT ( Stat1 +/+ ), Stat1 β/β , Stat1 α/α , and Stat1 −/− mice were infected i.p.

    Techniques Used: Mouse Assay, Infection

    Transcriptional activities of STAT1α and STAT1β overlap but are nonredundant. BMMϕ isolated from WT (+/+), Stat1 β/β (β/β), Stat1 α/α (α/α), and Stat1 −/−
    Figure Legend Snippet: Transcriptional activities of STAT1α and STAT1β overlap but are nonredundant. BMMϕ isolated from WT (+/+), Stat1 β/β (β/β), Stat1 α/α (α/α), and Stat1 −/−

    Techniques Used: Isolation

    10) Product Images from "Critical Role of the CXCL10/C-X-C Chemokine Receptor 3 Axis in Promoting Leukocyte Recruitment and Neuronal Injury during Traumatic Optic Neuropathy Induced by Optic Nerve Crush"

    Article Title: Critical Role of the CXCL10/C-X-C Chemokine Receptor 3 Axis in Promoting Leukocyte Recruitment and Neuronal Injury during Traumatic Optic Neuropathy Induced by Optic Nerve Crush

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2016.10.009

    Traumatic optic neuropathy (TON)-induced CXCL10 up-regulation is modulated by STAT signaling. A: Wild-type (WT) mice were subjected to TON, and proteins were isolated from retinas at indicated time points after TON. The phosphorylation levels of STAT1, STAT2, STAT3, STAT5, and STAT6 were evaluated by Western blotting. α-Tubulin was used as loading control. B and C: STAT1/STAT3 inhibitor Stattic or vehicle (Veh) was injected intravitreally into WT mice at 1 hour after the induction of TON. Retinas were collected at 4 hours after TON. B: Phosphorylated and total STAT1 and STAT3 were assessed by Western blotting. C: The expression of CXCL10 mRNA was analyzed by quantitative PCR. n = 5 mice ( A–C ). ∗ P
    Figure Legend Snippet: Traumatic optic neuropathy (TON)-induced CXCL10 up-regulation is modulated by STAT signaling. A: Wild-type (WT) mice were subjected to TON, and proteins were isolated from retinas at indicated time points after TON. The phosphorylation levels of STAT1, STAT2, STAT3, STAT5, and STAT6 were evaluated by Western blotting. α-Tubulin was used as loading control. B and C: STAT1/STAT3 inhibitor Stattic or vehicle (Veh) was injected intravitreally into WT mice at 1 hour after the induction of TON. Retinas were collected at 4 hours after TON. B: Phosphorylated and total STAT1 and STAT3 were assessed by Western blotting. C: The expression of CXCL10 mRNA was analyzed by quantitative PCR. n = 5 mice ( A–C ). ∗ P

    Techniques Used: Mouse Assay, Isolation, Western Blot, Injection, Expressing, Real-time Polymerase Chain Reaction

    11) Product Images from "A positive autoregulatory loop of Jak-STAT signaling controls the onset of astrogliogenesis"

    Article Title: A positive autoregulatory loop of Jak-STAT signaling controls the onset of astrogliogenesis

    Journal: Nature neuroscience

    doi: 10.1038/nn1440

    Sequential activation of the Jak-STAT pathway in vivo correlates with the timing of astrogliogenesis. ( a ) A fluorescent image of E15 cortical ventricular area from pNestin-GFP mice, demonstrating enriched GFP expression in the ventricular zone (VZ). ( b,c ) The ventricular zone (green) and non–ventricular zone (non-green) tissues were dissected from different developmental stages (E12, E16 or postnatal day (P) 0 from the pNestin-GFP transgenic mice under a fluorescent dissection microscope. Western blot analyses used a TuJ1 antibody that labels neuronal specific βIII tubulin, and a GFP antibody. β-actin blot indicates the loading control. ( d–f ) Western blot analyses of STAT activation and astrocyte differentiation in vivo at different developmental stages. After incubation with or without LIF (100 ng ml −1 ) for 20 min, lysates of green VZ/SVZ tissues from various developmental stages (E12, E14, P0 and P4) were probed with antibodies against tyrosine-phosphorylated STAT1 or STAT3 ( d,e ), astrocyte marker GFAP ( d ), gp130, LIFRβ ( f ), GAPDH ( e ), and β-actin ( d ). Lysates from P0 or P4 without 20-min LIF treatment were also enriched for active forms of STAT1 and STAT3. ( g ) RT-PCR analysis of gp130, Jak1 and astrocytic markers GFAP and S100β at different developmental stages.
    Figure Legend Snippet: Sequential activation of the Jak-STAT pathway in vivo correlates with the timing of astrogliogenesis. ( a ) A fluorescent image of E15 cortical ventricular area from pNestin-GFP mice, demonstrating enriched GFP expression in the ventricular zone (VZ). ( b,c ) The ventricular zone (green) and non–ventricular zone (non-green) tissues were dissected from different developmental stages (E12, E16 or postnatal day (P) 0 from the pNestin-GFP transgenic mice under a fluorescent dissection microscope. Western blot analyses used a TuJ1 antibody that labels neuronal specific βIII tubulin, and a GFP antibody. β-actin blot indicates the loading control. ( d–f ) Western blot analyses of STAT activation and astrocyte differentiation in vivo at different developmental stages. After incubation with or without LIF (100 ng ml −1 ) for 20 min, lysates of green VZ/SVZ tissues from various developmental stages (E12, E14, P0 and P4) were probed with antibodies against tyrosine-phosphorylated STAT1 or STAT3 ( d,e ), astrocyte marker GFAP ( d ), gp130, LIFRβ ( f ), GAPDH ( e ), and β-actin ( d ). Lysates from P0 or P4 without 20-min LIF treatment were also enriched for active forms of STAT1 and STAT3. ( g ) RT-PCR analysis of gp130, Jak1 and astrocytic markers GFAP and S100β at different developmental stages.

    Techniques Used: Activation Assay, In Vivo, Mouse Assay, Expressing, Transgenic Assay, Dissection, Microscopy, Western Blot, Incubation, Marker, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "STAT3 but Not STAT1 Is Required for Astrocyte Differentiation"

    Article Title: STAT3 but Not STAT1 Is Required for Astrocyte Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086851

    Expression of STAT and astrocyte markers in the spinal cord during CNS development. ( A) Western blot analysis of phospho-STAT1 (Ser 727) (p-STAT1), phospho-STAT3 (Tyr 705) (p-STAT3), STAT1, STAT3 and GFAP in the mouse embryonic CNS. α-Tubulin served as loading control. (B–I) Expression of STAT3 and NFIA in the spinal cord. STAT3 is present in E16.5 and E18.5 white matter astrocytes (D, E, arrowheads). NFIA expression is seen in migrating glial progenitor cells and overlaps with STAT3 expression in the marginal zone (F–I, arrowheads). (J–N) Expression of p-STAT1, p-STAT3, NFIA, GFAP and S100β in E18.5 spinal cords. P-STAT1 and p-STAT3 are present in astrocytes at the marginal zone (J–N, arrowheads). Note that motor neurons also express STAT3 and NFIA (B–I, K, L, asterisks). (O) Diagram of STAT expression in glial progenitors (GP) and astrocytes (A) in the developing spinal cord. Scale bar: in I, 100 µm for B–I; in N, 100 µm for J–N.
    Figure Legend Snippet: Expression of STAT and astrocyte markers in the spinal cord during CNS development. ( A) Western blot analysis of phospho-STAT1 (Ser 727) (p-STAT1), phospho-STAT3 (Tyr 705) (p-STAT3), STAT1, STAT3 and GFAP in the mouse embryonic CNS. α-Tubulin served as loading control. (B–I) Expression of STAT3 and NFIA in the spinal cord. STAT3 is present in E16.5 and E18.5 white matter astrocytes (D, E, arrowheads). NFIA expression is seen in migrating glial progenitor cells and overlaps with STAT3 expression in the marginal zone (F–I, arrowheads). (J–N) Expression of p-STAT1, p-STAT3, NFIA, GFAP and S100β in E18.5 spinal cords. P-STAT1 and p-STAT3 are present in astrocytes at the marginal zone (J–N, arrowheads). Note that motor neurons also express STAT3 and NFIA (B–I, K, L, asterisks). (O) Diagram of STAT expression in glial progenitors (GP) and astrocytes (A) in the developing spinal cord. Scale bar: in I, 100 µm for B–I; in N, 100 µm for J–N.

    Techniques Used: Expressing, Western Blot

    Structure-function analysis of STAT3 in astrocyte differentiation. (A) Diagrams of STAT3CA, STAT3YF, STAT3SA and STAT3β. CC, coiled-coil domain; DBD, DNA binding domain; Lk, linker domain; TAD, transcriptional activation domain. (B) Expression and phosphorylation of STAT3 wild-type or mutants assessed by Western blotting. Each construct was transfected into 293T cells, and the cells were stimulated with CNTF (100 ng/ml) for 10 min before harvesting. Expression of all the STAT mutants except STAT3YF was similar to that of wild-type, and they became phosphorylated in the presence of CNTF except STAT3YF. Expression of p-STAT3 in the control group is due to endogenous STAT3 activity. (C) Transactivation of the SBS8GFMP reporter in the presence of STAT derivatives. COS7 cell derivatives were incubated with CNTF (100 ng/ml) for 12 hrs. (D) Transactivation of the gfap promoter GF1L. E16.5 Stat1 KO; Stat3 cKO primary cortical progenitors were transfected with STATs and incubated with CNTF (100 ng/ml) for 12 hrs. (E) Assessment of p-STAT3 (Tyr 705), p-STAT1 (Tyr 701), STAT3, STAT1 and α-tubulin in the presence of CNTF (100 ng/ml) for 30 min and 90 min by Western blotting. (F) The binding between STAT and p300 in the presence of CNTF (100 ng/ml) tested by co-immunoprecipitation experiment. Error bars represents s.e.m. ** p
    Figure Legend Snippet: Structure-function analysis of STAT3 in astrocyte differentiation. (A) Diagrams of STAT3CA, STAT3YF, STAT3SA and STAT3β. CC, coiled-coil domain; DBD, DNA binding domain; Lk, linker domain; TAD, transcriptional activation domain. (B) Expression and phosphorylation of STAT3 wild-type or mutants assessed by Western blotting. Each construct was transfected into 293T cells, and the cells were stimulated with CNTF (100 ng/ml) for 10 min before harvesting. Expression of all the STAT mutants except STAT3YF was similar to that of wild-type, and they became phosphorylated in the presence of CNTF except STAT3YF. Expression of p-STAT3 in the control group is due to endogenous STAT3 activity. (C) Transactivation of the SBS8GFMP reporter in the presence of STAT derivatives. COS7 cell derivatives were incubated with CNTF (100 ng/ml) for 12 hrs. (D) Transactivation of the gfap promoter GF1L. E16.5 Stat1 KO; Stat3 cKO primary cortical progenitors were transfected with STATs and incubated with CNTF (100 ng/ml) for 12 hrs. (E) Assessment of p-STAT3 (Tyr 705), p-STAT1 (Tyr 701), STAT3, STAT1 and α-tubulin in the presence of CNTF (100 ng/ml) for 30 min and 90 min by Western blotting. (F) The binding between STAT and p300 in the presence of CNTF (100 ng/ml) tested by co-immunoprecipitation experiment. Error bars represents s.e.m. ** p

    Techniques Used: Binding Assay, Activation Assay, Expressing, Western Blot, Construct, Transfection, Activity Assay, Incubation, Immunoprecipitation

    13) Product Images from "Early induction of interferon-responsive mRNAs in Creutzfeldt-Jakob disease"

    Article Title: Early induction of interferon-responsive mRNAs in Creutzfeldt-Jakob disease

    Journal: Journal of neurovirology

    doi:

    Activation of kinases and transcription factors in CJD microglia. Representative western blots for IRF-3 ( A ), total and phosphorylated forms of the p44 and p42 MAP kinases ( B ), as well as phosphorylated STAT1 and total STAT1 α ( C ). The phospho-STAT1 antibody recognizes both the STAT1 α (p91) and STAT1 β (p84) forms of the molecule, which are generated by alternative splicing. Normalization to α -tubulin protein was used to control for differences in protein loading on the blots. Equivalent levels of total p44/p42 MAP kinases and STAT1 α in normal and CJD microglia confirm that any changes in the phosphospecific forms of these molecules are indeed the result of differential phosphorylation between these cell populations.
    Figure Legend Snippet: Activation of kinases and transcription factors in CJD microglia. Representative western blots for IRF-3 ( A ), total and phosphorylated forms of the p44 and p42 MAP kinases ( B ), as well as phosphorylated STAT1 and total STAT1 α ( C ). The phospho-STAT1 antibody recognizes both the STAT1 α (p91) and STAT1 β (p84) forms of the molecule, which are generated by alternative splicing. Normalization to α -tubulin protein was used to control for differences in protein loading on the blots. Equivalent levels of total p44/p42 MAP kinases and STAT1 α in normal and CJD microglia confirm that any changes in the phosphospecific forms of these molecules are indeed the result of differential phosphorylation between these cell populations.

    Techniques Used: Activation Assay, Western Blot, Generated

    14) Product Images from "Differential Delivery of Genomic Double-Stranded RNA Causes Reovirus Strain-Specific Differences in Interferon Regulatory Factor 3 Activation"

    Article Title: Differential Delivery of Genomic Double-Stranded RNA Causes Reovirus Strain-Specific Differences in Interferon Regulatory Factor 3 Activation

    Journal: Journal of Virology

    doi: 10.1128/JVI.01947-17

    rsT3D induces higher levels of IRF3 activation than rsT1L in SVECs. (A) SVECs were mock infected (M) or infected with rsT1L or rsT3D at an MOI of 1, 10, or 100 PFU/cell. Whole-cell lysates were prepared at the indicated times, and immunoblot analysis was performed to detect phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, or reovirus proteins. (B) SVECs were mock infected or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell, and cytoplasmic and nuclear fraction lysates were prepared at 6 h. Immunoblot analysis was performed to detect phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, α-tubulin, lamin A/C, or reovirus proteins. (C) SVECs were transfected with an IRF3 firefly luciferase reporter (p55C1BLuc) and a Renilla luciferase control. At 24 h, cells were mock infected, transfected with poly I·C (2 μg), or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell. Luciferase expression was measured at 24 h posttransfection. Results are presented as the mean ratio of firefly luciferase activity to Renilla luciferase activity for triplicate samples from three independent experiments ± SD. **, P
    Figure Legend Snippet: rsT3D induces higher levels of IRF3 activation than rsT1L in SVECs. (A) SVECs were mock infected (M) or infected with rsT1L or rsT3D at an MOI of 1, 10, or 100 PFU/cell. Whole-cell lysates were prepared at the indicated times, and immunoblot analysis was performed to detect phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, or reovirus proteins. (B) SVECs were mock infected or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell, and cytoplasmic and nuclear fraction lysates were prepared at 6 h. Immunoblot analysis was performed to detect phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, α-tubulin, lamin A/C, or reovirus proteins. (C) SVECs were transfected with an IRF3 firefly luciferase reporter (p55C1BLuc) and a Renilla luciferase control. At 24 h, cells were mock infected, transfected with poly I·C (2 μg), or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell. Luciferase expression was measured at 24 h posttransfection. Results are presented as the mean ratio of firefly luciferase activity to Renilla luciferase activity for triplicate samples from three independent experiments ± SD. **, P

    Techniques Used: Activation Assay, Infection, Transfection, Luciferase, Expressing, Activity Assay

    RIG-I, MDA5, MAVS, and TBK1 mediate reovirus-induced IRF3 phosphorylation. SVECs were transfected with negative-control siRNA or siRNA specific for MAVS (A), MAVS or TLR3 alone or in combination (B), RIG-I or MDA5 alone or in combination (C), or TBK1 or IKKε (E). At 48 h, cells were mock infected (M), infected with rsT3D (T3) or rsT1L (T1) at an MOI of 100 PFU/cell, or transfected with poly(I·C) (2 μg). At 6 h postinfection, whole-cell lysates were prepared and immunoblot analysis was performed for phosphorylated IRF3 (p-IRF3), total IRF3, RIG-I, MDA5, MAVS, β-actin, TBK1, IKKε, or reovirus proteins, as indicated. (D) At 48 h posttransfection with negative-control siRNA or TLR3-specific siRNA, TLR3 mRNA levels were quantified by RT-qPCR. Results are presented as the mean of triplicate samples ± SD. *, P
    Figure Legend Snippet: RIG-I, MDA5, MAVS, and TBK1 mediate reovirus-induced IRF3 phosphorylation. SVECs were transfected with negative-control siRNA or siRNA specific for MAVS (A), MAVS or TLR3 alone or in combination (B), RIG-I or MDA5 alone or in combination (C), or TBK1 or IKKε (E). At 48 h, cells were mock infected (M), infected with rsT3D (T3) or rsT1L (T1) at an MOI of 100 PFU/cell, or transfected with poly(I·C) (2 μg). At 6 h postinfection, whole-cell lysates were prepared and immunoblot analysis was performed for phosphorylated IRF3 (p-IRF3), total IRF3, RIG-I, MDA5, MAVS, β-actin, TBK1, IKKε, or reovirus proteins, as indicated. (D) At 48 h posttransfection with negative-control siRNA or TLR3-specific siRNA, TLR3 mRNA levels were quantified by RT-qPCR. Results are presented as the mean of triplicate samples ± SD. *, P

    Techniques Used: Transfection, Negative Control, Infection, Quantitative RT-PCR

    Reovirus disassembly is required for IRF-3 phosphorylation. (A) SVECs were infected with rsT1L (T1) or rsT3D (T3) at an MOI of 100 PFU/cell in the absence or presence of ammonium chloride or E64 at the indicated concentrations. At 6 h, immunoblot analysis was performed for phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, or reovirus proteins. (B) To generate ISVPs, 2 × 10 10 rsT1L or rsT3D particles were incubated with chymotrypsin (CHT). Proteins were resolved by SDS-PAGE and stained with Coomassie blue. Viral proteins are indicated to the right of the image. (C) SVECs were infected with rsT1L or rsT3D virions at an MOI of 100 PFU/cell or rsT1L or rsT3D ISVPs at an MOI of 10 PFU/cell. At 24 h, cells were fixed with methanol, stained with DAPI (blue) and reovirus polyclonal antiserum (green), and visualized by indirect immunofluorescence. (D) SVECs were infected with rsT1L or rsT3D virions or ISVPs at an MOI of 100 PFU/cell in the absence or presence of 20 mM ammonium chloride. At 6 h, immunoblot analysis was performed for phosphorylated IRF3, total IRF3, β-actin, or reovirus proteins.
    Figure Legend Snippet: Reovirus disassembly is required for IRF-3 phosphorylation. (A) SVECs were infected with rsT1L (T1) or rsT3D (T3) at an MOI of 100 PFU/cell in the absence or presence of ammonium chloride or E64 at the indicated concentrations. At 6 h, immunoblot analysis was performed for phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, or reovirus proteins. (B) To generate ISVPs, 2 × 10 10 rsT1L or rsT3D particles were incubated with chymotrypsin (CHT). Proteins were resolved by SDS-PAGE and stained with Coomassie blue. Viral proteins are indicated to the right of the image. (C) SVECs were infected with rsT1L or rsT3D virions at an MOI of 100 PFU/cell or rsT1L or rsT3D ISVPs at an MOI of 10 PFU/cell. At 24 h, cells were fixed with methanol, stained with DAPI (blue) and reovirus polyclonal antiserum (green), and visualized by indirect immunofluorescence. (D) SVECs were infected with rsT1L or rsT3D virions or ISVPs at an MOI of 100 PFU/cell in the absence or presence of 20 mM ammonium chloride. At 6 h, immunoblot analysis was performed for phosphorylated IRF3, total IRF3, β-actin, or reovirus proteins.

    Techniques Used: Infection, Incubation, SDS Page, Staining, Immunofluorescence

    rsT1L does not block entry-related IRF3 phosphorylation. SVECs were mock-infected (M), infected with rsT1L (T1) at an MOI of 100 PFU/cell, or transfected with poly I·C (2 μg). At 4 h postinfection, rsT1L-infected cells were transfected with poly I·C (2 μg). At 6 h postinfection, whole-cell lysates were prepared and proteins were separated by SDS-PAGE. Western blot analysis was performed for phosphorylated IRF3, total IRF3, RIG-I, MDA-5, β-actin, or reovirus proteins.
    Figure Legend Snippet: rsT1L does not block entry-related IRF3 phosphorylation. SVECs were mock-infected (M), infected with rsT1L (T1) at an MOI of 100 PFU/cell, or transfected with poly I·C (2 μg). At 4 h postinfection, rsT1L-infected cells were transfected with poly I·C (2 μg). At 6 h postinfection, whole-cell lysates were prepared and proteins were separated by SDS-PAGE. Western blot analysis was performed for phosphorylated IRF3, total IRF3, RIG-I, MDA-5, β-actin, or reovirus proteins.

    Techniques Used: Blocking Assay, Infection, Transfection, SDS Page, Western Blot, Multiple Displacement Amplification

    rsT3D elicits more NF-κB activation than rsT1L in SVECs. SVECs were left untreated (U) or were treated with TNF-α (TNF) for 15 min, mock infected (M), or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell. Whole-cell lysates were prepared at the indicated times, and immunoblot analysis was performed to detect phosphorylated IKKα/β (p-IKKα/β), IκBα, β-actin, or reovirus proteins. (B) SVECs were mock infected, infected with rsT1L or rsT3D at an MOI of 100 PFU/cell, or transfected with poly(I·C). At the indicated times, cytoplasmic and nuclear fraction lysates were prepared and immunoblot analysis was performed to detect p65, β-actin, α-tubulin, lamin A/C, or reovirus proteins. (C) Wild-type SVECs or MAVS-CRISPR SVECs were mock infected (M), treated with TNF-α (TNF) for 15 min, or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell. Whole-cell lysates were prepared at the indicated times, and immunoblot analysis was performed to detect phosphorylated IKKα/β (p-IKKα/β), total IKKα/β, IκBα, MAVS, β-actin, or reovirus proteins.
    Figure Legend Snippet: rsT3D elicits more NF-κB activation than rsT1L in SVECs. SVECs were left untreated (U) or were treated with TNF-α (TNF) for 15 min, mock infected (M), or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell. Whole-cell lysates were prepared at the indicated times, and immunoblot analysis was performed to detect phosphorylated IKKα/β (p-IKKα/β), IκBα, β-actin, or reovirus proteins. (B) SVECs were mock infected, infected with rsT1L or rsT3D at an MOI of 100 PFU/cell, or transfected with poly(I·C). At the indicated times, cytoplasmic and nuclear fraction lysates were prepared and immunoblot analysis was performed to detect p65, β-actin, α-tubulin, lamin A/C, or reovirus proteins. (C) Wild-type SVECs or MAVS-CRISPR SVECs were mock infected (M), treated with TNF-α (TNF) for 15 min, or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell. Whole-cell lysates were prepared at the indicated times, and immunoblot analysis was performed to detect phosphorylated IKKα/β (p-IKKα/β), total IKKα/β, IκBα, MAVS, β-actin, or reovirus proteins.

    Techniques Used: Activation Assay, Infection, Transfection, CRISPR

    Reovirus genomic RNA induces IRF3 phosphorylation in SVECs. (A) SVECs were transfected with 0.25 μg or 0.5 μg of rsT1L or rsT3D genomic RNA or with 2.0 μg of poly(I·C). (B) SVECs were transfected with siRNA specific for MAVS or TLR3 alone or in combination for 48 h prior to transfection with 0.5 μg of genomic RNA or poly(I·C) (2 μg). At 6 h, immunoblot analysis was performed for phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, or MAVS. **, P
    Figure Legend Snippet: Reovirus genomic RNA induces IRF3 phosphorylation in SVECs. (A) SVECs were transfected with 0.25 μg or 0.5 μg of rsT1L or rsT3D genomic RNA or with 2.0 μg of poly(I·C). (B) SVECs were transfected with siRNA specific for MAVS or TLR3 alone or in combination for 48 h prior to transfection with 0.5 μg of genomic RNA or poly(I·C) (2 μg). At 6 h, immunoblot analysis was performed for phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, or MAVS. **, P

    Techniques Used: Transfection

    Viral protein synthesis and RNA synthesis are not required for reovirus-induced IRF3 phosphorylation. (A) SVECs were mock infected (M) or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell in the absence or presence of ribavirin at the indicated concentrations. At 6 h, whole-cell lysates were prepared and immunoblot analysis was performed for phosphorylated-IRF3 (p-IRF3), total IRF3, β-actin, or reovirus proteins. (B) SVECs were infected with rsT1L or rsT3D at an MOI of 100 PFU/cell in the absence or presence of 40 μM ribavirin. At 6 h, total RNA was extracted and reovirus S4 mRNA levels were determined by RT-qPCR. Results are presented as the mean of triplicate samples ± SD and are representative of three independent experiments. *, P
    Figure Legend Snippet: Viral protein synthesis and RNA synthesis are not required for reovirus-induced IRF3 phosphorylation. (A) SVECs were mock infected (M) or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell in the absence or presence of ribavirin at the indicated concentrations. At 6 h, whole-cell lysates were prepared and immunoblot analysis was performed for phosphorylated-IRF3 (p-IRF3), total IRF3, β-actin, or reovirus proteins. (B) SVECs were infected with rsT1L or rsT3D at an MOI of 100 PFU/cell in the absence or presence of 40 μM ribavirin. At 6 h, total RNA was extracted and reovirus S4 mRNA levels were determined by RT-qPCR. Results are presented as the mean of triplicate samples ± SD and are representative of three independent experiments. *, P

    Techniques Used: Infection, Quantitative RT-PCR

    rsT1L and rsT3D core particles induce IRF3 phosphorylation. (A) To generate core particles, rsT1L and rsT3D virions were incubated with chymotrypsin (200 μg/ml) and cesium chloride (400 mM). A total of 2 × 10 10 particles were resolved by SDS-PAGE, and proteins were stained with Coomassie blue. Viral proteins are indicated to the right of the image. (B and C) SVECs were exposed to rsT1L or rsT3D cores at an MOI of 10,000 or 100,000 particles (prt)/cell with or without transfection reagent. (B) At 24 h, cells were fixed with methanol, stained with DAPI (blue) and reovirus polyclonal antiserum (green), and visualized by indirect immunofluorescence. (C) At 6 h, immunoblot analysis was performed for phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, or reovirus proteins.
    Figure Legend Snippet: rsT1L and rsT3D core particles induce IRF3 phosphorylation. (A) To generate core particles, rsT1L and rsT3D virions were incubated with chymotrypsin (200 μg/ml) and cesium chloride (400 mM). A total of 2 × 10 10 particles were resolved by SDS-PAGE, and proteins were stained with Coomassie blue. Viral proteins are indicated to the right of the image. (B and C) SVECs were exposed to rsT1L or rsT3D cores at an MOI of 10,000 or 100,000 particles (prt)/cell with or without transfection reagent. (B) At 24 h, cells were fixed with methanol, stained with DAPI (blue) and reovirus polyclonal antiserum (green), and visualized by indirect immunofluorescence. (C) At 6 h, immunoblot analysis was performed for phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, or reovirus proteins.

    Techniques Used: Incubation, SDS Page, Staining, Transfection, Immunofluorescence

    15) Product Images from "Activation of Protein Tyrosine Phosphatase Non-Receptor Type 2 by Spermidine Exerts Anti-Inflammatory Effects in Human THP-1 Monocytes and in a Mouse Model of Acute Colitis"

    Article Title: Activation of Protein Tyrosine Phosphatase Non-Receptor Type 2 by Spermidine Exerts Anti-Inflammatory Effects in Human THP-1 Monocytes and in a Mouse Model of Acute Colitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073703

    Effects of spermidine treatment on the phosphorylation levels of signal transducers and activators of transcription (STATs) and p38 mitogen-activated protein kinase (MAPK) in interferon-gamma (IFN-γ)-treated THP-1 cells. Representative Western blots and densitometry (n = 3) show phosphorylation status of ( a and b ) STAT1, ( c ) STAT3 and ( d ) p38 MAPK in THP-1 cells treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for ( a , c , and d ) 30 min or ( b ) 36 h. Blots were probed for β-actin to show equal protein loading. Significant differences compared to untreated cells (* = p
    Figure Legend Snippet: Effects of spermidine treatment on the phosphorylation levels of signal transducers and activators of transcription (STATs) and p38 mitogen-activated protein kinase (MAPK) in interferon-gamma (IFN-γ)-treated THP-1 cells. Representative Western blots and densitometry (n = 3) show phosphorylation status of ( a and b ) STAT1, ( c ) STAT3 and ( d ) p38 MAPK in THP-1 cells treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for ( a , c , and d ) 30 min or ( b ) 36 h. Blots were probed for β-actin to show equal protein loading. Significant differences compared to untreated cells (* = p

    Techniques Used: Western Blot

    Effect of spermidine and interferon-gamma (IFN-γ) treatment on protein tyrosine phosphatases (PTPN2 and PTP1B) expression in human monocytic THP-1 cells. THP-1 cells were treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for ( a , c ) 30 min or ( b , d ) 36 h. Whole-cell lysates were obtained and PTPN2 and PTP1B protein levels were analyzed by Western blot using β-actin as a loading control. The relative protein levels were assessed by densitometry (n = 3). Data are expressed as fold induction with respect to untreated cells. Asterisks indicate significant differences between treated and untreated cells (* = p
    Figure Legend Snippet: Effect of spermidine and interferon-gamma (IFN-γ) treatment on protein tyrosine phosphatases (PTPN2 and PTP1B) expression in human monocytic THP-1 cells. THP-1 cells were treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for ( a , c ) 30 min or ( b , d ) 36 h. Whole-cell lysates were obtained and PTPN2 and PTP1B protein levels were analyzed by Western blot using β-actin as a loading control. The relative protein levels were assessed by densitometry (n = 3). Data are expressed as fold induction with respect to untreated cells. Asterisks indicate significant differences between treated and untreated cells (* = p

    Techniques Used: Expressing, Western Blot

    16) Product Images from "T cell protein tyrosine phosphatase prevents STAT1 induction of claudin-2 expression in intestinal epithelial cells"

    Article Title: T cell protein tyrosine phosphatase prevents STAT1 induction of claudin-2 expression in intestinal epithelial cells

    Journal: Annals of the New York Academy of Sciences

    doi: 10.1111/nyas.13439

    Increased claudin-2 protein in TCPTP-deficient IECs is alleviated by STAT1 knockdown. (A and B) Soluble and insoluble fractions of HT-29 cell lysates were probed by western blotting to confirm significant knockdown of TCPTP in PTPN2-KD cells in both cytosolic and membrane cell compartments ( n = 3). Claudin-2 levels were significantly increased in both compartments ( n = 3). (C) Immunofluorescence staining for claudin-2 (indicated by arrowhead) in Con-shRNA and PTPN2-KD IECs grown on glass coverslips for 3 days (representative of three separate experiments). (D) Con-shRNA and PTPN2-KD HT-29 IEC were transfected with scrambled control siRNA (Csi) or STAT1 siRNA (STsi) and grown on plastic for 24 hours. Cells were treated with IFN-γ (1000 U/mL (100 ng/mL)) or control media (U) for 24 h before whole-cell lysis, western blotting, and densitometric analysis. (E) Phosphorylated STAT1 levels relative to total STAT1 were quantified by densitometry and normalized to β-actin ( n = 5). (F) Claudin-2 protein levels were quantified by densitometry and normalized to β-actin ( n = 5). Statistical significance was calculated using one-way analysis of variance followed by Student–Newman–Keuls post-test. * P
    Figure Legend Snippet: Increased claudin-2 protein in TCPTP-deficient IECs is alleviated by STAT1 knockdown. (A and B) Soluble and insoluble fractions of HT-29 cell lysates were probed by western blotting to confirm significant knockdown of TCPTP in PTPN2-KD cells in both cytosolic and membrane cell compartments ( n = 3). Claudin-2 levels were significantly increased in both compartments ( n = 3). (C) Immunofluorescence staining for claudin-2 (indicated by arrowhead) in Con-shRNA and PTPN2-KD IECs grown on glass coverslips for 3 days (representative of three separate experiments). (D) Con-shRNA and PTPN2-KD HT-29 IEC were transfected with scrambled control siRNA (Csi) or STAT1 siRNA (STsi) and grown on plastic for 24 hours. Cells were treated with IFN-γ (1000 U/mL (100 ng/mL)) or control media (U) for 24 h before whole-cell lysis, western blotting, and densitometric analysis. (E) Phosphorylated STAT1 levels relative to total STAT1 were quantified by densitometry and normalized to β-actin ( n = 5). (F) Claudin-2 protein levels were quantified by densitometry and normalized to β-actin ( n = 5). Statistical significance was calculated using one-way analysis of variance followed by Student–Newman–Keuls post-test. * P

    Techniques Used: Western Blot, Immunofluorescence, Staining, shRNA, Transfection, Lysis

    17) Product Images from "Differential Antagonism of Human Innate Immune Responses by Tick-Borne Phlebovirus Nonstructural Proteins"

    Article Title: Differential Antagonism of Human Innate Immune Responses by Tick-Borne Phlebovirus Nonstructural Proteins

    Journal: mSphere

    doi: 10.1128/mSphere.00234-17

    HRTV and SFTSV NSs, but not UUKV NSs, inhibits JAK/STAT IFN signaling. (A) ISRE reporter assay in the presence of tick-borne Phlebovirus NSs proteins. HEK293T cells were transfected with a Firefly luciferase reporter plasmid under the control of an ISRE promoter and a Renilla luciferase control plasmid in the presence of the indicated amounts of untagged or V5-tagged UUKV, HRTV, or SFTSV NSs proteins. The cells were stimulated with IFN-β (500 U/ml) 24 h posttransfection and lysed 18 h later for analysis. Induction was normalized against the induction control, whose results were assigned a value of 100%. The data represent results of three independent experiments performed in duplicate ( n = 3), presented as fold induction means ± SEM. RLU, relative light units. (B) Western blots of transfected cell lysates. (C and D) Cell lysates from HEK293T cells transiently expressing V5-tagged UUKV, HRTV, or SFTSV NSs were immunoprecipitated with an anti-V5 antibody and analyzed by Western blotting with STAT1 and STAT2 antibodies (C) or with a STAT3 antibody (D). (E) Phosphorylation of STAT1 and STAT2 in HEK293T cells upon treatment with recombinant IFN-β. HEK293T cells transiently expressing untagged or V5-tagged UUKV, HRTV, or SFTSV NSs were treated with recombinant IFN-β 24 h posttransfection. Upon 30 min of IFN-β treatment, the cell lysates were harvested and utilized for Western blotting of STAT2, STAT2 phosphorylated at Tyr690, STAT1, STAT1 phosphorylated at Ser727 or Tyr701, actin, and V5 with the appropriate antibodies. (F and G) HeLa cells were transfected with plasmids encoding V5-tagged HRTV or SFTSV NSs. At 24 h posttransfection, the cells were treated with IFN-β (1,000 U/ml) for 30 min, fixed, permeabilized, and probed with (V5) STAT1 (F) and STAT2 (G) antibodies to visualize the subcellular localization of endogenous STATs (red), V5-tagged NSs (green), and DAPI-stained nuclei (blue) by confocal microscopy. Scale bars indicate 20 μm. (H) HEK293T cells transiently expressing untagged or V5-tagged UUKV, HRTV, or SFTSV NSs proteins were treated with 50 ng IFN-γ. At 24 h posttreatment, total cellular RNA was isolated and subjected to RT-qPCR to examine mRNA levels of IP-10 and CXCL10. Fold increase was derived by normalizing relative mRNA levels of the target to GAPDH mRNA levels using a ΔΔ CT method.
    Figure Legend Snippet: HRTV and SFTSV NSs, but not UUKV NSs, inhibits JAK/STAT IFN signaling. (A) ISRE reporter assay in the presence of tick-borne Phlebovirus NSs proteins. HEK293T cells were transfected with a Firefly luciferase reporter plasmid under the control of an ISRE promoter and a Renilla luciferase control plasmid in the presence of the indicated amounts of untagged or V5-tagged UUKV, HRTV, or SFTSV NSs proteins. The cells were stimulated with IFN-β (500 U/ml) 24 h posttransfection and lysed 18 h later for analysis. Induction was normalized against the induction control, whose results were assigned a value of 100%. The data represent results of three independent experiments performed in duplicate ( n = 3), presented as fold induction means ± SEM. RLU, relative light units. (B) Western blots of transfected cell lysates. (C and D) Cell lysates from HEK293T cells transiently expressing V5-tagged UUKV, HRTV, or SFTSV NSs were immunoprecipitated with an anti-V5 antibody and analyzed by Western blotting with STAT1 and STAT2 antibodies (C) or with a STAT3 antibody (D). (E) Phosphorylation of STAT1 and STAT2 in HEK293T cells upon treatment with recombinant IFN-β. HEK293T cells transiently expressing untagged or V5-tagged UUKV, HRTV, or SFTSV NSs were treated with recombinant IFN-β 24 h posttransfection. Upon 30 min of IFN-β treatment, the cell lysates were harvested and utilized for Western blotting of STAT2, STAT2 phosphorylated at Tyr690, STAT1, STAT1 phosphorylated at Ser727 or Tyr701, actin, and V5 with the appropriate antibodies. (F and G) HeLa cells were transfected with plasmids encoding V5-tagged HRTV or SFTSV NSs. At 24 h posttransfection, the cells were treated with IFN-β (1,000 U/ml) for 30 min, fixed, permeabilized, and probed with (V5) STAT1 (F) and STAT2 (G) antibodies to visualize the subcellular localization of endogenous STATs (red), V5-tagged NSs (green), and DAPI-stained nuclei (blue) by confocal microscopy. Scale bars indicate 20 μm. (H) HEK293T cells transiently expressing untagged or V5-tagged UUKV, HRTV, or SFTSV NSs proteins were treated with 50 ng IFN-γ. At 24 h posttreatment, total cellular RNA was isolated and subjected to RT-qPCR to examine mRNA levels of IP-10 and CXCL10. Fold increase was derived by normalizing relative mRNA levels of the target to GAPDH mRNA levels using a ΔΔ CT method.

    Techniques Used: Reporter Assay, Transfection, Luciferase, Plasmid Preparation, Western Blot, Expressing, Immunoprecipitation, Recombinant, Staining, Confocal Microscopy, Isolation, Quantitative RT-PCR, Derivative Assay

    UUKV NSs interacts with MAVS. (A) HEK293T cells were cotransfected with a V5-tagged UUKV NSs-encoding plasmid along with FLAG-tagged RIG-I (N) MAVS, TBK1, IKKε, or untagged IRF3-5D-encoding plasmids. Transfected cell lysates were subjected to coimmunoprecipitation (co-IP) with beads conjugated to FLAG or IRF3 antibodies. V5-tagged UUKV NSs was detected through Western blotting with an anti-V5 antibody. (B) Reverse co-IP. HEK293T cells were mock transfected or transfected with V5-tagged UUKV NSs and FLAG-tagged RIG-I N (left panel) or MAVS (right panel). Cell lysates were subjected to co-IP with an anti-V5 antibody. FLAG-tagged RIG-I N and MAVS in the co-IP eluates were detected by Western blotting with an anti-FLAG antibody. (C) co-IP of UUKV NSs upon UUKV infection in the presence of FLAG-tagged RIG-I N or MAVS. HEK293T cells were mock infected or infected with UUKV at a high MOI (20 FFU/cell) for 8 h, followed by mock transfection or transfection of FLAG-tagged RIG-I N-encoding plasmids (left panel) or MAVS-encoding plasmids (right panel). At 30 h p.i., the cell lysates were subjected to co-IP using an anti-UUKV NSs antibody, followed by detection of FLAG-tagged RIG-I N or MAVS and UUKV NSs by Western blotting. (D) Subcellular localization of UUKV NSs-V5 and FLAG-tagged MAVS. HEK293T cells were mock transfected or transfected with UUKV NSs-V5 or FLAG-tagged MAVS or both. At 24 h posttransfection, the cells were fixed, permeabilized, and probed with V5 (red) and FLAG (green) antibodies. DAPI-stained nuclei (blue) and subcellular localization of the proteins were analyzed by confocal microscopy. Intensity profile graphs are shown at the bottom of the image ( x axis, distance [in micrometers]; y axis, intensity). Scale bars indicate 10 μm. IP, immunoprecipitation; WB, Western blotting; WCL, whole-cell lysate.
    Figure Legend Snippet: UUKV NSs interacts with MAVS. (A) HEK293T cells were cotransfected with a V5-tagged UUKV NSs-encoding plasmid along with FLAG-tagged RIG-I (N) MAVS, TBK1, IKKε, or untagged IRF3-5D-encoding plasmids. Transfected cell lysates were subjected to coimmunoprecipitation (co-IP) with beads conjugated to FLAG or IRF3 antibodies. V5-tagged UUKV NSs was detected through Western blotting with an anti-V5 antibody. (B) Reverse co-IP. HEK293T cells were mock transfected or transfected with V5-tagged UUKV NSs and FLAG-tagged RIG-I N (left panel) or MAVS (right panel). Cell lysates were subjected to co-IP with an anti-V5 antibody. FLAG-tagged RIG-I N and MAVS in the co-IP eluates were detected by Western blotting with an anti-FLAG antibody. (C) co-IP of UUKV NSs upon UUKV infection in the presence of FLAG-tagged RIG-I N or MAVS. HEK293T cells were mock infected or infected with UUKV at a high MOI (20 FFU/cell) for 8 h, followed by mock transfection or transfection of FLAG-tagged RIG-I N-encoding plasmids (left panel) or MAVS-encoding plasmids (right panel). At 30 h p.i., the cell lysates were subjected to co-IP using an anti-UUKV NSs antibody, followed by detection of FLAG-tagged RIG-I N or MAVS and UUKV NSs by Western blotting. (D) Subcellular localization of UUKV NSs-V5 and FLAG-tagged MAVS. HEK293T cells were mock transfected or transfected with UUKV NSs-V5 or FLAG-tagged MAVS or both. At 24 h posttransfection, the cells were fixed, permeabilized, and probed with V5 (red) and FLAG (green) antibodies. DAPI-stained nuclei (blue) and subcellular localization of the proteins were analyzed by confocal microscopy. Intensity profile graphs are shown at the bottom of the image ( x axis, distance [in micrometers]; y axis, intensity). Scale bars indicate 10 μm. IP, immunoprecipitation; WB, Western blotting; WCL, whole-cell lysate.

    Techniques Used: Plasmid Preparation, Transfection, Co-Immunoprecipitation Assay, Western Blot, Infection, Staining, Confocal Microscopy, Immunoprecipitation

    HRTV NSs interacts with TBK1. (A) HEK293T cells were cotransfected with a V5-tagged HRTV NSs-encoding plasmid along with FLAG-tagged RIG-I (N), MAVS, TBK1, or IKKε or untagged IRF3-5D-encoding plasmids. Transfected lysates were subjected to coimmunoprecipitation with beads conjugated to FLAG or IRF3 antibodies. V5-tagged HRTV NSs was detected through Western blotting with an anti-V5 antibody. (B) Immunoprecipitation of HRTV NSs in infected cells. Cell lysates of A549 cells infected with HRTV (MOI 10 FFU/cell) were subjected to immunoprecipitation with an anti-HRTV NSs antibody. Western blotting was performed on IP eluates to detect the presence of TBK1 and HRTV NSs in HRTV-infected cells. (C) HEK293T cells were cotransfected with plasmids expressing untagged or V5-tagged HRTV or SFTSV NSs and FLAG-tagged TBK1 to induce interferon induction. At 24 h posttransfection, cell lysates were harvested and utilized for Western blotting of TBK1, TBK1 phosphorylated at Ser172, tubulin, and V5 with the appropriate antibodies. WB, Western blot. (D) Indirect immunofluorescent staining of HEK293T cells transiently expressing TBK1-FLAG and V5-tagged HRTV or SFTSV NSs and probed with anti-TBK1 and anti-V5 antibodies 24 h posttransfection. Subcellular localization of NSs proteins (red), TBK1 (green), and nuclei stained with DAPI (blue) was analyzed by confocal microscopy. Intensity profile graphs are shown to the right of the image. Scale bars indicate 20 μm.
    Figure Legend Snippet: HRTV NSs interacts with TBK1. (A) HEK293T cells were cotransfected with a V5-tagged HRTV NSs-encoding plasmid along with FLAG-tagged RIG-I (N), MAVS, TBK1, or IKKε or untagged IRF3-5D-encoding plasmids. Transfected lysates were subjected to coimmunoprecipitation with beads conjugated to FLAG or IRF3 antibodies. V5-tagged HRTV NSs was detected through Western blotting with an anti-V5 antibody. (B) Immunoprecipitation of HRTV NSs in infected cells. Cell lysates of A549 cells infected with HRTV (MOI 10 FFU/cell) were subjected to immunoprecipitation with an anti-HRTV NSs antibody. Western blotting was performed on IP eluates to detect the presence of TBK1 and HRTV NSs in HRTV-infected cells. (C) HEK293T cells were cotransfected with plasmids expressing untagged or V5-tagged HRTV or SFTSV NSs and FLAG-tagged TBK1 to induce interferon induction. At 24 h posttransfection, cell lysates were harvested and utilized for Western blotting of TBK1, TBK1 phosphorylated at Ser172, tubulin, and V5 with the appropriate antibodies. WB, Western blot. (D) Indirect immunofluorescent staining of HEK293T cells transiently expressing TBK1-FLAG and V5-tagged HRTV or SFTSV NSs and probed with anti-TBK1 and anti-V5 antibodies 24 h posttransfection. Subcellular localization of NSs proteins (red), TBK1 (green), and nuclei stained with DAPI (blue) was analyzed by confocal microscopy. Intensity profile graphs are shown to the right of the image. Scale bars indicate 20 μm.

    Techniques Used: Plasmid Preparation, Transfection, Western Blot, Immunoprecipitation, Infection, Expressing, Staining, Confocal Microscopy

    Subcellular localization of UUKV, HRTV, and SFTSV NSs. The subcellular localization of UUKV (A), HRTV (B), or SFTSV (C) NSs proteins was analyzed by confocal microscopy. The indicated cell lines were infected with UUKV, HRTV, and SFTSV at an MOI of 3 FFU/cell (or were mock infected), fixed at 24 h p.i., permeabilized, and probed with the appropriate NSs antibody. NSs proteins (green) and cell nuclei (blue) were visualized by confocal microscopy. (D) The subcellular localization of V5-tagged NSs proteins was also investigated. HEK293T cells were transfected with plasmids encoding UUKV, HRTV, or SFTSV NSs proteins tagged at their C terminus with a V5 tag. At 24 h posttransfection, cells were fixed, permeabilized, and probed with an anti-V5 antibody. DAPI-stained nuclei (blue) and the V5-tagged NSs proteins (red) were visualized by confocal microscopy. Scale bars indicate 20 μm.
    Figure Legend Snippet: Subcellular localization of UUKV, HRTV, and SFTSV NSs. The subcellular localization of UUKV (A), HRTV (B), or SFTSV (C) NSs proteins was analyzed by confocal microscopy. The indicated cell lines were infected with UUKV, HRTV, and SFTSV at an MOI of 3 FFU/cell (or were mock infected), fixed at 24 h p.i., permeabilized, and probed with the appropriate NSs antibody. NSs proteins (green) and cell nuclei (blue) were visualized by confocal microscopy. (D) The subcellular localization of V5-tagged NSs proteins was also investigated. HEK293T cells were transfected with plasmids encoding UUKV, HRTV, or SFTSV NSs proteins tagged at their C terminus with a V5 tag. At 24 h posttransfection, cells were fixed, permeabilized, and probed with an anti-V5 antibody. DAPI-stained nuclei (blue) and the V5-tagged NSs proteins (red) were visualized by confocal microscopy. Scale bars indicate 20 μm.

    Techniques Used: Confocal Microscopy, Infection, Transfection, Staining

    18) Product Images from "Disease Tolerance Mediated by Phosphorylated Indoleamine-2,3 Dioxygenase Confers Resistance to a Primary Fungal Pathogen"

    Article Title: Disease Tolerance Mediated by Phosphorylated Indoleamine-2,3 Dioxygenase Confers Resistance to a Primary Fungal Pathogen

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01522

    Indoleamine-2,3 dioxygenase (IDO) is regulated by TGF-β signaling in A/J dendritic cells (DCs). 1-Methyl- dl -tryptophan (1MT)-treated or -untreated B10.A and A/J mice were infected i.t. with 1 × 10 6 Paracoccidiodes brasiliensis yeasts and 2 weeks after infection total lung inflammatory cells were obtained and DCs purified by anti-CD11c magnetic beads. (A) The relative expression of mRNA of TGF-β, Smad2, Smad 3, SHIP, SHP-1, SHP-2, IFN-α, and IFN-β was measured by real-time PCR. (B) Smad2, Ship, Shp-2, IKK-α and IDO1, and pIDO protein expression was assessed by western blot in supernatants of lysed DCs. (C) Proteins were estimated by analyzing the intensity of each band normalized by β-tubulin, used as control. Densitometry of bands was performed using ImageQuant TL 8.1 software. Values are the mean ± SEM of three independent experiments; the asterisks represent statistically significant differences between treatments (* p
    Figure Legend Snippet: Indoleamine-2,3 dioxygenase (IDO) is regulated by TGF-β signaling in A/J dendritic cells (DCs). 1-Methyl- dl -tryptophan (1MT)-treated or -untreated B10.A and A/J mice were infected i.t. with 1 × 10 6 Paracoccidiodes brasiliensis yeasts and 2 weeks after infection total lung inflammatory cells were obtained and DCs purified by anti-CD11c magnetic beads. (A) The relative expression of mRNA of TGF-β, Smad2, Smad 3, SHIP, SHP-1, SHP-2, IFN-α, and IFN-β was measured by real-time PCR. (B) Smad2, Ship, Shp-2, IKK-α and IDO1, and pIDO protein expression was assessed by western blot in supernatants of lysed DCs. (C) Proteins were estimated by analyzing the intensity of each band normalized by β-tubulin, used as control. Densitometry of bands was performed using ImageQuant TL 8.1 software. Values are the mean ± SEM of three independent experiments; the asterisks represent statistically significant differences between treatments (* p

    Techniques Used: Mouse Assay, Infection, Purification, Magnetic Beads, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software

    Indoleamine-2,3 dioxygenase (IDO) is regulated by IFN-γ signaling in B10.A dendritic cells (DCs). 1-Methyl- dl -tryptophan (1MT) treated or untreated B10.A and A/J mice were infected i.t. with 1 × 10 6 yeast cells of P. brasiliensis , and 2 weeks after infection total lung inflammatory cells were obtained and DCs purified with anti-CD11c magnetic beads. (A) The relative expression of mRNA of IFN-γ, Janus kinase 1 (Jak1), signal transducer and activator of transcription (STAT1), IL-6, suppressor of cytokine signaling 3 (SOCS3), and nitric oxide synthase (NOS2) was measured by real-time PCR. (B) Presence of Jak1, Stat1, IKK-β, and IDO1 proteins was assessed by western blot in supernatants of lysed DCs. Proteins were estimated by analyzing the intensity of each band normalized by β-tubulin, used as control. (C) Densitometry of bands was performed using ImageQuant TL 8.1 software. The asterisks represent statistically significant differences between treatments (* p
    Figure Legend Snippet: Indoleamine-2,3 dioxygenase (IDO) is regulated by IFN-γ signaling in B10.A dendritic cells (DCs). 1-Methyl- dl -tryptophan (1MT) treated or untreated B10.A and A/J mice were infected i.t. with 1 × 10 6 yeast cells of P. brasiliensis , and 2 weeks after infection total lung inflammatory cells were obtained and DCs purified with anti-CD11c magnetic beads. (A) The relative expression of mRNA of IFN-γ, Janus kinase 1 (Jak1), signal transducer and activator of transcription (STAT1), IL-6, suppressor of cytokine signaling 3 (SOCS3), and nitric oxide synthase (NOS2) was measured by real-time PCR. (B) Presence of Jak1, Stat1, IKK-β, and IDO1 proteins was assessed by western blot in supernatants of lysed DCs. Proteins were estimated by analyzing the intensity of each band normalized by β-tubulin, used as control. (C) Densitometry of bands was performed using ImageQuant TL 8.1 software. The asterisks represent statistically significant differences between treatments (* p

    Techniques Used: Mouse Assay, Infection, Purification, Magnetic Beads, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software

    19) Product Images from "The Peptidylarginine Deiminase Inhibitor Cl-Amidine Suppresses Inducible Nitric Oxide Synthase Expression in Dendritic Cells"

    Article Title: The Peptidylarginine Deiminase Inhibitor Cl-Amidine Suppresses Inducible Nitric Oxide Synthase Expression in Dendritic Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18112258

    Mild suppression of COX-2 expression and PGE 2 production by Cl-amidine treatment. ( A ) LPS (0.1 μg/mL)-treated or -untreated DCs (1 × 10 7 cells) were cultured in the absence or presence of Cl-amidine (50, 100, and 200 μM) for 24 h. COX-2 expression was measured by Western blotting with an anti-COX-2 antibody. β-actin was used as the loading control. ( B ) Columns represent the relative intensity of COX-2 from three independent experiments. ( C ) DCs (1 × 10 5 cells) were stimulated with LPS (0.1 μg/mL), with or without Cl-amidine (50, 100, or 200 μM), for 24 h. PGE 2 production was measured as described in the Materials and Methods (mean ± SD; n = 3). * p
    Figure Legend Snippet: Mild suppression of COX-2 expression and PGE 2 production by Cl-amidine treatment. ( A ) LPS (0.1 μg/mL)-treated or -untreated DCs (1 × 10 7 cells) were cultured in the absence or presence of Cl-amidine (50, 100, and 200 μM) for 24 h. COX-2 expression was measured by Western blotting with an anti-COX-2 antibody. β-actin was used as the loading control. ( B ) Columns represent the relative intensity of COX-2 from three independent experiments. ( C ) DCs (1 × 10 5 cells) were stimulated with LPS (0.1 μg/mL), with or without Cl-amidine (50, 100, or 200 μM), for 24 h. PGE 2 production was measured as described in the Materials and Methods (mean ± SD; n = 3). * p

    Techniques Used: Expressing, Cell Culture, Western Blot

    20) Product Images from "Protective Effect of Panax notoginseng Root Water Extract against Influenza A Virus Infection by Enhancing Antiviral Interferon-Mediated Immune Responses and Natural Killer Cell Activity"

    Article Title: Protective Effect of Panax notoginseng Root Water Extract against Influenza A Virus Infection by Enhancing Antiviral Interferon-Mediated Immune Responses and Natural Killer Cell Activity

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01542

    Induction of pro-inflammatory cytokines and activation of type-I interferon (IFN) by Panax notoginseng root (PNR) in vitro . (A–C) RAW 264.7 cells were treated with DMEM containing 10% fetal bovine serum (FBS) alone, 1,000 U/mL recombinant mouse IFN-β, or 100 µg/mL PNR and incubated at 37°C and 5% CO 2 . Supernatant from each group was harvested at 0 and 24 h and clarified by centrifugation at 2,500 g for 10 min at 4°C. Clarified supernatants were dispensed into murine interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IFN-β capture antibody-coated enzyme-linked immunosorbent assay plates to measure cytokine secretion. The test was performed in duplicate. (D) Western blotting was performed using the whole-cell lysates of macrophage-type cells treated with or without PNR (1, 10, and 100 µg/mL) to assess the expression of the nonphosphorylated and phosphorylated forms of IRF3, TANK-binding kinase 1 (TBK1), STAT1, and β-actin over time. Similar results were obtained, and the experiment was performed three times independently. Bar graph (mean ± SEM) statistics were determined using two-way ANOVA with Bonferroni’s correction (posttest), *** P
    Figure Legend Snippet: Induction of pro-inflammatory cytokines and activation of type-I interferon (IFN) by Panax notoginseng root (PNR) in vitro . (A–C) RAW 264.7 cells were treated with DMEM containing 10% fetal bovine serum (FBS) alone, 1,000 U/mL recombinant mouse IFN-β, or 100 µg/mL PNR and incubated at 37°C and 5% CO 2 . Supernatant from each group was harvested at 0 and 24 h and clarified by centrifugation at 2,500 g for 10 min at 4°C. Clarified supernatants were dispensed into murine interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IFN-β capture antibody-coated enzyme-linked immunosorbent assay plates to measure cytokine secretion. The test was performed in duplicate. (D) Western blotting was performed using the whole-cell lysates of macrophage-type cells treated with or without PNR (1, 10, and 100 µg/mL) to assess the expression of the nonphosphorylated and phosphorylated forms of IRF3, TANK-binding kinase 1 (TBK1), STAT1, and β-actin over time. Similar results were obtained, and the experiment was performed three times independently. Bar graph (mean ± SEM) statistics were determined using two-way ANOVA with Bonferroni’s correction (posttest), *** P

    Techniques Used: Activation Assay, In Vitro, Recombinant, Incubation, Centrifugation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Binding Assay

    21) Product Images from "Signal Transducer and Activator of Transcription-3 Maintains the Stemness of Radial Glia at Mid-Neurogenesis"

    Article Title: Signal Transducer and Activator of Transcription-3 Maintains the Stemness of Radial Glia at Mid-Neurogenesis

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2119-14.2015

    STAT3 expression in mouse embryonic brains. A , Expression of STAT3, STAT1, nestin, Sox2, GFAP, and α-tubulin in embryonic mouse brains assessed by Western blotting. The asterisk marks a splice variant of STAT1. B , C , STAT3 is expressed in the VZ of E10.5 brains marked by Nestin. D–H , At E14.5, Stat3 transcripts and protein are located in the VZ in which Sox2 and Pax6 are expressed. The IPC marker Tbr2 is more broadly expressed. I , STAT3 expression (red) partially overlaps with that of nestin (green) in E13.5 brains. J , Higher-magnification views of I . K–O , STAT3 protein or STAT3 reporter expression is merged with Sox2, Pax6, Cux1, Tbr2, or Ki67. P , Percentages of STAT3 reporter-labeled cells that express the indicated markers ( n = 2–4 embryos, n = 3–5 sections per group). Q , Differential expression of CAG–RFP and STAT3 reporter in the coelectroporated cortex. R–U , The SBS8–H2BdGFP reporter and CAG–RFP were transfected into HEK293T cells. The cells were incubated with LIF for 1 d before harvest. The SBS8–H2BdGFP reporter was activated in response to LIF or when STAT3 CA was cotransfected. V–X , oRG cells and phospho-vimentin + cells (green) in the oSVZ do not express STAT3 (red; W ). Mitotic RG labeled by phospho-vimentin on the apical side of the VZ express STAT3 in their nuclei and radial processes ( X , arrows). Y , A schematic diagram summarizing the expression of STAT3 and other markers among progenitors. For simplicity, only asymmetrically dividing radial glial cells are shown. NE, Neuroepithelium; SNP, short neural precursor; N L , lower-layer neurons; N U , upper-layer neurons. Error bars represent SEM. * p
    Figure Legend Snippet: STAT3 expression in mouse embryonic brains. A , Expression of STAT3, STAT1, nestin, Sox2, GFAP, and α-tubulin in embryonic mouse brains assessed by Western blotting. The asterisk marks a splice variant of STAT1. B , C , STAT3 is expressed in the VZ of E10.5 brains marked by Nestin. D–H , At E14.5, Stat3 transcripts and protein are located in the VZ in which Sox2 and Pax6 are expressed. The IPC marker Tbr2 is more broadly expressed. I , STAT3 expression (red) partially overlaps with that of nestin (green) in E13.5 brains. J , Higher-magnification views of I . K–O , STAT3 protein or STAT3 reporter expression is merged with Sox2, Pax6, Cux1, Tbr2, or Ki67. P , Percentages of STAT3 reporter-labeled cells that express the indicated markers ( n = 2–4 embryos, n = 3–5 sections per group). Q , Differential expression of CAG–RFP and STAT3 reporter in the coelectroporated cortex. R–U , The SBS8–H2BdGFP reporter and CAG–RFP were transfected into HEK293T cells. The cells were incubated with LIF for 1 d before harvest. The SBS8–H2BdGFP reporter was activated in response to LIF or when STAT3 CA was cotransfected. V–X , oRG cells and phospho-vimentin + cells (green) in the oSVZ do not express STAT3 (red; W ). Mitotic RG labeled by phospho-vimentin on the apical side of the VZ express STAT3 in their nuclei and radial processes ( X , arrows). Y , A schematic diagram summarizing the expression of STAT3 and other markers among progenitors. For simplicity, only asymmetrically dividing radial glial cells are shown. NE, Neuroepithelium; SNP, short neural precursor; N L , lower-layer neurons; N U , upper-layer neurons. Error bars represent SEM. * p

    Techniques Used: Expressing, Western Blot, Variant Assay, Marker, Labeling, Transfection, Incubation

    22) 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

    23) Product Images from "Hemojuvelin regulates the innate immune response to peritoneal bacterial infection in mice"

    Article Title: Hemojuvelin regulates the innate immune response to peritoneal bacterial infection in mice

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2017.28

    Hjv expression is upregulated in macrophages following stimulation with heat-killed E. coli , and Hjv expression facilitates iNOS signaling in vitro . ( a ) Hjv mRNA was measured in macrophages 0, 1, 3, 6, 12 and 24 h after stimulation with heat-killed E. coli (10 8 CFU) and is expressed normalized to β-actin mRNA, ** P
    Figure Legend Snippet: Hjv expression is upregulated in macrophages following stimulation with heat-killed E. coli , and Hjv expression facilitates iNOS signaling in vitro . ( a ) Hjv mRNA was measured in macrophages 0, 1, 3, 6, 12 and 24 h after stimulation with heat-killed E. coli (10 8 CFU) and is expressed normalized to β-actin mRNA, ** P

    Techniques Used: Expressing, In Vitro

    24) Product Images from "The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis"

    Article Title: The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis

    Journal: Cellular Signalling

    doi: 10.1016/j.cellsig.2019.03.024

    Immunohistochemical analysis of IFITM1/3 protein expression in cervical cancers using the Mab-MHK. Formalin-fixed, paraffin-embedded cervical carcinoma tissue was processed as stated in the Experimental Procedures using the Mab-MHK that binds to shared epitopes in the N-terminal domains of IFITM1 and IFITM3 (Supplementary Fig. 1A, B). Representative images highlight grades of IFITM1/3 protein expression; (A). high expression in squamous cell carcinoma, (B). medium expression in squamous cell carcinoma, (C). absence of expression in squamous cell carcinoma, (D). high expression in adenocarcinoma, and (E). expression in basal stem cell layer in normal tissue . (F). (Top panel); Quantification of IFITM1/3 protein expression in cervical cancer in relation to histology type and (Bottom panel) IFITM1/3 protein expression in cervical cancer in relation to lymph node metastasis, excluding neuroendocrine. (G). Immunoblotting of protein expression in the parental SiHa cells after IFNγ treatment for the indicated time points. Proteins evaluated include; STAT1; β-actin, IRF1, and IFITM1/3.
    Figure Legend Snippet: Immunohistochemical analysis of IFITM1/3 protein expression in cervical cancers using the Mab-MHK. Formalin-fixed, paraffin-embedded cervical carcinoma tissue was processed as stated in the Experimental Procedures using the Mab-MHK that binds to shared epitopes in the N-terminal domains of IFITM1 and IFITM3 (Supplementary Fig. 1A, B). Representative images highlight grades of IFITM1/3 protein expression; (A). high expression in squamous cell carcinoma, (B). medium expression in squamous cell carcinoma, (C). absence of expression in squamous cell carcinoma, (D). high expression in adenocarcinoma, and (E). expression in basal stem cell layer in normal tissue . (F). (Top panel); Quantification of IFITM1/3 protein expression in cervical cancer in relation to histology type and (Bottom panel) IFITM1/3 protein expression in cervical cancer in relation to lymph node metastasis, excluding neuroendocrine. (G). Immunoblotting of protein expression in the parental SiHa cells after IFNγ treatment for the indicated time points. Proteins evaluated include; STAT1; β-actin, IRF1, and IFITM1/3.

    Techniques Used: Immunohistochemistry, Expressing, Formalin-fixed Paraffin-Embedded

    Validation of the IFITM3 guide RNA. (A). Gene structure of the IFITM3 gene highlighting its two coding exons and one intron. The genomic region surrounding the gRNA target site (with PAM site and homology region in red) for IFITM3 was amplified and subjected to DNA sequencing. The raw DNA sequencing reads in the IFITM3 gene are highlighted in (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) The IFITM3 gene has two insertions that result in a poly-proline frame-shift creating a stop codon (C). (D). Immunoblotting was performed using Mab-MHK with lysates from SiHa cells (lanes 1 and 2); IFITM1 single null cells (lanes 3 and 4); and IFITM1/IFITM3 double null cells (lanes 5 and 6). The cells were either untreated or treated with IFNγ (100 ng/ml) for 24 h and processed for immunoblotting with either a β-actin antibody or Mab-MHK.
    Figure Legend Snippet: Validation of the IFITM3 guide RNA. (A). Gene structure of the IFITM3 gene highlighting its two coding exons and one intron. The genomic region surrounding the gRNA target site (with PAM site and homology region in red) for IFITM3 was amplified and subjected to DNA sequencing. The raw DNA sequencing reads in the IFITM3 gene are highlighted in (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) The IFITM3 gene has two insertions that result in a poly-proline frame-shift creating a stop codon (C). (D). Immunoblotting was performed using Mab-MHK with lysates from SiHa cells (lanes 1 and 2); IFITM1 single null cells (lanes 3 and 4); and IFITM1/IFITM3 double null cells (lanes 5 and 6). The cells were either untreated or treated with IFNγ (100 ng/ml) for 24 h and processed for immunoblotting with either a β-actin antibody or Mab-MHK.

    Techniques Used: Amplification, DNA Sequencing

    Identification of IFITM1 interacting proteins in IFNγ treated cells. (A). SBP vector or SBP-tagged IFITM1 were transfected into parental SiHa cells and treated with IFNγ. Cells were lysed (L) and subjected to immunoprecipitation (PD) and evaluated by immunoblotting with Mab-MHK that can detect untagged or SBP-tagged IFITM1. (B). A 4-quadrant plot showing the relative quantification for binding proteins corresponding to fold change of cells transfected with SBP-IFITM1 versus SBP-empty vector and affinity purified with IFN-γ for 24 h (X-axis) or without IFN-γ stimulation (Y-axis). (C). Table with selected binding proteins detected by mass spectrometry for SBP-IFITM1 enrichment without or with IFN-γ stimulation (from Supplementary Table 3). (D). Cells (parental SiHa; lane 1; or IFITM1/IFITM3 double null (lane 2) were transfected with empty vector (as in 8A) and treated with IFNγ. Samples were processed by immunoblotting with antibodies to ISG15. Free, monomeric ISG15 protein is highlighted, as well as conjugated ISG15. Free ISG15 protein is expressed at similar levels in both cells whilst conjugated ISG15 is attenuated in the IFITM1/IFITM3 double null cell (lane 2 vs 1). This suggests that ISG15ylation protein synthesis is linked to covalent conjugation under these conditions, under conditions where attenuated ISG15 protein synthesis was observed using pulse SILAC ( Fig. 5 ). Antibodies to β-actin and IFITM1/3 proteins were used as loading controls, as indicated. (E). Free and conjugated ISG15 were quantified using ImageJ software. The relative units (in A.U.) define expression as a function of free or conjugated ISG15 in parental and IFITM1/IFITM3 double null cells. The relative change in ISG15 conjugation over free ISG15 was 5.58 A.U. in parental cells. The relative change in ISG15 conjugation over free ISG15 was 3.63 A.U. in IFITM1/3 double null cells.
    Figure Legend Snippet: Identification of IFITM1 interacting proteins in IFNγ treated cells. (A). SBP vector or SBP-tagged IFITM1 were transfected into parental SiHa cells and treated with IFNγ. Cells were lysed (L) and subjected to immunoprecipitation (PD) and evaluated by immunoblotting with Mab-MHK that can detect untagged or SBP-tagged IFITM1. (B). A 4-quadrant plot showing the relative quantification for binding proteins corresponding to fold change of cells transfected with SBP-IFITM1 versus SBP-empty vector and affinity purified with IFN-γ for 24 h (X-axis) or without IFN-γ stimulation (Y-axis). (C). Table with selected binding proteins detected by mass spectrometry for SBP-IFITM1 enrichment without or with IFN-γ stimulation (from Supplementary Table 3). (D). Cells (parental SiHa; lane 1; or IFITM1/IFITM3 double null (lane 2) were transfected with empty vector (as in 8A) and treated with IFNγ. Samples were processed by immunoblotting with antibodies to ISG15. Free, monomeric ISG15 protein is highlighted, as well as conjugated ISG15. Free ISG15 protein is expressed at similar levels in both cells whilst conjugated ISG15 is attenuated in the IFITM1/IFITM3 double null cell (lane 2 vs 1). This suggests that ISG15ylation protein synthesis is linked to covalent conjugation under these conditions, under conditions where attenuated ISG15 protein synthesis was observed using pulse SILAC ( Fig. 5 ). Antibodies to β-actin and IFITM1/3 proteins were used as loading controls, as indicated. (E). Free and conjugated ISG15 were quantified using ImageJ software. The relative units (in A.U.) define expression as a function of free or conjugated ISG15 in parental and IFITM1/IFITM3 double null cells. The relative change in ISG15 conjugation over free ISG15 was 5.58 A.U. in parental cells. The relative change in ISG15 conjugation over free ISG15 was 3.63 A.U. in IFITM1/3 double null cells.

    Techniques Used: Plasmid Preparation, Transfection, Immunoprecipitation, Binding Assay, Affinity Purification, Mass Spectrometry, Conjugation Assay, Software, Expressing

    25) Product Images from "Interferons Transcriptionally Up-Regulate MLKL Expression in Cancer Cells"

    Article Title: Interferons Transcriptionally Up-Regulate MLKL Expression in Cancer Cells

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.11.002

    Type I and type II IFNs increase MLKL expression in different cancer cell lines. EFM-192A cells were treated with 1.5 ng/ml IFNα, β or γ for 24 hours (A), HeLa cells (B), MV4–11 (C) and MDA-MB-231 cells (D) were treated with 20 ng/ml IFNα, β or γ for 24 hours. Protein expression of MLKL, STAT1 and β-Actin was analyzed by Western blotting. (E and F) Western blots in A and B were quantified, and protein levels of MLKL are shown normalized to β-Actin expression of at least three independent experiments.
    Figure Legend Snippet: Type I and type II IFNs increase MLKL expression in different cancer cell lines. EFM-192A cells were treated with 1.5 ng/ml IFNα, β or γ for 24 hours (A), HeLa cells (B), MV4–11 (C) and MDA-MB-231 cells (D) were treated with 20 ng/ml IFNα, β or γ for 24 hours. Protein expression of MLKL, STAT1 and β-Actin was analyzed by Western blotting. (E and F) Western blots in A and B were quantified, and protein levels of MLKL are shown normalized to β-Actin expression of at least three independent experiments.

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot

    Caspase activity is dispensable for IFNγ-induced MLKL expression. EFM-192A cells were treated with 1.5 ng/ml IFNγ and/or 20 μM zVAD.fmk for 24 hours. Protein expression of MLKL, phospho-STAT1 (pSTAT1), STAT1 and β-Actin was analyzed by Western blotting.
    Figure Legend Snippet: Caspase activity is dispensable for IFNγ-induced MLKL expression. EFM-192A cells were treated with 1.5 ng/ml IFNγ and/or 20 μM zVAD.fmk for 24 hours. Protein expression of MLKL, phospho-STAT1 (pSTAT1), STAT1 and β-Actin was analyzed by Western blotting.

    Techniques Used: Activity Assay, Expressing, Western Blot

    Inhibition of transcription prevents IFNγ-induced MLKL expression. (A) EFM-192A cells were treated with 1.5 ng/ml IFNγ for indicated time points with or without pretreatment with 100 nM Actinomycin D for 2 hours. Protein expression of MLKL, IRF1, pSTAT1, STAT1 and β-Actin was analyzed by Western blotting after indicated time points. (B) mRNA levels of MLKL were quantified via RT-PCR 9 hours after IFNγ treatment and are shown as fold increase to untreated control cells with mean and SEM of at least three independent experiments performed in duplicate; * P
    Figure Legend Snippet: Inhibition of transcription prevents IFNγ-induced MLKL expression. (A) EFM-192A cells were treated with 1.5 ng/ml IFNγ for indicated time points with or without pretreatment with 100 nM Actinomycin D for 2 hours. Protein expression of MLKL, IRF1, pSTAT1, STAT1 and β-Actin was analyzed by Western blotting after indicated time points. (B) mRNA levels of MLKL were quantified via RT-PCR 9 hours after IFNγ treatment and are shown as fold increase to untreated control cells with mean and SEM of at least three independent experiments performed in duplicate; * P

    Techniques Used: Inhibition, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

    26) Product Images from "ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response"

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05559-w

    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
    Figure Legend Snippet: ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

    Techniques Used: Transfection, Incubation, In Vitro, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction

    27) Product Images from "HDAC Inhibitor-Mediated Beta-Cell Protection Against Cytokine-Induced Toxicity Is STAT1 Tyr701 Phosphorylation Independent"

    Article Title: HDAC Inhibitor-Mediated Beta-Cell Protection Against Cytokine-Induced Toxicity Is STAT1 Tyr701 Phosphorylation Independent

    Journal: Journal of Interferon & Cytokine Research

    doi: 10.1089/jir.2014.0022

    HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation or STAT1 acetylation, but knockdown of HDAC1 increases IFN-γ-induced Tyr701 STAT1 phosphorylation. (A) INS-1 cells were preincubated (24 h) with givinostat
    Figure Legend Snippet: HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation or STAT1 acetylation, but knockdown of HDAC1 increases IFN-γ-induced Tyr701 STAT1 phosphorylation. (A) INS-1 cells were preincubated (24 h) with givinostat

    Techniques Used: Inhibition

    Interferon (IFN)-γ-induced Cxcl9 and iNos mRNA expression is reduced by histone deacetylase (HDAC) inhibition. INS-1 cells were preincubated with givinostat (125 nM) or vehicle (1 h) before exposure to IFN-γ at either a
    Figure Legend Snippet: Interferon (IFN)-γ-induced Cxcl9 and iNos mRNA expression is reduced by histone deacetylase (HDAC) inhibition. INS-1 cells were preincubated with givinostat (125 nM) or vehicle (1 h) before exposure to IFN-γ at either a

    Techniques Used: Expressing, Histone Deacetylase Assay, Inhibition

    IFN-γ induces Tyr701 STAT1 phosphorylation in a time- and dose-dependent manner. INS-1 cells were exposed to IFN-γ at either a low (0.1 ng/mL) or high (1.3 ng/mL) concentration. Cells were lysed at the indicated times and
    Figure Legend Snippet: IFN-γ induces Tyr701 STAT1 phosphorylation in a time- and dose-dependent manner. INS-1 cells were exposed to IFN-γ at either a low (0.1 ng/mL) or high (1.3 ng/mL) concentration. Cells were lysed at the indicated times and

    Techniques Used: Concentration Assay

    Nonselective HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation. INS-1 cells were preincubated (1 h) with givinostat (125 nM) before exposure to IFN-γ (1.3 ng/mL) for 15 or 30 min
    Figure Legend Snippet: Nonselective HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation. INS-1 cells were preincubated (1 h) with givinostat (125 nM) before exposure to IFN-γ (1.3 ng/mL) for 15 or 30 min

    Techniques Used: Inhibition

    28) Product Images from "Interferons Transcriptionally Up-Regulate MLKL Expression in Cancer Cells"

    Article Title: Interferons Transcriptionally Up-Regulate MLKL Expression in Cancer Cells

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.11.002

    Type I and type II IFNs increase MLKL expression in different cancer cell lines. EFM-192A cells were treated with 1.5 ng/ml IFNα, β or γ for 24 hours (A), HeLa cells (B), MV4–11 (C) and MDA-MB-231 cells (D) were treated with 20 ng/ml IFNα, β or γ for 24 hours. Protein expression of MLKL, STAT1 and β-Actin was analyzed by Western blotting. (E and F) Western blots in A and B were quantified, and protein levels of MLKL are shown normalized to β-Actin expression of at least three independent experiments.
    Figure Legend Snippet: Type I and type II IFNs increase MLKL expression in different cancer cell lines. EFM-192A cells were treated with 1.5 ng/ml IFNα, β or γ for 24 hours (A), HeLa cells (B), MV4–11 (C) and MDA-MB-231 cells (D) were treated with 20 ng/ml IFNα, β or γ for 24 hours. Protein expression of MLKL, STAT1 and β-Actin was analyzed by Western blotting. (E and F) Western blots in A and B were quantified, and protein levels of MLKL are shown normalized to β-Actin expression of at least three independent experiments.

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot

    Caspase activity is dispensable for IFNγ-induced MLKL expression. EFM-192A cells were treated with 1.5 ng/ml IFNγ and/or 20 μM zVAD.fmk for 24 hours. Protein expression of MLKL, phospho-STAT1 (pSTAT1), STAT1 and β-Actin was analyzed by Western blotting.
    Figure Legend Snippet: Caspase activity is dispensable for IFNγ-induced MLKL expression. EFM-192A cells were treated with 1.5 ng/ml IFNγ and/or 20 μM zVAD.fmk for 24 hours. Protein expression of MLKL, phospho-STAT1 (pSTAT1), STAT1 and β-Actin was analyzed by Western blotting.

    Techniques Used: Activity Assay, Expressing, Western Blot

    Inhibition of transcription prevents IFNγ-induced MLKL expression. (A) EFM-192A cells were treated with 1.5 ng/ml IFNγ for indicated time points with or without pretreatment with 100 nM Actinomycin D for 2 hours. Protein expression of MLKL, IRF1, pSTAT1, STAT1 and β-Actin was analyzed by Western blotting after indicated time points. (B) mRNA levels of MLKL were quantified via RT-PCR 9 hours after IFNγ treatment and are shown as fold increase to untreated control cells with mean and SEM of at least three independent experiments performed in duplicate; * P
    Figure Legend Snippet: Inhibition of transcription prevents IFNγ-induced MLKL expression. (A) EFM-192A cells were treated with 1.5 ng/ml IFNγ for indicated time points with or without pretreatment with 100 nM Actinomycin D for 2 hours. Protein expression of MLKL, IRF1, pSTAT1, STAT1 and β-Actin was analyzed by Western blotting after indicated time points. (B) mRNA levels of MLKL were quantified via RT-PCR 9 hours after IFNγ treatment and are shown as fold increase to untreated control cells with mean and SEM of at least three independent experiments performed in duplicate; * P

    Techniques Used: Inhibition, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

    29) Product Images from "A positive autoregulatory loop of Jak-STAT signaling controls the onset of astrogliogenesis"

    Article Title: A positive autoregulatory loop of Jak-STAT signaling controls the onset of astrogliogenesis

    Journal: Nature neuroscience

    doi: 10.1038/nn1440

    Sequential activation of the Jak-STAT pathway in vivo correlates with the timing of astrogliogenesis. ( a ) A fluorescent image of E15 cortical ventricular area from pNestin-GFP mice, demonstrating enriched GFP expression in the ventricular zone (VZ). ( b,c ) The ventricular zone (green) and non–ventricular zone (non-green) tissues were dissected from different developmental stages (E12, E16 or postnatal day (P) 0 from the pNestin-GFP transgenic mice under a fluorescent dissection microscope. Western blot analyses used a TuJ1 antibody that labels neuronal specific βIII tubulin, and a GFP antibody. β-actin blot indicates the loading control. ( d–f ) Western blot analyses of STAT activation and astrocyte differentiation in vivo at different developmental stages. After incubation with or without LIF (100 ng ml −1 ) for 20 min, lysates of green VZ/SVZ tissues from various developmental stages (E12, E14, P0 and P4) were probed with antibodies against tyrosine-phosphorylated STAT1 or STAT3 ( d,e ), astrocyte marker GFAP ( d ), gp130, LIFRβ ( f ), GAPDH ( e ), and β-actin ( d ). Lysates from P0 or P4 without 20-min LIF treatment were also enriched for active forms of STAT1 and STAT3. ( g ) RT-PCR analysis of gp130, Jak1 and astrocytic markers GFAP and S100β at different developmental stages.
    Figure Legend Snippet: Sequential activation of the Jak-STAT pathway in vivo correlates with the timing of astrogliogenesis. ( a ) A fluorescent image of E15 cortical ventricular area from pNestin-GFP mice, demonstrating enriched GFP expression in the ventricular zone (VZ). ( b,c ) The ventricular zone (green) and non–ventricular zone (non-green) tissues were dissected from different developmental stages (E12, E16 or postnatal day (P) 0 from the pNestin-GFP transgenic mice under a fluorescent dissection microscope. Western blot analyses used a TuJ1 antibody that labels neuronal specific βIII tubulin, and a GFP antibody. β-actin blot indicates the loading control. ( d–f ) Western blot analyses of STAT activation and astrocyte differentiation in vivo at different developmental stages. After incubation with or without LIF (100 ng ml −1 ) for 20 min, lysates of green VZ/SVZ tissues from various developmental stages (E12, E14, P0 and P4) were probed with antibodies against tyrosine-phosphorylated STAT1 or STAT3 ( d,e ), astrocyte marker GFAP ( d ), gp130, LIFRβ ( f ), GAPDH ( e ), and β-actin ( d ). Lysates from P0 or P4 without 20-min LIF treatment were also enriched for active forms of STAT1 and STAT3. ( g ) RT-PCR analysis of gp130, Jak1 and astrocytic markers GFAP and S100β at different developmental stages.

    Techniques Used: Activation Assay, In Vivo, Mouse Assay, Expressing, Transgenic Assay, Dissection, Microscopy, Western Blot, Incubation, Marker, Reverse Transcription Polymerase Chain Reaction

    30) Product Images from "The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis"

    Article Title: The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis

    Journal: Cellular Signalling

    doi: 10.1016/j.cellsig.2019.03.024

    Identification of IFITM1 interacting proteins in IFNγ treated cells. (A). SBP vector or SBP-tagged IFITM1 were transfected into parental SiHa cells and treated with IFNγ. Cells were lysed (L) and subjected to immunoprecipitation (PD) and evaluated by immunoblotting with Mab-MHK that can detect untagged or SBP-tagged IFITM1. (B). A 4-quadrant plot showing the relative quantification for binding proteins corresponding to fold change of cells transfected with SBP-IFITM1 versus SBP-empty vector and affinity purified with IFN-γ for 24 h (X-axis) or without IFN-γ stimulation (Y-axis). (C). Table with selected binding proteins detected by mass spectrometry for SBP-IFITM1 enrichment without or with IFN-γ stimulation (from Supplementary Table 3). (D). Cells (parental SiHa; lane 1; or IFITM1/IFITM3 double null (lane 2) were transfected with empty vector (as in 8A) and treated with IFNγ. Samples were processed by immunoblotting with antibodies to ISG15. Free, monomeric ISG15 protein is highlighted, as well as conjugated ISG15. Free ISG15 protein is expressed at similar levels in both cells whilst conjugated ISG15 is attenuated in the IFITM1/IFITM3 double null cell (lane 2 vs 1). This suggests that ISG15ylation protein synthesis is linked to covalent conjugation under these conditions, under conditions where attenuated ISG15 protein synthesis was observed using pulse SILAC ( Fig. 5 ). Antibodies to β-actin and IFITM1/3 proteins were used as loading controls, as indicated. (E). Free and conjugated ISG15 were quantified using ImageJ software. The relative units (in A.U.) define expression as a function of free or conjugated ISG15 in parental and IFITM1/IFITM3 double null cells. The relative change in ISG15 conjugation over free ISG15 was 5.58 A.U. in parental cells. The relative change in ISG15 conjugation over free ISG15 was 3.63 A.U. in IFITM1/3 double null cells.
    Figure Legend Snippet: Identification of IFITM1 interacting proteins in IFNγ treated cells. (A). SBP vector or SBP-tagged IFITM1 were transfected into parental SiHa cells and treated with IFNγ. Cells were lysed (L) and subjected to immunoprecipitation (PD) and evaluated by immunoblotting with Mab-MHK that can detect untagged or SBP-tagged IFITM1. (B). A 4-quadrant plot showing the relative quantification for binding proteins corresponding to fold change of cells transfected with SBP-IFITM1 versus SBP-empty vector and affinity purified with IFN-γ for 24 h (X-axis) or without IFN-γ stimulation (Y-axis). (C). Table with selected binding proteins detected by mass spectrometry for SBP-IFITM1 enrichment without or with IFN-γ stimulation (from Supplementary Table 3). (D). Cells (parental SiHa; lane 1; or IFITM1/IFITM3 double null (lane 2) were transfected with empty vector (as in 8A) and treated with IFNγ. Samples were processed by immunoblotting with antibodies to ISG15. Free, monomeric ISG15 protein is highlighted, as well as conjugated ISG15. Free ISG15 protein is expressed at similar levels in both cells whilst conjugated ISG15 is attenuated in the IFITM1/IFITM3 double null cell (lane 2 vs 1). This suggests that ISG15ylation protein synthesis is linked to covalent conjugation under these conditions, under conditions where attenuated ISG15 protein synthesis was observed using pulse SILAC ( Fig. 5 ). Antibodies to β-actin and IFITM1/3 proteins were used as loading controls, as indicated. (E). Free and conjugated ISG15 were quantified using ImageJ software. The relative units (in A.U.) define expression as a function of free or conjugated ISG15 in parental and IFITM1/IFITM3 double null cells. The relative change in ISG15 conjugation over free ISG15 was 5.58 A.U. in parental cells. The relative change in ISG15 conjugation over free ISG15 was 3.63 A.U. in IFITM1/3 double null cells.

    Techniques Used: Plasmid Preparation, Transfection, Immunoprecipitation, Binding Assay, Affinity Purification, Mass Spectrometry, Conjugation Assay, Software, Expressing

    31) Product Images from "The interaction between mesenchymal stem cells and steroids during inflammation"

    Article Title: The interaction between mesenchymal stem cells and steroids during inflammation

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.537

    Dex blocked the expression of inflammatory cytokine-induced iNOS and IDO through inhibiting STAT1 phosphorylation. Cultured mouse MSCs or human MSCs were supplemented with the indicated combinations of IFN- γ  and TNF- α  (10 ng/ml each), with or without Dex for 24 h. ( a ) Nitrates were assayed in mouse MSC supernatants. ( b ) IDO mRNA in human MSCs was determined using real-time PCR. ( c ) Cultured mouse MSCs were supplemented with IFN- γ  and TNF- α  (2 ng/ml each), and graded dosages of Dex. STAT1 phosphorylation and iNOS expression at 30 min and 24 h were detected by western blot analysis. ( d ) Similarly, human MSCs were supplemented with IFN- γ  and TNF- α  (0.5 ng/ml each) with or without Dex. STAT1 phosphorylation and IDO expression at 30 min and 24 h were examined. ( e ) Mouse MSCs were stimulated with IFN- γ  and TNF- α  (10 ng/ml each) for 12 h, in the presence of graded doses of Dex. Levels of mRNA for CCL2, CCL5, CXCL9, and CXCL10 were detected and normalized to  β -actin. ( f ) Similarly, CCL2, CXCL9, CXCL10, and CXCL11 mRNA were detected in human MSCs, after stimulation with IFN- γ  and TNF- α  (10 ng/ml each) for 12 h in the presence of graded doses of Dex. Values are shown as mean±S.E.M. Representative of four independent experiments
    Figure Legend Snippet: Dex blocked the expression of inflammatory cytokine-induced iNOS and IDO through inhibiting STAT1 phosphorylation. Cultured mouse MSCs or human MSCs were supplemented with the indicated combinations of IFN- γ and TNF- α (10 ng/ml each), with or without Dex for 24 h. ( a ) Nitrates were assayed in mouse MSC supernatants. ( b ) IDO mRNA in human MSCs was determined using real-time PCR. ( c ) Cultured mouse MSCs were supplemented with IFN- γ and TNF- α (2 ng/ml each), and graded dosages of Dex. STAT1 phosphorylation and iNOS expression at 30 min and 24 h were detected by western blot analysis. ( d ) Similarly, human MSCs were supplemented with IFN- γ and TNF- α (0.5 ng/ml each) with or without Dex. STAT1 phosphorylation and IDO expression at 30 min and 24 h were examined. ( e ) Mouse MSCs were stimulated with IFN- γ and TNF- α (10 ng/ml each) for 12 h, in the presence of graded doses of Dex. Levels of mRNA for CCL2, CCL5, CXCL9, and CXCL10 were detected and normalized to β -actin. ( f ) Similarly, CCL2, CXCL9, CXCL10, and CXCL11 mRNA were detected in human MSCs, after stimulation with IFN- γ and TNF- α (10 ng/ml each) for 12 h in the presence of graded doses of Dex. Values are shown as mean±S.E.M. Representative of four independent experiments

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

    32) Product Images from "STAT1β Is Not Dominant Negative and Is Capable of Contributing to Gamma Interferon-Dependent Innate Immunity"

    Article Title: STAT1β Is Not Dominant Negative and Is Capable of Contributing to Gamma Interferon-Dependent Innate Immunity

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00295-14

    STAT1β shows prolonged tyrosine 701 phosphorylation. (A to D) BMMϕ derived from WT (+/+), Stat1 β/β (β/β), and Stat1 α/α (α/α) mice were stimulated with IFN-β (A) or
    Figure Legend Snippet: STAT1β shows prolonged tyrosine 701 phosphorylation. (A to D) BMMϕ derived from WT (+/+), Stat1 β/β (β/β), and Stat1 α/α (α/α) mice were stimulated with IFN-β (A) or

    Techniques Used: Derivative Assay, Mouse Assay

    STAT1β shows prolonged nuclear localization and prolonged promoter binding after IFN-γ treatment compared to STAT1α. (A) BMMϕ derived from WT (+/+), Stat1 β/β (β/β), and Stat1 α/α
    Figure Legend Snippet: STAT1β shows prolonged nuclear localization and prolonged promoter binding after IFN-γ treatment compared to STAT1α. (A) BMMϕ derived from WT (+/+), Stat1 β/β (β/β), and Stat1 α/α

    Techniques Used: Binding Assay, Derivative Assay

    STAT1α and STAT1β can mediate type I and type III IFN-dependent antiviral immunity in vivo . (A) EMCV (50 PFU/mouse) was administered i.p. to WT ( Stat1 +/+ ), Stat1 β/β , Stat1 α/α , and Stat1 −/−
    Figure Legend Snippet: STAT1α and STAT1β can mediate type I and type III IFN-dependent antiviral immunity in vivo . (A) EMCV (50 PFU/mouse) was administered i.p. to WT ( Stat1 +/+ ), Stat1 β/β , Stat1 α/α , and Stat1 −/−

    Techniques Used: In Vivo

    STAT1β is transcriptionally active in response to IFN-β and IFN-γ. BMMϕ isolated from WT (+/+), Stat1 β/β (β/β), Stat1 α/α (α/α), and Stat1 −/−
    Figure Legend Snippet: STAT1β is transcriptionally active in response to IFN-β and IFN-γ. BMMϕ isolated from WT (+/+), Stat1 β/β (β/β), Stat1 α/α (α/α), and Stat1 −/−

    Techniques Used: Isolation

    STAT1α and STAT1β show differential efficiencies in immune defense against MCMV and L. monocytogenes infections. (A) WT ( Stat1 +/+ ), Stat1 β/β , Stat1 α/α , and Stat1 −/− mice were infected i.p.
    Figure Legend Snippet: STAT1α and STAT1β show differential efficiencies in immune defense against MCMV and L. monocytogenes infections. (A) WT ( Stat1 +/+ ), Stat1 β/β , Stat1 α/α , and Stat1 −/− mice were infected i.p.

    Techniques Used: Mouse Assay, Infection

    Transcriptional activities of STAT1α and STAT1β overlap but are nonredundant. BMMϕ isolated from WT (+/+), Stat1 β/β (β/β), Stat1 α/α (α/α), and Stat1 −/−
    Figure Legend Snippet: Transcriptional activities of STAT1α and STAT1β overlap but are nonredundant. BMMϕ isolated from WT (+/+), Stat1 β/β (β/β), Stat1 α/α (α/α), and Stat1 −/−

    Techniques Used: Isolation

    33) Product Images from "Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses"

    Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiv255

    Stimulation with type I interferon (IFN) induces IFN-induced transmembrane (IFITM) protein expression in human macrophages. Monocyte-derived macrophages were incubated with the indicated IFNs for 24 hours or left untreated and expression of STAT1, phosphorylated STAT1, β-actin, IFITM1, and IFITM2/3 was assessed by Western blot analysis. As controls, 293T cells were transfected with empty plasmid or expression plasmids for IFITM1–3. Similar results were obtained in 3 separate experiments. Abbreviation: MDM, monocyte-derived macrophages.
    Figure Legend Snippet: Stimulation with type I interferon (IFN) induces IFN-induced transmembrane (IFITM) protein expression in human macrophages. Monocyte-derived macrophages were incubated with the indicated IFNs for 24 hours or left untreated and expression of STAT1, phosphorylated STAT1, β-actin, IFITM1, and IFITM2/3 was assessed by Western blot analysis. As controls, 293T cells were transfected with empty plasmid or expression plasmids for IFITM1–3. Similar results were obtained in 3 separate experiments. Abbreviation: MDM, monocyte-derived macrophages.

    Techniques Used: Expressing, Derivative Assay, Incubation, Western Blot, Transfection, Plasmid Preparation

    34) Product Images from "A cytomegaloviral protein reveals a dual role for STAT2 in IFN-? signaling and antiviral responses"

    Article Title: A cytomegaloviral protein reveals a dual role for STAT2 in IFN-? signaling and antiviral responses

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20041401

    IFN- γ –induced increase of STAT2-P in ΔM27-infected cells and its impact for the inhibition of MCMV replication. (A) IFNAR1-deficient MEFs were either mock infected or infected with ΔM27-MCMV or MCMV-WT (10 PFU/cell each) for 24 h before exposed to 500 U/ml IFN-γ for the indicated time. Equivalent amounts of nucleoplasmic lysates were subjected to SDS-PAGE and analyzed by Western blot for p-Tyr 689 STAT2, and reprobed for STAT2 and β-actin. (B) Comparative analysis of MCMV replication in MEF lacking components of the IFN signaling cascade. The indicated cells were incubated with 500 U/ml IFN-α or 500 U/ml IFN-γ for 48 h or left untreated before being infected with MCMV-WT or ΔM27-MCMV (0.01 PFU/cell each). The efficiency of MCMV replication is expressed as the ratio of virus yield at 96 h p.i., as determined by a standard plaque assay.
    Figure Legend Snippet: IFN- γ –induced increase of STAT2-P in ΔM27-infected cells and its impact for the inhibition of MCMV replication. (A) IFNAR1-deficient MEFs were either mock infected or infected with ΔM27-MCMV or MCMV-WT (10 PFU/cell each) for 24 h before exposed to 500 U/ml IFN-γ for the indicated time. Equivalent amounts of nucleoplasmic lysates were subjected to SDS-PAGE and analyzed by Western blot for p-Tyr 689 STAT2, and reprobed for STAT2 and β-actin. (B) Comparative analysis of MCMV replication in MEF lacking components of the IFN signaling cascade. The indicated cells were incubated with 500 U/ml IFN-α or 500 U/ml IFN-γ for 48 h or left untreated before being infected with MCMV-WT or ΔM27-MCMV (0.01 PFU/cell each). The efficiency of MCMV replication is expressed as the ratio of virus yield at 96 h p.i., as determined by a standard plaque assay.

    Techniques Used: Infection, Inhibition, SDS Page, Western Blot, Incubation, Plaque Assay

    pM27 selectively affects STAT2. (A) pM27 down-regulates STAT2. C57BL/6-MEFs were either mock infected or infected with MCMV-WT, ΔM27-MCMV, or M27HA (10 PFU/cell each) for 24 h. Equivalent amounts of cell lysates were subjected to SDS-PAGE and analyzed by Western blot for STAT2, STAT1, IRF9/p48, IRF1, MCMV IE1/pp89, and β-actin. (B) NIH 3T3 were either mock infected, infected with VV-WT as a control, or infected with rVVM27FL (5 PFU/cell each). Cell lysates were prepared 16 h p.i. and analyzed by Western blot for STAT2, STAT1, pM27, and β-actin. (C) pM27 forms a complex with STAT2. STAT2 −/− fibroblasts were infected with rVV expressing M27FL or STAT2-HA, or coinfected with both viruses (3 PFU/cell each). Cell lysates were prepared 8 h p.i. using an EMSA buffer, split into two aliquots, and subjected to immunoprecipitation using anti-HA and anti-FLAG antibodies, respectively. Immunoprecipitates were analyzed by Western blot for STAT2, pM27, and β-actin.
    Figure Legend Snippet: pM27 selectively affects STAT2. (A) pM27 down-regulates STAT2. C57BL/6-MEFs were either mock infected or infected with MCMV-WT, ΔM27-MCMV, or M27HA (10 PFU/cell each) for 24 h. Equivalent amounts of cell lysates were subjected to SDS-PAGE and analyzed by Western blot for STAT2, STAT1, IRF9/p48, IRF1, MCMV IE1/pp89, and β-actin. (B) NIH 3T3 were either mock infected, infected with VV-WT as a control, or infected with rVVM27FL (5 PFU/cell each). Cell lysates were prepared 16 h p.i. and analyzed by Western blot for STAT2, STAT1, pM27, and β-actin. (C) pM27 forms a complex with STAT2. STAT2 −/− fibroblasts were infected with rVV expressing M27FL or STAT2-HA, or coinfected with both viruses (3 PFU/cell each). Cell lysates were prepared 8 h p.i. using an EMSA buffer, split into two aliquots, and subjected to immunoprecipitation using anti-HA and anti-FLAG antibodies, respectively. Immunoprecipitates were analyzed by Western blot for STAT2, pM27, and β-actin.

    Techniques Used: Infection, SDS Page, Western Blot, Expressing, Immunoprecipitation

    Phosphorylation of STAT2 in response to IFN- γ . (A) Primary IFNAR1-deficient MEFs were either mock infected or infected with MCMV-WT or ΔM27-MCMV (10 PFU/cell each) for 24 h before being exposed to 500 U/ml IFN-γ for the indicated time. Equivalent amounts of cell lysates were subjected to SDS-PAGE and analyzed by Western blot for p-Tyr 689 STAT2, and reprobed for STAT2, p-Tyr 701 STAT1, STAT1, and β-actin. (B) Primary IFNAR1- and IFNGR-deficient MEFs were either left untreated or treated with IFN-α (100 U/ml and 10 U/ml, respectively) or 500 U/ml IFN-γ for 20 min. Equivalent amounts of cell lysates were analyzed by Western blot for p-Tyr 689 STAT2 and reprobed for STAT2 and β-actin. (C) NIH 3T3 fibroblasts and J774 macrophages were either left untreated or treated with 50 U/ml IFN-α or 500 U/ml IFN-γ for 20 min. 50 NU/ml type I neutralizing antibodies were added as indicated to remove endogenously produced IFN-β. Equivalent amounts of cell lysates were analyzed by Western blot for p-Tyr 689 STAT2, and reprobed for STAT2, p-Tyr 701 STAT1, and β-actin. (D) NIH 3T3 fibroblasts and IFNAR-deficient MEFs were not exposed or exposed to 500 U/ml IFN-γ or 50 U/ml IFN-α for 20 min. Equal protein amounts from 1:1 mixtures of nuclear and cytoplasmic cell extracts were evaluated by EMSA with an ISRE probe (reference 47 ). Supershifting was performed by the addition of a STAT2-specific antibody before incubation with the probe. The mobility of ISGF3 is indicated in the right margin.
    Figure Legend Snippet: Phosphorylation of STAT2 in response to IFN- γ . (A) Primary IFNAR1-deficient MEFs were either mock infected or infected with MCMV-WT or ΔM27-MCMV (10 PFU/cell each) for 24 h before being exposed to 500 U/ml IFN-γ for the indicated time. Equivalent amounts of cell lysates were subjected to SDS-PAGE and analyzed by Western blot for p-Tyr 689 STAT2, and reprobed for STAT2, p-Tyr 701 STAT1, STAT1, and β-actin. (B) Primary IFNAR1- and IFNGR-deficient MEFs were either left untreated or treated with IFN-α (100 U/ml and 10 U/ml, respectively) or 500 U/ml IFN-γ for 20 min. Equivalent amounts of cell lysates were analyzed by Western blot for p-Tyr 689 STAT2 and reprobed for STAT2 and β-actin. (C) NIH 3T3 fibroblasts and J774 macrophages were either left untreated or treated with 50 U/ml IFN-α or 500 U/ml IFN-γ for 20 min. 50 NU/ml type I neutralizing antibodies were added as indicated to remove endogenously produced IFN-β. Equivalent amounts of cell lysates were analyzed by Western blot for p-Tyr 689 STAT2, and reprobed for STAT2, p-Tyr 701 STAT1, and β-actin. (D) NIH 3T3 fibroblasts and IFNAR-deficient MEFs were not exposed or exposed to 500 U/ml IFN-γ or 50 U/ml IFN-α for 20 min. Equal protein amounts from 1:1 mixtures of nuclear and cytoplasmic cell extracts were evaluated by EMSA with an ISRE probe (reference 47 ). Supershifting was performed by the addition of a STAT2-specific antibody before incubation with the probe. The mobility of ISGF3 is indicated in the right margin.

    Techniques Used: Infection, SDS Page, Western Blot, Produced, Incubation

    35) Product Images from "Phosphorylation of the Stat1 transactivating domain is required for the response to type I interferons"

    Article Title: Phosphorylation of the Stat1 transactivating domain is required for the response to type I interferons

    Journal: EMBO Reports

    doi: 10.1038/sj.embor.embor802

    Expression and activation of mutant signal tranducer and activator of transcription 1. ( A ) Isolation of fibroblast clones expressing signal tranducer and activator of transcription 1-β (Stat1-β). Stat1-deficient fibroblasts were transfected with a plasmid driving the expression of Stat1-β, selection was carried out, and cells were cloned by a limiting dilution procedure. Levels of Stat1-β were analysed by western blotting using a monoclonal antibody that recognizes the amino terminus (Stat1N). For normalization, the blot was reprobed with an antibody (pan-ERK) against extracellular signal-regulated protein kinases 1 and 2 (ERKs 1 and 2). The results from two clones that expressed Stat1-β (clones β1 and β8) and, for comparison, clones that expressed Stat1-α or the Stat1-S727A mutant are shown. ( B ) Stat1 tyrosine phosphorylation in response to interferon-β (IFN-β). Fibroblasts that expressed Stat1-α, Stat1-β or Stat1-S727A were stimulated with IFN-β for the durations indicated, and Stat1 phosphorylation at Tyr701 (pY701) was analysed by western blotting using a phospho-specific antiserum. For normalization, the blots were reprobed with a monoclonal antibodies that recognize either the N terminus or the carboxyl terminus of Stat1. ( C ) Formation of the interferon-stimulated gene factor 3 (Isgf3) complex. Fibroblasts expressing Stat1-α, Stat1-β or Stat1-S727A were treated for the durations indicated with IFN-β. The presence of Isgf3 in cell extracts was analysed by an electrophoretic mobility-shift assay using DNA containing an interferon-stimulated response-element sequence as a probe.
    Figure Legend Snippet: Expression and activation of mutant signal tranducer and activator of transcription 1. ( A ) Isolation of fibroblast clones expressing signal tranducer and activator of transcription 1-β (Stat1-β). Stat1-deficient fibroblasts were transfected with a plasmid driving the expression of Stat1-β, selection was carried out, and cells were cloned by a limiting dilution procedure. Levels of Stat1-β were analysed by western blotting using a monoclonal antibody that recognizes the amino terminus (Stat1N). For normalization, the blot was reprobed with an antibody (pan-ERK) against extracellular signal-regulated protein kinases 1 and 2 (ERKs 1 and 2). The results from two clones that expressed Stat1-β (clones β1 and β8) and, for comparison, clones that expressed Stat1-α or the Stat1-S727A mutant are shown. ( B ) Stat1 tyrosine phosphorylation in response to interferon-β (IFN-β). Fibroblasts that expressed Stat1-α, Stat1-β or Stat1-S727A were stimulated with IFN-β for the durations indicated, and Stat1 phosphorylation at Tyr701 (pY701) was analysed by western blotting using a phospho-specific antiserum. For normalization, the blots were reprobed with a monoclonal antibodies that recognize either the N terminus or the carboxyl terminus of Stat1. ( C ) Formation of the interferon-stimulated gene factor 3 (Isgf3) complex. Fibroblasts expressing Stat1-α, Stat1-β or Stat1-S727A were treated for the durations indicated with IFN-β. The presence of Isgf3 in cell extracts was analysed by an electrophoretic mobility-shift assay using DNA containing an interferon-stimulated response-element sequence as a probe.

    Techniques Used: Expressing, Activation Assay, Mutagenesis, Isolation, Clone Assay, Transfection, Plasmid Preparation, Selection, Western Blot, Electrophoretic Mobility Shift Assay, Sequencing

    Requirement of the signal tranducer and activator of transcription 1 transactivating domain and its phosphorylation at Ser727 for the expression of interferon-β-induced genes. Stat1-deficient fibroblasts, and cells that express Stat1-α, Stat1-β or Stat1-S727A were treated with interferon-β (IFN-β) for 4 h or 6 h. Expression of the Irf1 , Gbp1 and Mx1 genes was determined by quantitative real-time PCR of reverse-transcribed messenger RNA. For normalization to input quantities, the amount of Hprt mRNA was determined. ( A ) Inducibility in response to treatment with IFN-β (that is, the ratio of expression in IFN-β-treated and untreated cells) after 4 h and 6 h in one of three independent experiments. ( B ) The results of three independent experiments are shown, expressed as the percentage induction ± s.d. in cells that express Stat1-β, Stat1-S727A or that express no Stat1, compared with the values measured in cells that express Stat1-α (100%).
    Figure Legend Snippet: Requirement of the signal tranducer and activator of transcription 1 transactivating domain and its phosphorylation at Ser727 for the expression of interferon-β-induced genes. Stat1-deficient fibroblasts, and cells that express Stat1-α, Stat1-β or Stat1-S727A were treated with interferon-β (IFN-β) for 4 h or 6 h. Expression of the Irf1 , Gbp1 and Mx1 genes was determined by quantitative real-time PCR of reverse-transcribed messenger RNA. For normalization to input quantities, the amount of Hprt mRNA was determined. ( A ) Inducibility in response to treatment with IFN-β (that is, the ratio of expression in IFN-β-treated and untreated cells) after 4 h and 6 h in one of three independent experiments. ( B ) The results of three independent experiments are shown, expressed as the percentage induction ± s.d. in cells that express Stat1-β, Stat1-S727A or that express no Stat1, compared with the values measured in cells that express Stat1-α (100%).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effect of the signal tranducer and activator of transcription 1-S727A mutation on the expression of the endogenous Mx1 and Gbp1 genes during transient transfection. Stat1-deficient fibroblasts were co-transfected with Stat1-α or Stat1-S727A and a plasmid that drives expression of the β-galactosidase gene (β- Gal ). Some of the transfected cells were used for measuring β-Gal activity in cell extracts to ensure comparable transfection efficiencies. The remaining cells were either left untreated or were treated with interferon-β (IFN-β) for 4 h, after which total RNA was isolated and processed for real-time quantitative PCR analysis of the expression of the Mx1 and Gbp1 genes. Inducibility is calculated as the ratio of expression measured in IFN-β-treated and untreated cells.
    Figure Legend Snippet: Effect of the signal tranducer and activator of transcription 1-S727A mutation on the expression of the endogenous Mx1 and Gbp1 genes during transient transfection. Stat1-deficient fibroblasts were co-transfected with Stat1-α or Stat1-S727A and a plasmid that drives expression of the β-galactosidase gene (β- Gal ). Some of the transfected cells were used for measuring β-Gal activity in cell extracts to ensure comparable transfection efficiencies. The remaining cells were either left untreated or were treated with interferon-β (IFN-β) for 4 h, after which total RNA was isolated and processed for real-time quantitative PCR analysis of the expression of the Mx1 and Gbp1 genes. Inducibility is calculated as the ratio of expression measured in IFN-β-treated and untreated cells.

    Techniques Used: Mutagenesis, Expressing, Transfection, Plasmid Preparation, Activity Assay, Isolation, Real-time Polymerase Chain Reaction

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    Inhibition of the AMPK/mTOR pathway attenuates the upregulation of CD133 by CD90 in cell lines and fresh liver cancer specimen A. The phosphorylation of mTOR and AMPK in the transfectants was determined by western blotting. The quantitative data represent mean ± SD ( n = 3). CD133 mRNA B. and protein expression C. in the transfectants were determined by quantitative RT-PCR and western blotting respectively after <t>rapamycin</t> or <t>AICAR</t> treatment for 24 hours. D. The transfectants were plated in soft agar and treated with the indicated inhibitors. Colonies were monitored for 14 days and quantified using Image-Pro Plus software. Data represent mean ± SEM ( n = 3). E. The CD90 transfectant was subcutaneously injected to NOD/SCID mice. The mice were intraperitoneally injected with indicated sorafenib or OSU-CG5 after 14 days and the drug was administrated to mice every other day. P value was calculated using two-way anova analysis and * indicated P
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    Inhibition of the AMPK/mTOR pathway attenuates the upregulation of CD133 by CD90 in cell lines and fresh liver cancer specimen A. The phosphorylation of mTOR and AMPK in the transfectants was determined by western blotting. The quantitative data represent mean ± SD ( n = 3). CD133 mRNA B. and protein expression C. in the transfectants were determined by quantitative RT-PCR and western blotting respectively after rapamycin or AICAR treatment for 24 hours. D. The transfectants were plated in soft agar and treated with the indicated inhibitors. Colonies were monitored for 14 days and quantified using Image-Pro Plus software. Data represent mean ± SEM ( n = 3). E. The CD90 transfectant was subcutaneously injected to NOD/SCID mice. The mice were intraperitoneally injected with indicated sorafenib or OSU-CG5 after 14 days and the drug was administrated to mice every other day. P value was calculated using two-way anova analysis and * indicated P

    Journal: Oncotarget

    Article Title: Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer

    doi:

    Figure Lengend Snippet: Inhibition of the AMPK/mTOR pathway attenuates the upregulation of CD133 by CD90 in cell lines and fresh liver cancer specimen A. The phosphorylation of mTOR and AMPK in the transfectants was determined by western blotting. The quantitative data represent mean ± SD ( n = 3). CD133 mRNA B. and protein expression C. in the transfectants were determined by quantitative RT-PCR and western blotting respectively after rapamycin or AICAR treatment for 24 hours. D. The transfectants were plated in soft agar and treated with the indicated inhibitors. Colonies were monitored for 14 days and quantified using Image-Pro Plus software. Data represent mean ± SEM ( n = 3). E. The CD90 transfectant was subcutaneously injected to NOD/SCID mice. The mice were intraperitoneally injected with indicated sorafenib or OSU-CG5 after 14 days and the drug was administrated to mice every other day. P value was calculated using two-way anova analysis and * indicated P

    Article Snippet: Rapamycin and AICAR were purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Inhibition, Western Blot, Expressing, Quantitative RT-PCR, Software, Transfection, Injection, Mouse Assay