bcl 2  (Roche)


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  • 92

    Structured Review

    Roche bcl 2
    Proteolytic degradation of SERCA and Bcl-2Δ21 and protection by protease inhibitors ( A – C , F ) Rat skeletal-muscle SR (0.6 mg/ml) was incubated in absence (samples 2–5) or presence (samples 7–10) of 1 mM PMSF. Samples without (2, 4, 7, 9) and with 75 μg/ml Bcl-2Δ21 (3, 5, 8, 10) were incubated at 37 °C for 0 (2, 3, 7, 8) or 2.5 h (4, 5, 9, 10) in the STE buffer and separated by SDS/PAGE ( A ), analysed for Ca 2+ -ATPase activity ( B ), and monitored by Western blotting with anti-SERCA1 ( C ) or <t>anti-Bcl-2</t> ( F ) antibodies. In ( A , C , F ), lanes 1 and 6 show molecular-mass standards. Samples 2–5 and 7–10 are labelled under the respective lanes (in A , C , F ) or columns ( B ). Bands named PP and labelled by arrows indicate proteolytic degradation products. ( D ) SDS/PAGE analysis of Bcl-2Δ21 (150 μg/ml in STE buffer) incubated without protease inhibitors for 1 (lane 2) and 3 h (lane 3) at 37 °C or stored at 4 °C for 1 (lane 5), 2 (lane 6) or 3 days (lane 7). Bands named PP and labelled by arrows indicate proteolytic degradation products. Lanes 1 and 4 show molecular-mass-standards. ( E ) MALDI–TOF spectrum of Bcl-2Δ21 after storage for 3 days at 4 °C (corresponds to lane 7 in D ).
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    Images

    1) Product Images from "Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)"

    Article Title: Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)

    Journal: Biochemical Journal

    doi: 10.1042/BJ20040187

    Proteolytic degradation of SERCA and Bcl-2Δ21 and protection by protease inhibitors ( A – C , F ) Rat skeletal-muscle SR (0.6 mg/ml) was incubated in absence (samples 2–5) or presence (samples 7–10) of 1 mM PMSF. Samples without (2, 4, 7, 9) and with 75 μg/ml Bcl-2Δ21 (3, 5, 8, 10) were incubated at 37 °C for 0 (2, 3, 7, 8) or 2.5 h (4, 5, 9, 10) in the STE buffer and separated by SDS/PAGE ( A ), analysed for Ca 2+ -ATPase activity ( B ), and monitored by Western blotting with anti-SERCA1 ( C ) or anti-Bcl-2 ( F ) antibodies. In ( A , C , F ), lanes 1 and 6 show molecular-mass standards. Samples 2–5 and 7–10 are labelled under the respective lanes (in A , C , F ) or columns ( B ). Bands named PP and labelled by arrows indicate proteolytic degradation products. ( D ) SDS/PAGE analysis of Bcl-2Δ21 (150 μg/ml in STE buffer) incubated without protease inhibitors for 1 (lane 2) and 3 h (lane 3) at 37 °C or stored at 4 °C for 1 (lane 5), 2 (lane 6) or 3 days (lane 7). Bands named PP and labelled by arrows indicate proteolytic degradation products. Lanes 1 and 4 show molecular-mass-standards. ( E ) MALDI–TOF spectrum of Bcl-2Δ21 after storage for 3 days at 4 °C (corresponds to lane 7 in D ).
    Figure Legend Snippet: Proteolytic degradation of SERCA and Bcl-2Δ21 and protection by protease inhibitors ( A – C , F ) Rat skeletal-muscle SR (0.6 mg/ml) was incubated in absence (samples 2–5) or presence (samples 7–10) of 1 mM PMSF. Samples without (2, 4, 7, 9) and with 75 μg/ml Bcl-2Δ21 (3, 5, 8, 10) were incubated at 37 °C for 0 (2, 3, 7, 8) or 2.5 h (4, 5, 9, 10) in the STE buffer and separated by SDS/PAGE ( A ), analysed for Ca 2+ -ATPase activity ( B ), and monitored by Western blotting with anti-SERCA1 ( C ) or anti-Bcl-2 ( F ) antibodies. In ( A , C , F ), lanes 1 and 6 show molecular-mass standards. Samples 2–5 and 7–10 are labelled under the respective lanes (in A , C , F ) or columns ( B ). Bands named PP and labelled by arrows indicate proteolytic degradation products. ( D ) SDS/PAGE analysis of Bcl-2Δ21 (150 μg/ml in STE buffer) incubated without protease inhibitors for 1 (lane 2) and 3 h (lane 3) at 37 °C or stored at 4 °C for 1 (lane 5), 2 (lane 6) or 3 days (lane 7). Bands named PP and labelled by arrows indicate proteolytic degradation products. Lanes 1 and 4 show molecular-mass-standards. ( E ) MALDI–TOF spectrum of Bcl-2Δ21 after storage for 3 days at 4 °C (corresponds to lane 7 in D ).

    Techniques Used: Incubation, SDS Page, Activity Assay, Western Blot

    2) Product Images from "V‐ATPase inhibition overcomes trastuzumab resistance in breast cancer), V‐ATPase inhibition overcomes trastuzumab resistance in breast cancer"

    Article Title: V‐ATPase inhibition overcomes trastuzumab resistance in breast cancer), V‐ATPase inhibition overcomes trastuzumab resistance in breast cancer

    Journal: Molecular Oncology

    doi: 10.1016/j.molonc.2013.08.011

    Archazolid leads to accumulation of HER2 in endocytotic vesicles. Colocalization of HER2 (red) with lysosomes (Lamp‐1, green), autophagosomes (LC3, green) or endosomes (EEA1, green) after treatment with 10 nM archazolid for 24 h
    Figure Legend Snippet: Archazolid leads to accumulation of HER2 in endocytotic vesicles. Colocalization of HER2 (red) with lysosomes (Lamp‐1, green), autophagosomes (LC3, green) or endosomes (EEA1, green) after treatment with 10 nM archazolid for 24 h

    Techniques Used:

    Archazolid induces apoptosis in trastuzumab‐resistant cells. (A) JIMT‐1 cells were treated with increasing doses of archazolid or 20 μg/ml trastuzumab for 24 and 48 h and apoptosis was assessed by propidium iodide
    Figure Legend Snippet: Archazolid induces apoptosis in trastuzumab‐resistant cells. (A) JIMT‐1 cells were treated with increasing doses of archazolid or 20 μg/ml trastuzumab for 24 and 48 h and apoptosis was assessed by propidium iodide

    Techniques Used:

    Decreased tumor growth in SCID mice is coherent with decreased nuclear Ki67 expression and increased HER2 internalization of archazolid treated mice. (A) Growth of xenograft tumors is significantly inhibited by bi‐daily archazolid treatment (3 ng/g),
    Figure Legend Snippet: Decreased tumor growth in SCID mice is coherent with decreased nuclear Ki67 expression and increased HER2 internalization of archazolid treated mice. (A) Growth of xenograft tumors is significantly inhibited by bi‐daily archazolid treatment (3 ng/g),

    Techniques Used: Mouse Assay, Expressing

    Archazolid leads to accumulation of HER2 in the cytosol. (A) JIMT‐1 and SKBR3 cells were treated with 1 and 10 nM archazolid for 24 or 48 h and HER2 level was measured on the cell surface by flow cytometry (*p ≤ 0,01,
    Figure Legend Snippet: Archazolid leads to accumulation of HER2 in the cytosol. (A) JIMT‐1 and SKBR3 cells were treated with 1 and 10 nM archazolid for 24 or 48 h and HER2 level was measured on the cell surface by flow cytometry (*p ≤ 0,01,

    Techniques Used: Flow Cytometry, Cytometry

    Archazolid leads to growth inhibition in trastuzumab‐resistant tumor cells. (A) Western blot analysis of HER2 expression in SKBR3, JIMT‐1 and MCF‐7 cells. SKBR3 and JIMT‐1 cells were treated with increasing doses of trastuzumab
    Figure Legend Snippet: Archazolid leads to growth inhibition in trastuzumab‐resistant tumor cells. (A) Western blot analysis of HER2 expression in SKBR3, JIMT‐1 and MCF‐7 cells. SKBR3 and JIMT‐1 cells were treated with increasing doses of trastuzumab

    Techniques Used: Inhibition, Western Blot, Expressing

    Archazolid impairs HER2‐related signaling. (A) SKBR3 cells were treated with increasing doses of trastuzumab or archazolid for 24 h and phosphorylation of AKT and P70S6K was analyzed by western blot. (B) JIMT‐1 cells were treated
    Figure Legend Snippet: Archazolid impairs HER2‐related signaling. (A) SKBR3 cells were treated with increasing doses of trastuzumab or archazolid for 24 h and phosphorylation of AKT and P70S6K was analyzed by western blot. (B) JIMT‐1 cells were treated

    Techniques Used: Western Blot

    3) Product Images from "Characterization of a core fragment of the rhesus monkey TRIM5? protein"

    Article Title: Characterization of a core fragment of the rhesus monkey TRIM5? protein

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-12-1

    Oligomerization state of BCCL2 variants in mammalian cells . A . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins with V5 epitope tags were analyzed by 12% SDS-PAGE and Western blotted with an HRP-conjugated anti-V5 antibody. B . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins were treated with the indicated concentrations of glutaraldehyde and then boiled in Laemmli buffer and analyzed by SDS-PAGE and Western blotting, as described above. The positions of the molecular-weight markers in kD are shown. The arrows indicate the positions of monomers (M), dimers (D) and higher-order oligomers (H-O).
    Figure Legend Snippet: Oligomerization state of BCCL2 variants in mammalian cells . A . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins with V5 epitope tags were analyzed by 12% SDS-PAGE and Western blotted with an HRP-conjugated anti-V5 antibody. B . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins were treated with the indicated concentrations of glutaraldehyde and then boiled in Laemmli buffer and analyzed by SDS-PAGE and Western blotting, as described above. The positions of the molecular-weight markers in kD are shown. The arrows indicate the positions of monomers (M), dimers (D) and higher-order oligomers (H-O).

    Techniques Used: Expressing, Mutagenesis, SDS Page, Western Blot, Molecular Weight

    Electron microscopy of the LLER protein . A . The BCCL2 LLER protein purified by nickel-affinity, anion-exchange, and gel-filtration chromatography was applied to glow-discharged carbon grids. After staining with 1% uranyl formate, the grids were examined with a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company) at 100 kV. B . The cryoelectron microscopic images of the LLER protein were taken at a magnification of 150,000 × and at a defocus of 3~5 μm with a Tecnai F20 field-emission gun electron microscope operating at 200 kV. The proteins that were purified as described above were embedded in a thin ice film on a Quantifoil grid, using an FEI Vitrobot, a robot that swiftly plunges the protein-loaded grid into liquid ethane. The images were low-pass filtered with background noises removed (right column). The bars in the left-hand images are 20 nm. C . The Peak 2 fraction of the LLER protein was incubated with an anti-His 6 antibody and imaged by single-particle cryoelectron microscopy, as described above. Representative images of the LLER protein alone (panel 1), the antibody alone (panel 2), and the LLER protein complexed with one or two antibody molecules (panels 3 and 4, respectively) are shown.
    Figure Legend Snippet: Electron microscopy of the LLER protein . A . The BCCL2 LLER protein purified by nickel-affinity, anion-exchange, and gel-filtration chromatography was applied to glow-discharged carbon grids. After staining with 1% uranyl formate, the grids were examined with a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company) at 100 kV. B . The cryoelectron microscopic images of the LLER protein were taken at a magnification of 150,000 × and at a defocus of 3~5 μm with a Tecnai F20 field-emission gun electron microscope operating at 200 kV. The proteins that were purified as described above were embedded in a thin ice film on a Quantifoil grid, using an FEI Vitrobot, a robot that swiftly plunges the protein-loaded grid into liquid ethane. The images were low-pass filtered with background noises removed (right column). The bars in the left-hand images are 20 nm. C . The Peak 2 fraction of the LLER protein was incubated with an anti-His 6 antibody and imaged by single-particle cryoelectron microscopy, as described above. Representative images of the LLER protein alone (panel 1), the antibody alone (panel 2), and the LLER protein complexed with one or two antibody molecules (panels 3 and 4, respectively) are shown.

    Techniques Used: Electron Microscopy, Purification, Filtration, Chromatography, Staining, Microscopy, Incubation

    Secondary structure and melting temperature of the LLER protein . A . The far-UV spectra (195 - 245 nm) of the LLER protein were recorded with an Aviv circular dichroism (CD) spectrometer at the indicated temperatures. The percentage of alpha-helical content at the various temperatures was calculated and is shown in the key. B . The melting curve of the BCCL2 protein was generated by plotting the alpha-helical content as a function of temperature. Note the biphasic shape of the curve. C . Approximately 2 μg of the BCCL2 protein was incubated at the indicated temperatures for 5 minutes prior to the addition of 1 mM glutaraldehyde. Incubation at the same temperature was continued for another 8 minutes, after which the reaction was quenched by addition of excess 0.1 M Tris-HCl, pH 7.5 buffer. A control reaction without the addition of glutaraldehyde was also performed at each temperature. The reaction mixtures were boiled in Laemmli buffer and analyzed on a 4-12% SDS-polyacrylamide gel. M, molecular weight markers.
    Figure Legend Snippet: Secondary structure and melting temperature of the LLER protein . A . The far-UV spectra (195 - 245 nm) of the LLER protein were recorded with an Aviv circular dichroism (CD) spectrometer at the indicated temperatures. The percentage of alpha-helical content at the various temperatures was calculated and is shown in the key. B . The melting curve of the BCCL2 protein was generated by plotting the alpha-helical content as a function of temperature. Note the biphasic shape of the curve. C . Approximately 2 μg of the BCCL2 protein was incubated at the indicated temperatures for 5 minutes prior to the addition of 1 mM glutaraldehyde. Incubation at the same temperature was continued for another 8 minutes, after which the reaction was quenched by addition of excess 0.1 M Tris-HCl, pH 7.5 buffer. A control reaction without the addition of glutaraldehyde was also performed at each temperature. The reaction mixtures were boiled in Laemmli buffer and analyzed on a 4-12% SDS-polyacrylamide gel. M, molecular weight markers.

    Techniques Used: Generated, Incubation, Molecular Weight

    Effect of B-box 2 changes on BCCL2 expression and solubility . The wild-type (wt) BCCL2 protein and the indicated B-box 2 mutants were expressed in E. coli. The bacteria were lysed and the lysates centrifuged at 4000 × g for 10 minutes. The supernatants were loaded onto a Ni +2 -NTA affinity column; the proteins eluted with 300 mM imidazole were analyzed by SDS-PAGE and Coomassie Blue staining.
    Figure Legend Snippet: Effect of B-box 2 changes on BCCL2 expression and solubility . The wild-type (wt) BCCL2 protein and the indicated B-box 2 mutants were expressed in E. coli. The bacteria were lysed and the lysates centrifuged at 4000 × g for 10 minutes. The supernatants were loaded onto a Ni +2 -NTA affinity column; the proteins eluted with 300 mM imidazole were analyzed by SDS-PAGE and Coomassie Blue staining.

    Techniques Used: Expressing, Solubility, Affinity Column, SDS Page, Staining

    Comparison of the size-exclusion chromatography profiles of the wild-type and mutant BCCL2 proteins . A . Purified wild-type (wt) and mutant BCCL2 proteins were loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The protein peaks 1 (P1) and 2 (P2) are indicated. The positions at which the globular proteins standards thyroglobulin (670 kD) and bovine gamma-globulin (158 kD) were eluted in a parallel run are indicated. B . Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (bottom panel). The 25- and 37-kD molecular weight markers (M) are shown in the left-most lanes. An aliquot of the LLWL protein loaded on the gel-filtration column was also analyzed (Load). The positions of peaks P1 and P2 are noted.
    Figure Legend Snippet: Comparison of the size-exclusion chromatography profiles of the wild-type and mutant BCCL2 proteins . A . Purified wild-type (wt) and mutant BCCL2 proteins were loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The protein peaks 1 (P1) and 2 (P2) are indicated. The positions at which the globular proteins standards thyroglobulin (670 kD) and bovine gamma-globulin (158 kD) were eluted in a parallel run are indicated. B . Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (bottom panel). The 25- and 37-kD molecular weight markers (M) are shown in the left-most lanes. An aliquot of the LLWL protein loaded on the gel-filtration column was also analyzed (Load). The positions of peaks P1 and P2 are noted.

    Techniques Used: Size-exclusion Chromatography, Mutagenesis, Purification, Filtration, Flow Cytometry, Staining, Molecular Weight

    Purification of the BCCL2 protein expressed in bacteria . A . Bacterial cells expressing the BCCL2 protein were lysed and the homogenates subjected to purification approaches. In lane 1, the soluble BCCL2 protein was purified by Ni +2 -NTA metal-affinity chromatography. Lane 2 shows the insoluble pellet obtained after lysis of the bacteria with lysis buffer. The proteins in each sample were resolved by SDS-PAGE and Coomassie Blue staining. B . The affinity-purified BCCL2 protein was loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The OD 280 of the eluted protein is plotted (blue line). The profile of the globular protein standards (thyroglobulin (670 kD), bovine gamma-globulin (158 kD), chicken ovalbumin (44 kD), equine myoglobin (17 kD) and vitamin B12 (1.35 kD) is shown in red. Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue. An aliquot of the BCCL2 protein sample loaded on the gel-filtration column was analyzed (Load), along with the molecular-weight markers (M). C . The affinity-purified BCCL2 protein was loaded onto a Hi-trap Q anion-exchange column and eluted at a flow rate of 0.5 ml/min (left panel). The fractions from the column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (right panel).
    Figure Legend Snippet: Purification of the BCCL2 protein expressed in bacteria . A . Bacterial cells expressing the BCCL2 protein were lysed and the homogenates subjected to purification approaches. In lane 1, the soluble BCCL2 protein was purified by Ni +2 -NTA metal-affinity chromatography. Lane 2 shows the insoluble pellet obtained after lysis of the bacteria with lysis buffer. The proteins in each sample were resolved by SDS-PAGE and Coomassie Blue staining. B . The affinity-purified BCCL2 protein was loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The OD 280 of the eluted protein is plotted (blue line). The profile of the globular protein standards (thyroglobulin (670 kD), bovine gamma-globulin (158 kD), chicken ovalbumin (44 kD), equine myoglobin (17 kD) and vitamin B12 (1.35 kD) is shown in red. Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue. An aliquot of the BCCL2 protein sample loaded on the gel-filtration column was analyzed (Load), along with the molecular-weight markers (M). C . The affinity-purified BCCL2 protein was loaded onto a Hi-trap Q anion-exchange column and eluted at a flow rate of 0.5 ml/min (left panel). The fractions from the column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (right panel).

    Techniques Used: Purification, Expressing, Affinity Chromatography, Lysis, SDS Page, Staining, Affinity Purification, Filtration, Flow Cytometry, Molecular Weight

    4) Product Images from "Molecular Basis for the Association of Microcephalin (MCPH1) Protein with the Cell Division Cycle Protein 27 (Cdc27) Subunit of the Anaphase-promoting Complex *"

    Article Title: Molecular Basis for the Association of Microcephalin (MCPH1) Protein with the Cell Division Cycle Protein 27 (Cdc27) Subunit of the Anaphase-promoting Complex *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.307868

    Validation and bi-directional mapping of the MCPH1-Cdc27 interaction. A , immunoprecipitation ( IP ) experiments performed in 293T cells co-transfected with GFP-tagged Cdc27 and Myc-tagged MCPH1. Ms IgG CTL refers to an IgG non-specific immunoprecipitation control. WB , Western blot. B , immunoprecipitation with anti-MCPH1 antibody and Western blot with anti-Cdc27 antibody in the presence and absence of proteasome inhibitor MG132. C , cell lysates pre-treated with λ-phosphatase ( ppase ) or λ-phosphatase and EDTA (phosphatase inhibitor) were immunoprecipitated for Cdc27 (GFP) and immunoblotted ( WB ) for MCPH1 (Myc). D , Western blotting for MCPH1 (Myc) and Cdc27 (GFP) protein levels in the presence and absence of 10 Gy of ionizing radiation (IR). E , immunoprecipitation ( IP ) for MCPH1 (Myc) and Western blotting for Cdc27 (GFP) in response to 10 Gy of IR and/or λ-phosphatase ( ppase ) showing that the MCPH1-Cdc27 interaction is phosphorylation dependent.
    Figure Legend Snippet: Validation and bi-directional mapping of the MCPH1-Cdc27 interaction. A , immunoprecipitation ( IP ) experiments performed in 293T cells co-transfected with GFP-tagged Cdc27 and Myc-tagged MCPH1. Ms IgG CTL refers to an IgG non-specific immunoprecipitation control. WB , Western blot. B , immunoprecipitation with anti-MCPH1 antibody and Western blot with anti-Cdc27 antibody in the presence and absence of proteasome inhibitor MG132. C , cell lysates pre-treated with λ-phosphatase ( ppase ) or λ-phosphatase and EDTA (phosphatase inhibitor) were immunoprecipitated for Cdc27 (GFP) and immunoblotted ( WB ) for MCPH1 (Myc). D , Western blotting for MCPH1 (Myc) and Cdc27 (GFP) protein levels in the presence and absence of 10 Gy of ionizing radiation (IR). E , immunoprecipitation ( IP ) for MCPH1 (Myc) and Western blotting for Cdc27 (GFP) in response to 10 Gy of IR and/or λ-phosphatase ( ppase ) showing that the MCPH1-Cdc27 interaction is phosphorylation dependent.

    Techniques Used: Immunoprecipitation, Transfection, Mass Spectrometry, CTL Assay, Western Blot

    5) Product Images from "B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model of Limb Girdle Muscular Dystrophy 2I"

    Article Title: B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model of Limb Girdle Muscular Dystrophy 2I

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2016.05.021

    Change in myofiber size distribution after overexpression of GALGT2 in FKRP P448Lneo − mice. The gastroc, quad, and TA muscles of rAAVrh74.MCK.GALGT2-treated and -untreated FKRP P448Lneo − mice were analyzed at 1, 3, and 6 months after injection.
    Figure Legend Snippet: Change in myofiber size distribution after overexpression of GALGT2 in FKRP P448Lneo − mice. The gastroc, quad, and TA muscles of rAAVrh74.MCK.GALGT2-treated and -untreated FKRP P448Lneo − mice were analyzed at 1, 3, and 6 months after injection.

    Techniques Used: Over Expression, Mouse Assay, Injection

    Quantification of immunoglobulin G (IgG) uptake after overexpression of GALGT2 in FKRP P448Lneo − mice. The gastroc, quad, and TA muscles of FKRP P448Lneo − mutant mice were injected with rAAVrh74.MCK.GALGT2 (treated) or PBS (untreated)
    Figure Legend Snippet: Quantification of immunoglobulin G (IgG) uptake after overexpression of GALGT2 in FKRP P448Lneo − mice. The gastroc, quad, and TA muscles of FKRP P448Lneo − mutant mice were injected with rAAVrh74.MCK.GALGT2 (treated) or PBS (untreated)

    Techniques Used: Over Expression, Mouse Assay, Mutagenesis, Injection

    CT glycan staining in rAAVrh74.MCK.GALGT2-treated FKRP P448Lneo − muscle. A: The gastroc, quad, and TA muscles of FKRP P448Lneo − mice were injected with rAAVrh74.MCK.GALGT2 (treated) and analyzed at 1, 3, and 6 months after injection compared
    Figure Legend Snippet: CT glycan staining in rAAVrh74.MCK.GALGT2-treated FKRP P448Lneo − muscle. A: The gastroc, quad, and TA muscles of FKRP P448Lneo − mice were injected with rAAVrh74.MCK.GALGT2 (treated) and analyzed at 1, 3, and 6 months after injection compared

    Techniques Used: Staining, Mouse Assay, Injection

    Immunostaining for dystrophin and laminin α2 surrogates in GALGT2-overexpressing FKRP P448Lneo − muscle. FKRP P448Lneo − muscles (gastroc) were injected with rAAVrh74.MCK.GALGT2 and analyzed at 3 months after injection. Cross-sections
    Figure Legend Snippet: Immunostaining for dystrophin and laminin α2 surrogates in GALGT2-overexpressing FKRP P448Lneo − muscle. FKRP P448Lneo − muscles (gastroc) were injected with rAAVrh74.MCK.GALGT2 and analyzed at 3 months after injection. Cross-sections

    Techniques Used: Immunostaining, Injection

    Assessment of α-dystroglycan glycosylation after GALGT2 overexpression in FKRP P448Lneo − muscle. A: Gastrocnemius muscles from FKRP P448Lneo − mice were treated with rAAVrh74.MCK.GALGT2 (treated) or PBS (untreated). At 3 months
    Figure Legend Snippet: Assessment of α-dystroglycan glycosylation after GALGT2 overexpression in FKRP P448Lneo − muscle. A: Gastrocnemius muscles from FKRP P448Lneo − mice were treated with rAAVrh74.MCK.GALGT2 (treated) or PBS (untreated). At 3 months

    Techniques Used: Over Expression, Mouse Assay

    Inhibition of muscle pathology by GALGT2 overexpression in FKRP P448Lneo − mice. The gastroc, quad, and TA muscles of FKRP P448Lneo − mice were treated with rAAVrh74.MCK.GALGT2 at 2 to 4 weeks of age, analyzed at 1, 3, and 6 months after
    Figure Legend Snippet: Inhibition of muscle pathology by GALGT2 overexpression in FKRP P448Lneo − mice. The gastroc, quad, and TA muscles of FKRP P448Lneo − mice were treated with rAAVrh74.MCK.GALGT2 at 2 to 4 weeks of age, analyzed at 1, 3, and 6 months after

    Techniques Used: Inhibition, Over Expression, Mouse Assay

    GALGT2 and CT glycan expression after intramuscular injection of rAAVrh74.MCK.GALGT2 in FKRP P448Lneo − mice. The gastroc, quad, and TA muscles of P448L FKRP mutant mice were injected with rAAVrh74.MCK.GALGT2 (treated) or PBS (untreated) and compared
    Figure Legend Snippet: GALGT2 and CT glycan expression after intramuscular injection of rAAVrh74.MCK.GALGT2 in FKRP P448Lneo − mice. The gastroc, quad, and TA muscles of P448L FKRP mutant mice were injected with rAAVrh74.MCK.GALGT2 (treated) or PBS (untreated) and compared

    Techniques Used: Expressing, Injection, Mouse Assay, Mutagenesis

    Immunostaining for natively glycosylated α-dystroglycan and for embryonic myosin in GALGT2-overexpressing FKRP P448Lneo − muscles. Data shown are from rAAVrh74.MCK.GALGT2-injected (treated) or PBS-injected (untreated) gastrocnemius muscles
    Figure Legend Snippet: Immunostaining for natively glycosylated α-dystroglycan and for embryonic myosin in GALGT2-overexpressing FKRP P448Lneo − muscles. Data shown are from rAAVrh74.MCK.GALGT2-injected (treated) or PBS-injected (untreated) gastrocnemius muscles

    Techniques Used: Immunostaining, Injection

    6) Product Images from "The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice"

    Article Title: The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice

    Journal: Biochemical Journal

    doi: 10.1042/BJ20121070

    The highly phosphorylated species of Cx43 were reduced in the mammary gland of Cx43 I130T/+ mice at parturition, whereas co-expressed mammary gland connexins remain unchanged ( A ) Western blot analysis of Cx43 revealed significantly reduced levels of the highly phosphorylated species (P) of Cx43, whereas the primarily unphosphorylated (P0) species remained similar to that found in littermate control mice. Values are mean levels of total, P0 and P relative to β-actin±S.E.M. (* P
    Figure Legend Snippet: The highly phosphorylated species of Cx43 were reduced in the mammary gland of Cx43 I130T/+ mice at parturition, whereas co-expressed mammary gland connexins remain unchanged ( A ) Western blot analysis of Cx43 revealed significantly reduced levels of the highly phosphorylated species (P) of Cx43, whereas the primarily unphosphorylated (P0) species remained similar to that found in littermate control mice. Values are mean levels of total, P0 and P relative to β-actin±S.E.M. (* P

    Techniques Used: Mouse Assay, Western Blot

    Lactating Cx43 I130T/+ mice can produce and eject milk into ducts upon oxytocin stimulation ( A and B ) Western blot analysis revealed that Cx43 I130T/+ mice express the common milk proteins WAP and β-casein, similar to controls. Values are mean expression±S.E.M. ( C and D ). An oxytocin-induced milk-ejection assay revealed that lactating Cx43 I130T/+ mice respond to exogenous oxytocin to deliver milk into ducts (arrowheads). Images in ( D ) represent the same gland field before and after (separated by arrows) the addition of oxytocin. n =4.
    Figure Legend Snippet: Lactating Cx43 I130T/+ mice can produce and eject milk into ducts upon oxytocin stimulation ( A and B ) Western blot analysis revealed that Cx43 I130T/+ mice express the common milk proteins WAP and β-casein, similar to controls. Values are mean expression±S.E.M. ( C and D ). An oxytocin-induced milk-ejection assay revealed that lactating Cx43 I130T/+ mice respond to exogenous oxytocin to deliver milk into ducts (arrowheads). Images in ( D ) represent the same gland field before and after (separated by arrows) the addition of oxytocin. n =4.

    Techniques Used: Mouse Assay, Western Blot, Expressing

    7) Product Images from "Investigating CFTR and KCa3.1 Protein/Protein Interactions"

    Article Title: Investigating CFTR and KCa3.1 Protein/Protein Interactions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153665

    Co-immunoprecipitation of endogenous CFTR and KCa3.1 proteins extracted from CFBE airway cells. Immunoblots showing CFTR and KCa3.1 proteins extracted from CFBE bronchial cells expressing wt-CFTR (A, B) and F508del-CFTR (C, D). Membranes were blotted with anti-CFTR (mAb 596 from CFFT, 1:1000, A, C) and anti-KCa3.1 (Alomone, 1:300, B, D) antibodies. Endogenous expression of CFTR and KCa3.1 proteins in the CFBE-wt and CFBE-ΔF508 cell lysates are presented in lane “Total Lysate”. Immunoprecipitation of endogenous CFTR using anti-CFTR antibody followed by co-immunoprecipitation of KCa3.1 is illustrated in lane IP CFTR (B, D), while immunoprecipitation of endogenous KCa3.1 (using anti-KCa3.1 antibody) followed by co-immunoprecitation of CFTR is shown in lane IP KCa3.1 (A, C). Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with CFTR and KCa3.1 antibodies, respectively.
    Figure Legend Snippet: Co-immunoprecipitation of endogenous CFTR and KCa3.1 proteins extracted from CFBE airway cells. Immunoblots showing CFTR and KCa3.1 proteins extracted from CFBE bronchial cells expressing wt-CFTR (A, B) and F508del-CFTR (C, D). Membranes were blotted with anti-CFTR (mAb 596 from CFFT, 1:1000, A, C) and anti-KCa3.1 (Alomone, 1:300, B, D) antibodies. Endogenous expression of CFTR and KCa3.1 proteins in the CFBE-wt and CFBE-ΔF508 cell lysates are presented in lane “Total Lysate”. Immunoprecipitation of endogenous CFTR using anti-CFTR antibody followed by co-immunoprecipitation of KCa3.1 is illustrated in lane IP CFTR (B, D), while immunoprecipitation of endogenous KCa3.1 (using anti-KCa3.1 antibody) followed by co-immunoprecitation of CFTR is shown in lane IP KCa3.1 (A, C). Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with CFTR and KCa3.1 antibodies, respectively.

    Techniques Used: Immunoprecipitation, Western Blot, Expressing

    8) Product Images from "F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2"

    Article Title: F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    Journal: Scientific Reports

    doi: 10.1038/srep29834

    Subcellular location of NLRP3 inflammasome requires F-actin but not active polymerization. ( A,B ) Primed THP-1 cells were activated with ( A ) ATP and ( B ) nigericin after pretreatment with cytochalasin D (CytoD) or latrunculin B (LatB). Cytosolic (CYT) and cytoskeletal (CSK) fractions were analyzed by Western blot. The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of subcellular location of ASC and NLRP3 in primed THP-1 cells treated or not with cytochalasin D (CytoD) and latrunculin B (LatB) prior to activation with ATP or nigericin for 6 h; Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments.
    Figure Legend Snippet: Subcellular location of NLRP3 inflammasome requires F-actin but not active polymerization. ( A,B ) Primed THP-1 cells were activated with ( A ) ATP and ( B ) nigericin after pretreatment with cytochalasin D (CytoD) or latrunculin B (LatB). Cytosolic (CYT) and cytoskeletal (CSK) fractions were analyzed by Western blot. The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of subcellular location of ASC and NLRP3 in primed THP-1 cells treated or not with cytochalasin D (CytoD) and latrunculin B (LatB) prior to activation with ATP or nigericin for 6 h; Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments.

    Techniques Used: Western Blot, Confocal Microscopy, Activation Assay

    F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .
    Figure Legend Snippet: F-actin downregulates NLRP3 inflammasome activity. ( A ) Actin polymerization was performed with 9.6 μM of pyrene-labeled G-actin containing ATP- or nigericin-activated THP-1 cells lysate. The polymerization was initiated by addition of actin polymerization buffer (arrow). Representative experiments out of 3 are presented. ( B ) Area under the curve (AUC) represented in ( A ) was calculated using GraphPad Prism version 6. Data are means ± SEM of at least 3 independent. ( C,D ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and then activated by ATP ( C ) and nigericin ( D ) for 6 h. Data are means ± SEM of at least 3 independent. ( E,F ) IL-1β production in culture supernatants of LPS-primed primary human monocytes pretreated with increasing doses of cytochalasin D (CytoD) or latrunculin B (LatB) and stimulated or not with ATP ( E ) or nigericin ( F ) for 15 min. Data are means ± SEM of at least 3 independent. ( G,H ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); total, uncleaved caspase-1 in cell lysate is presented in bottom panels. The cropped blots were run under the same experimental conditions. The asterisk indicates nonspecific crossreactive bands. Data are means ± SEM of at least 4 independent experiments (upper panels) or representative of 3 independent experiments (bottom panels). Statistical significance was determined by Mann-Whitney U analysis. See also Figures S1 and S2 .

    Techniques Used: Activity Assay, Labeling, Enzyme-linked Immunosorbent Assay, Western Blot, MANN-WHITNEY

    Ca 2+ increase the NLRP3 inflammasome activation by enhancing the severing of F-actin by FliI. ( A ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of CaCl 2 (Ca 2+ ) and stimulated with nigericin. Data are means ± SEM of at least 3 independent. ( B ) Caspase-1 in supernatants (SN) of THP-1 cells pretreated with 1.6 mM CaCl 2 and activated by nigericin as indicated and assessed by ELISA. Data are means ± SEM of at least 3 independent. ( C ) Actin depolymerization was performed with 0.2 μg/ml of pyrene-labeled F-actin containing nigericin-activated THP-1 cells lysate. Representative pictures of 3 independent experiments. ( D ) Area under the curve (AUC) represented in ( C ) was calculated using GraphPad Prism version 6. Data are represented as mean ± SEM of at least 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. See Figure S4 .
    Figure Legend Snippet: Ca 2+ increase the NLRP3 inflammasome activation by enhancing the severing of F-actin by FliI. ( A ) IL-1β production in culture supernatants of primed THP-1 cells pretreated with increasing doses of CaCl 2 (Ca 2+ ) and stimulated with nigericin. Data are means ± SEM of at least 3 independent. ( B ) Caspase-1 in supernatants (SN) of THP-1 cells pretreated with 1.6 mM CaCl 2 and activated by nigericin as indicated and assessed by ELISA. Data are means ± SEM of at least 3 independent. ( C ) Actin depolymerization was performed with 0.2 μg/ml of pyrene-labeled F-actin containing nigericin-activated THP-1 cells lysate. Representative pictures of 3 independent experiments. ( D ) Area under the curve (AUC) represented in ( C ) was calculated using GraphPad Prism version 6. Data are represented as mean ± SEM of at least 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. See Figure S4 .

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Labeling, MANN-WHITNEY

    FliI and LRRFIP2 enable inhibition of NLRP3 inflammasome activity via co-localization with F-actin. ( A ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( B ) Western blot analysis of FliI and LRRFIP2 expression in THP-1 cells stably transduced with lentivirus carrying FliI and LRRFIP2 shRNA. Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells transduced with FliI and LRRFIP2 shRNA. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( D ) IL-1β production in culture supernatants of THP-1 cells transduced with FliI and LRRFIP2 shRNA and unstimulated or stimulated with ATP and nigericin. Data are means ± SEM of at least 3 independent. ( E ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); the presence of caspase-1 was assessed by Western blot into cells lysate. Data are represented as mean ± SEM of at least 3 independent experiments or representative of 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. The cropped blots were run under the same experimental conditions. See Figure S3 .
    Figure Legend Snippet: FliI and LRRFIP2 enable inhibition of NLRP3 inflammasome activity via co-localization with F-actin. ( A ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( B ) Western blot analysis of FliI and LRRFIP2 expression in THP-1 cells stably transduced with lentivirus carrying FliI and LRRFIP2 shRNA. Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells transduced with FliI and LRRFIP2 shRNA. Blue, nuclei. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Representative pictures of 3 independent experiments. ( D ) IL-1β production in culture supernatants of THP-1 cells transduced with FliI and LRRFIP2 shRNA and unstimulated or stimulated with ATP and nigericin. Data are means ± SEM of at least 3 independent. ( E ) Caspase-1 in supernatants (SN) of THP-1 cells treated as indicated and assessed by ELISA (upper panels) and Western blot (bottom panels); the presence of caspase-1 was assessed by Western blot into cells lysate. Data are represented as mean ± SEM of at least 3 independent experiments or representative of 3 independent experiments. Statistical significance was determined by Mann-Whitney U analysis. The cropped blots were run under the same experimental conditions. See Figure S3 .

    Techniques Used: Inhibition, Activity Assay, Confocal Microscopy, Western Blot, Expressing, Stable Transfection, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Schematic representation of NLRP3 inflammasome regulation. TLRs activation or PMA (Signal 1) induce the expression of pro-IL-1β, pro-IL-18 and NLRP3. Signal 2 provided by ATP or nigericin induces NLRP3 inflammasome assembly through a dynein- and microtubules-dependent transport. Then, we postulate that the increase of intracellular Ca 2+ concentration through channels such as TRPM7, TRPV2 and/or InsP 3 R, enhances the ability of FliI to sever F-actin and thus abrogates LRRFIP2-FliI-dependent NLRP3 inflammasome inhibition increasing IL-1β and IL-18 production. The colored part of the picture is the present study.
    Figure Legend Snippet: Schematic representation of NLRP3 inflammasome regulation. TLRs activation or PMA (Signal 1) induce the expression of pro-IL-1β, pro-IL-18 and NLRP3. Signal 2 provided by ATP or nigericin induces NLRP3 inflammasome assembly through a dynein- and microtubules-dependent transport. Then, we postulate that the increase of intracellular Ca 2+ concentration through channels such as TRPM7, TRPV2 and/or InsP 3 R, enhances the ability of FliI to sever F-actin and thus abrogates LRRFIP2-FliI-dependent NLRP3 inflammasome inhibition increasing IL-1β and IL-18 production. The colored part of the picture is the present study.

    Techniques Used: Activation Assay, Expressing, Concentration Assay, Inhibition

    NLRP3 inflammasome interacts with F-actin in ATP- and nigericin-treated THP-1 cells. ( A,B ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h. Cytosolic (CYT) and cytoskeletal (CSK) fractions of ( A ) ATP- and ( B ) nigericin-activated cells were subjected to Western blot and analyzed for the presence of ASC, NLRP3, pro-IL-1β and vimentin (control for cytoskeletal fraction). The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells; nuclei are stained in blue. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Data are representative of 3 independent experiments. ( D ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h, immunoprecipitated with anti-actin and subjected to Western blot and analyzed for the presence of ASC, NLRP3 and actin. The cropped blots were run under the same experimental conditions; Data are representative of 2 independent experiments.
    Figure Legend Snippet: NLRP3 inflammasome interacts with F-actin in ATP- and nigericin-treated THP-1 cells. ( A,B ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h. Cytosolic (CYT) and cytoskeletal (CSK) fractions of ( A ) ATP- and ( B ) nigericin-activated cells were subjected to Western blot and analyzed for the presence of ASC, NLRP3, pro-IL-1β and vimentin (control for cytoskeletal fraction). The cropped blots were run under the same experimental conditions; Data are representative of 3 independent experiments. ( C ) Confocal microscopy of ATP- and nigericin-activated THP-1 cells; nuclei are stained in blue. Outlined areas are enlarged in top right corners. Scale bars, 10 μm. Data are representative of 3 independent experiments. ( D ) Primed THP-1 cells were activated with 5 mM ATP or 1 μM nigericin (NIG) for 6 h, immunoprecipitated with anti-actin and subjected to Western blot and analyzed for the presence of ASC, NLRP3 and actin. The cropped blots were run under the same experimental conditions; Data are representative of 2 independent experiments.

    Techniques Used: Western Blot, Confocal Microscopy, Staining, Immunoprecipitation

    9) Product Images from "β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation"

    Article Title: β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation

    Journal: Cancer Cell International

    doi: 10.1186/s12935-019-1029-1

    Dlg5 is associated with β-TrCP. a The interaction protein network of AEBP2 revealed by the BioGRID database. b 293T cell were transfected with Flag-Dlg5 and HA-β-TrCP for 36 h. Flag-Dlg5 protein complex was immunoprecipitated with Flag M2 beads. The Flag-DLG5 immunoprecipitate was detected by western blot using indicated antibodies. c SMMC-7721 cell lysate was subjected to immunoprecipitation by anti-Dlg5 antibody. Immunoprecipitates was detected by western blot using indicated antibodies
    Figure Legend Snippet: Dlg5 is associated with β-TrCP. a The interaction protein network of AEBP2 revealed by the BioGRID database. b 293T cell were transfected with Flag-Dlg5 and HA-β-TrCP for 36 h. Flag-Dlg5 protein complex was immunoprecipitated with Flag M2 beads. The Flag-DLG5 immunoprecipitate was detected by western blot using indicated antibodies. c SMMC-7721 cell lysate was subjected to immunoprecipitation by anti-Dlg5 antibody. Immunoprecipitates was detected by western blot using indicated antibodies

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    β-TrCP regulates the stability and ubiquitination of Dlg5. a Western blot analysis of SMMC-7721 cells transfected with indicated doses of Flag-β-TrCP. b Western blot analysis of SMMC-7721 cells transfected with siRNAs against control or β-TrCP. c SMMC-7721 cells transfected with siRNAs against control or β-TrCP for 24 h, then 20 μg/ml cycloheximide (CHX) was added for the indicated time course. Cell extracts were subjected to western blot with the indicated antibodies. d SMMC-7721 cells transfected with siRNAs against control or β-TrCP for 36 h, cell lysate was subjected to immunoprecipitation by anti-Dlg5 antibody. Immunoprecipitates was detected by western blot using indicated antibodies. e Alignment of amino acids corresponding to the DSGxxxE sequence with Dlg5 orthologs. f 293T cells were co-transfected Flag-β-TrCP with HA-Dlg5 WT or HA-Dlg5 S730A for 36 h, cell lysate was subjected to immunoprecipitation by anti-Flag antibody. Immunoprecipitates was detected by western blot using indicated antibodies. g 293T cells transfected with indicated plasmids for 36 h, cell lysate was subjected to western blot detection. h 293T cells were co-transfected Flag-β-TrCP and his-K48-ub with HA-Dlg5 WT or HA-Dlg5 S730A for 36 h, cell lysate was subjected to immunoprecipitation by Ni+ purification. Immunoprecipitate was detected by western blot using indicated antibodies
    Figure Legend Snippet: β-TrCP regulates the stability and ubiquitination of Dlg5. a Western blot analysis of SMMC-7721 cells transfected with indicated doses of Flag-β-TrCP. b Western blot analysis of SMMC-7721 cells transfected with siRNAs against control or β-TrCP. c SMMC-7721 cells transfected with siRNAs against control or β-TrCP for 24 h, then 20 μg/ml cycloheximide (CHX) was added for the indicated time course. Cell extracts were subjected to western blot with the indicated antibodies. d SMMC-7721 cells transfected with siRNAs against control or β-TrCP for 36 h, cell lysate was subjected to immunoprecipitation by anti-Dlg5 antibody. Immunoprecipitates was detected by western blot using indicated antibodies. e Alignment of amino acids corresponding to the DSGxxxE sequence with Dlg5 orthologs. f 293T cells were co-transfected Flag-β-TrCP with HA-Dlg5 WT or HA-Dlg5 S730A for 36 h, cell lysate was subjected to immunoprecipitation by anti-Flag antibody. Immunoprecipitates was detected by western blot using indicated antibodies. g 293T cells transfected with indicated plasmids for 36 h, cell lysate was subjected to western blot detection. h 293T cells were co-transfected Flag-β-TrCP and his-K48-ub with HA-Dlg5 WT or HA-Dlg5 S730A for 36 h, cell lysate was subjected to immunoprecipitation by Ni+ purification. Immunoprecipitate was detected by western blot using indicated antibodies

    Techniques Used: Western Blot, Transfection, Immunoprecipitation, Sequencing, Purification

    Dlg5 is regulated by the ubiquitin proteasome system via an SCF ubiquitin ligase complex. a Western blot analysis of SMMC-7721 cells treated with DMSO or 10 μM MG132 for 4 h. b Western blot analysis of HepG-2 cells treated with DMSO or 10 μM MG132 for 4 h. c 293T cells were transfected with Flag-Con or Flag-Dlg5 for 36 h, cells were then treated with 10 μM MG132 for 4 h and subjected to western blot analysis with indicated antibodies. d Western blot analysis of 293T cells treated with indicated doses of MLN4924 for 4 h. e Western blot analysis of 293T cells transfected with DN-Cullin1, DN-Cullin2, DN-Cullin3, DN-Cullin4A or DN-Cullin4B plasmid, respectively. f Western blot analysis of SMMC-7721 cells transfected with siRNAs against control or Cullin1. g 293T cells were transfected with Flag-Con or Flag-Dlg5 for 36 h, cell lysate was subjected to immunoprecipitation by FlagM2 antibody. Immunoprecipitates were detected by western blot using indicated antibodies
    Figure Legend Snippet: Dlg5 is regulated by the ubiquitin proteasome system via an SCF ubiquitin ligase complex. a Western blot analysis of SMMC-7721 cells treated with DMSO or 10 μM MG132 for 4 h. b Western blot analysis of HepG-2 cells treated with DMSO or 10 μM MG132 for 4 h. c 293T cells were transfected with Flag-Con or Flag-Dlg5 for 36 h, cells were then treated with 10 μM MG132 for 4 h and subjected to western blot analysis with indicated antibodies. d Western blot analysis of 293T cells treated with indicated doses of MLN4924 for 4 h. e Western blot analysis of 293T cells transfected with DN-Cullin1, DN-Cullin2, DN-Cullin3, DN-Cullin4A or DN-Cullin4B plasmid, respectively. f Western blot analysis of SMMC-7721 cells transfected with siRNAs against control or Cullin1. g 293T cells were transfected with Flag-Con or Flag-Dlg5 for 36 h, cell lysate was subjected to immunoprecipitation by FlagM2 antibody. Immunoprecipitates were detected by western blot using indicated antibodies

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation

    10) Product Images from "The non-structural protein μNS of piscine orthoreovirus (PRV) forms viral factory-like structures"

    Article Title: The non-structural protein μNS of piscine orthoreovirus (PRV) forms viral factory-like structures

    Journal: Veterinary Research

    doi: 10.1186/s13567-015-0302-0

    Co-transfections with μNS. EPC cells transfected with constructs encoding σNS, μ2 and λ1 and co-transfected with µNS. The cells were processed for confocal microscopy 48 hpt. A EPC cells transfected with σNS alone and cotransfected with μNS. B EPC cells transfected with μ2 alone and cotransfected with μNS. C EPC cells transfected with λ1 alone and cotransfected with μNS.
    Figure Legend Snippet: Co-transfections with μNS. EPC cells transfected with constructs encoding σNS, μ2 and λ1 and co-transfected with µNS. The cells were processed for confocal microscopy 48 hpt. A EPC cells transfected with σNS alone and cotransfected with μNS. B EPC cells transfected with μ2 alone and cotransfected with μNS. C EPC cells transfected with λ1 alone and cotransfected with μNS.

    Techniques Used: Transfection, Construct, Confocal Microscopy

    Co-transfections with truncated μNS variants. EPC cells transfected with pcDNA3.1-μNS-Δ743-752, pcDNA3.1-μNS-Δ736-752, pcDNA3.1-μNS-Δ1-401 and pcDNA3.1-μNS-Δ402-752 processed for fluorescence microscopy 48 hpt. A EPC cells expressing μNSΔ743-752 alone and co-expressed with σNS. B EPC cells expressing μNSΔ736-752 alone and co-expressed with σNS. C EPC cells expressing μNSΔ402-752 alone and co-expressed with σNS. D EPC cells expressing μNSΔ1-401 alone and co-expressed with σNS.
    Figure Legend Snippet: Co-transfections with truncated μNS variants. EPC cells transfected with pcDNA3.1-μNS-Δ743-752, pcDNA3.1-μNS-Δ736-752, pcDNA3.1-μNS-Δ1-401 and pcDNA3.1-μNS-Δ402-752 processed for fluorescence microscopy 48 hpt. A EPC cells expressing μNSΔ743-752 alone and co-expressed with σNS. B EPC cells expressing μNSΔ736-752 alone and co-expressed with σNS. C EPC cells expressing μNSΔ402-752 alone and co-expressed with σNS. D EPC cells expressing μNSΔ1-401 alone and co-expressed with σNS.

    Techniques Used: Transfection, Fluorescence, Microscopy, Expressing

    11) Product Images from "Viral targeting of TFIIB impairs de novo polymerase II recruitment and affects antiviral immunity"

    Article Title: Viral targeting of TFIIB impairs de novo polymerase II recruitment and affects antiviral immunity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006980

    Thogoto virus ML protein inhibits IFN-α/β production by interacting with TFIIB. A) Volcano plot of proteins enriched in ML vs. M pulldown in HEK293 cells and identified by AP-LC-MS/MS. HA-tagged M or ML proteins were overexpressed in 4 biological replicates. B) Schematic representation of ML protein and its mutants not binding TFIIB or CAPN15. C) IP of GST-tagged M or ML (wt and mut) and co-IP of FLAG-tagged CAPN15 and TFIIB transiently overexpressed in HEK293 cells. Western blot is a representative of two independent experiments with similar results. D) IFN-α/β levels after infection with THOV wt, ΔML or mutant ML(SW) 24 h.p.i. SN from infected HeLa cells were applied to 293T Mx1-luc. *—p value
    Figure Legend Snippet: Thogoto virus ML protein inhibits IFN-α/β production by interacting with TFIIB. A) Volcano plot of proteins enriched in ML vs. M pulldown in HEK293 cells and identified by AP-LC-MS/MS. HA-tagged M or ML proteins were overexpressed in 4 biological replicates. B) Schematic representation of ML protein and its mutants not binding TFIIB or CAPN15. C) IP of GST-tagged M or ML (wt and mut) and co-IP of FLAG-tagged CAPN15 and TFIIB transiently overexpressed in HEK293 cells. Western blot is a representative of two independent experiments with similar results. D) IFN-α/β levels after infection with THOV wt, ΔML or mutant ML(SW) 24 h.p.i. SN from infected HeLa cells were applied to 293T Mx1-luc. *—p value

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Infection, Mutagenesis

    12) Product Images from "General Anesthetics Inhibit Erythropoietin Induction under Hypoxic Conditions in the Mouse Brain"

    Article Title: General Anesthetics Inhibit Erythropoietin Induction under Hypoxic Conditions in the Mouse Brain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029378

    Expression analysis of HIF-1α, HIF-2α, and ARNT protein in primary cultured astrocytes by immunoblotting. Primary cultured astrocytes were incubated under hypoxic (1% O 2 ) conditions with or without 1.5% isoflurane, 1 mM pentobarbital, or 1 mM ketamine for 4 hours. Whole cell lysates were analyzed for HIF-1α, HIF-2α, ARNT, GFAP, NeuN and β-actin protein expression by immunoblot assay. Figures are representative of at least three independent experiments.
    Figure Legend Snippet: Expression analysis of HIF-1α, HIF-2α, and ARNT protein in primary cultured astrocytes by immunoblotting. Primary cultured astrocytes were incubated under hypoxic (1% O 2 ) conditions with or without 1.5% isoflurane, 1 mM pentobarbital, or 1 mM ketamine for 4 hours. Whole cell lysates were analyzed for HIF-1α, HIF-2α, ARNT, GFAP, NeuN and β-actin protein expression by immunoblot assay. Figures are representative of at least three independent experiments.

    Techniques Used: Expressing, Cell Culture, Incubation

    13) Product Images from "Amyloid-β with isomerized Asp7 cytotoxicity is coupled to protein phosphorylation"

    Article Title: Amyloid-β with isomerized Asp7 cytotoxicity is coupled to protein phosphorylation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21815-x

    Effects of Aβ42 and isoAβ42 (10 μM, 6 h) treatment on the levels of phosphoserine-containing proteins. After incubation with the amyloid peptides, SH-SY5Y cells were lysed, the proteins were separated using 8% SDS-PAGE, and antibodies against phosphoserine were used to identify phosphoserine-containing protein bands.
    Figure Legend Snippet: Effects of Aβ42 and isoAβ42 (10 μM, 6 h) treatment on the levels of phosphoserine-containing proteins. After incubation with the amyloid peptides, SH-SY5Y cells were lysed, the proteins were separated using 8% SDS-PAGE, and antibodies against phosphoserine were used to identify phosphoserine-containing protein bands.

    Techniques Used: Incubation, SDS Page

    Effects of Aβ42 and isoAβ42 (10 μM, 6 h) treatment on procaspase 3 levels in SH-SY5Y cells. After peptide treatment, the cells were lysed, the isolated proteins were separated by SDS-PAGE, caspase 3 and actin were detected by Western blot using the appropriate antibodies. The bars represent procaspase 3 expression changes. Each value represents the mean ± SD of at least three independent experiments; *p
    Figure Legend Snippet: Effects of Aβ42 and isoAβ42 (10 μM, 6 h) treatment on procaspase 3 levels in SH-SY5Y cells. After peptide treatment, the cells were lysed, the isolated proteins were separated by SDS-PAGE, caspase 3 and actin were detected by Western blot using the appropriate antibodies. The bars represent procaspase 3 expression changes. Each value represents the mean ± SD of at least three independent experiments; *p

    Techniques Used: Isolation, SDS Page, Western Blot, Expressing

    Effect of Aβ42 and isoAβ42 (10 μM, 6 h) treatment on the total tau protein level. After incubation with the amyloid peptides, the SH-SY5Y cells were lysed, the proteins in the cell lysate were separated by SDS-PAGE, and total tau was detected by Western blot using antibodies against total tau. The total tau level is expressed as a percentage of the tau level in the control group, which was not treated with Aβs. The bars represent the changes in the total tau level. Each value is the mean ± SD of at least three independent experiments; *p
    Figure Legend Snippet: Effect of Aβ42 and isoAβ42 (10 μM, 6 h) treatment on the total tau protein level. After incubation with the amyloid peptides, the SH-SY5Y cells were lysed, the proteins in the cell lysate were separated by SDS-PAGE, and total tau was detected by Western blot using antibodies against total tau. The total tau level is expressed as a percentage of the tau level in the control group, which was not treated with Aβs. The bars represent the changes in the total tau level. Each value is the mean ± SD of at least three independent experiments; *p

    Techniques Used: Incubation, SDS Page, Western Blot

    Effect of Aβ42 and isoAβ42 treatment (10 μM, 24 h) on the total tau level and the phosphorylated tau level in SH-SY5Y cells. After incubation with Aβ42 or isoAβ42, the cells were lysed, and the proteins were separated by SDS-PAGE and detected by Western blot using antibodies against total tau, phosphorylated S396 tau, phosphorylated T231 tau or phosphorylated S262 tau ( A ). The bars represent the changes in the total tau level ( B ), phosphorylated S396 tau ( C ), phosphorylated T231 tau ( D ) or phosphorylated S262 tau ( E ), expressed as a percentage of the total tau level in the control group, which was not treated with Aβ. Changes to tau phosphorylation levels were normalized to its total amount. Each value is the mean ± SD of at least three independent experiments; *p
    Figure Legend Snippet: Effect of Aβ42 and isoAβ42 treatment (10 μM, 24 h) on the total tau level and the phosphorylated tau level in SH-SY5Y cells. After incubation with Aβ42 or isoAβ42, the cells were lysed, and the proteins were separated by SDS-PAGE and detected by Western blot using antibodies against total tau, phosphorylated S396 tau, phosphorylated T231 tau or phosphorylated S262 tau ( A ). The bars represent the changes in the total tau level ( B ), phosphorylated S396 tau ( C ), phosphorylated T231 tau ( D ) or phosphorylated S262 tau ( E ), expressed as a percentage of the total tau level in the control group, which was not treated with Aβ. Changes to tau phosphorylation levels were normalized to its total amount. Each value is the mean ± SD of at least three independent experiments; *p

    Techniques Used: Incubation, SDS Page, Western Blot

    Visualization of changes in the isoelectric points of tubulin proteins after SH-SY5Y cells were treated with Aβ42 and isoAβ42 peptides (10 μM, 6 h). 35 S-labelled proteins from control SH-SY5Y cells ( I ) and cells treated with Aβ42 ( II ) or isoAβ42 ( III ) were separated by 2D electrophoresis. The double arrow shows *the position of the acid end of β-tubulin in the control cells and ** the position of α-tubulin in the control cells. Protein identification: 1 - α-tubulin; 2a - β-tubulin (TBB5); 2b - β-tubulin (TBB4); 3-– actin (ACTB).
    Figure Legend Snippet: Visualization of changes in the isoelectric points of tubulin proteins after SH-SY5Y cells were treated with Aβ42 and isoAβ42 peptides (10 μM, 6 h). 35 S-labelled proteins from control SH-SY5Y cells ( I ) and cells treated with Aβ42 ( II ) or isoAβ42 ( III ) were separated by 2D electrophoresis. The double arrow shows *the position of the acid end of β-tubulin in the control cells and ** the position of α-tubulin in the control cells. Protein identification: 1 - α-tubulin; 2a - β-tubulin (TBB5); 2b - β-tubulin (TBB4); 3-– actin (ACTB).

    Techniques Used: Two-Dimensional Gel Electrophoresis

    Western blot analysis of 2D-gels from SH-SY5Y control cells and SH-SY5Y cells treated with Aβ42 and isoAβ42 (10 μM, 6 h). ( A ) A merged image of anti-actin, anti-matrin 3, anti-total tau, anti-β-tubulin, anti-α-tubulin and anti-HSP90 antibodies (1 - α-tubulin, 2 - β-tubulin, 3 - matrin 3, 4 - actin, 5 - total tau, 6 – HSP90). ( B ) Anti-phosphoserine antibodies (1 - α-tubulin, 2 - β-tubulin, 3 - HNRH1, 4 - matrin 3).
    Figure Legend Snippet: Western blot analysis of 2D-gels from SH-SY5Y control cells and SH-SY5Y cells treated with Aβ42 and isoAβ42 (10 μM, 6 h). ( A ) A merged image of anti-actin, anti-matrin 3, anti-total tau, anti-β-tubulin, anti-α-tubulin and anti-HSP90 antibodies (1 - α-tubulin, 2 - β-tubulin, 3 - matrin 3, 4 - actin, 5 - total tau, 6 – HSP90). ( B ) Anti-phosphoserine antibodies (1 - α-tubulin, 2 - β-tubulin, 3 - HNRH1, 4 - matrin 3).

    Techniques Used: Western Blot

    Effects of Aβ42 and isoAβ42 (10 µM) on SH-SY5Y cells. ( A ) Visualization of the cells using fluorescent microscopy. Left, untreated cells. Centre, cells treated with Aβ42. Right, cells treated with isoAβ42. Arrows indicate the following: 1 - apoptotic cells stained with Hoechst 33342; 2 - living cells; 3 - necrotic cells stained with propidium iodide. Scale is 20 µm. ( B ) Number of apoptotic cells in the cell population. Control - untreated cells; isoAβ42 and Aβ42 - cells treated with isoAβ42 and Aβ42 for 24 h, respectively. ( C ) Viability of the cells in percent, revealed by MTT-test treated with Aβ42 or isoAβ42 relative to control without treatment at 48 h. Each value represents mean ± SD of at least three independent experiments performed in quadruplicate; *p
    Figure Legend Snippet: Effects of Aβ42 and isoAβ42 (10 µM) on SH-SY5Y cells. ( A ) Visualization of the cells using fluorescent microscopy. Left, untreated cells. Centre, cells treated with Aβ42. Right, cells treated with isoAβ42. Arrows indicate the following: 1 - apoptotic cells stained with Hoechst 33342; 2 - living cells; 3 - necrotic cells stained with propidium iodide. Scale is 20 µm. ( B ) Number of apoptotic cells in the cell population. Control - untreated cells; isoAβ42 and Aβ42 - cells treated with isoAβ42 and Aβ42 for 24 h, respectively. ( C ) Viability of the cells in percent, revealed by MTT-test treated with Aβ42 or isoAβ42 relative to control without treatment at 48 h. Each value represents mean ± SD of at least three independent experiments performed in quadruplicate; *p

    Techniques Used: Microscopy, Staining, MTT Assay

    Visualization of the changes in protein phosphorylation levels after SH-SY5Y cells incubation with Aβ42 or isoAβ42 (10 μM, 6 h). Extracted proteins were separated by 2D electrophoresis. In the second dimension, proteins were separated on 11% SDS-PAGE, followed by Pro-Q diamond phosphoprotein staining and after image acquisition were restained with Comassie R -250 ( A ). 1 – α-tubulin, 2 - β-tubulin, 3- matrin 3. Densitometric analysis was performed using Fiji ImageJ software. Phosphorylated matrin 3 ( B ), α-tubulin ( C ) and β-tubulin ( D ) stained with Pro-Q diamond phosphoprotein stain were normalized to its total amount stained with Coomassie R-250. Each value is the mean expressed as a percentage of the protein phosphorylation level in the control group ± SD of at least three independent experiments; *p
    Figure Legend Snippet: Visualization of the changes in protein phosphorylation levels after SH-SY5Y cells incubation with Aβ42 or isoAβ42 (10 μM, 6 h). Extracted proteins were separated by 2D electrophoresis. In the second dimension, proteins were separated on 11% SDS-PAGE, followed by Pro-Q diamond phosphoprotein staining and after image acquisition were restained with Comassie R -250 ( A ). 1 – α-tubulin, 2 - β-tubulin, 3- matrin 3. Densitometric analysis was performed using Fiji ImageJ software. Phosphorylated matrin 3 ( B ), α-tubulin ( C ) and β-tubulin ( D ) stained with Pro-Q diamond phosphoprotein stain were normalized to its total amount stained with Coomassie R-250. Each value is the mean expressed as a percentage of the protein phosphorylation level in the control group ± SD of at least three independent experiments; *p

    Techniques Used: Incubation, Two-Dimensional Gel Electrophoresis, SDS Page, Staining, Software

    14) Product Images from "SUV39H2 methylates and stabilizes LSD1 by inhibiting polyubiquitination in human cancer cells"

    Article Title: SUV39H2 methylates and stabilizes LSD1 by inhibiting polyubiquitination in human cancer cells

    Journal: Oncotarget

    doi:

    Methylation of LSD1 at lysine 322 is critical for its binding to CoREST, and regulating targeted genes A. 293T cells were individually transfected with FLAG-Mock, FLAG-LSD1 or FLAG-LSD1mutant (K322R) vectors, and cell extracts were immunoprecipitated with anti-FLAG M2 agarose beads. B. Expression levels of LSD1 proteins were examined in stably expressing wild-type LSD1 or mutant LSD1 (K322R) HeLa cells. C. The mRNA levels of CDKN1A and SOX2 were quantified by real-time PCR. All error bars indicate SEM of four independent experiments. P -values were calculated using Student's t -test (* P
    Figure Legend Snippet: Methylation of LSD1 at lysine 322 is critical for its binding to CoREST, and regulating targeted genes A. 293T cells were individually transfected with FLAG-Mock, FLAG-LSD1 or FLAG-LSD1mutant (K322R) vectors, and cell extracts were immunoprecipitated with anti-FLAG M2 agarose beads. B. Expression levels of LSD1 proteins were examined in stably expressing wild-type LSD1 or mutant LSD1 (K322R) HeLa cells. C. The mRNA levels of CDKN1A and SOX2 were quantified by real-time PCR. All error bars indicate SEM of four independent experiments. P -values were calculated using Student's t -test (* P

    Techniques Used: Methylation, Binding Assay, Transfection, Immunoprecipitation, Expressing, Stable Transfection, Mutagenesis, Real-time Polymerase Chain Reaction

    Lys 322 on LSD1 methylation by SUV39H2 both in vitro and in vivo A. In vitro methyltransferase assay indicated that LSD1 peptide (amino acid residues 313-330) was methylated by SUV39H2 but not Lys 322-substituted LSD1 peptide (K322R). Amounts of loading proteins were evaluated by staining the MemCode TM Reversible Protein Stain (Thermo Fisher Scientific). B. Amino acid sequence indicated that the methylation site Lys 322 was highly conserved across species. C. Methylation of LSD1 in human cells was confirmed by in vivo labeling experiment. 293T cells were transfected with FLAG-LSD1-WT or FLAG-LSD1-K322R in the presence of HA-SUV39H2 and treated with methionine-free medium, including cycloheximide and chloramphenicol. They were then labeled with L-[methyl- 3 H] methionine for 3 hours. Cell lysates were immunoprecipitated with FLAG-M2 agarose, and methylated LSD1 was visualized by fluorography. The membrane was immunoblotted with an anti-FLAG (an internal control) antibody.
    Figure Legend Snippet: Lys 322 on LSD1 methylation by SUV39H2 both in vitro and in vivo A. In vitro methyltransferase assay indicated that LSD1 peptide (amino acid residues 313-330) was methylated by SUV39H2 but not Lys 322-substituted LSD1 peptide (K322R). Amounts of loading proteins were evaluated by staining the MemCode TM Reversible Protein Stain (Thermo Fisher Scientific). B. Amino acid sequence indicated that the methylation site Lys 322 was highly conserved across species. C. Methylation of LSD1 in human cells was confirmed by in vivo labeling experiment. 293T cells were transfected with FLAG-LSD1-WT or FLAG-LSD1-K322R in the presence of HA-SUV39H2 and treated with methionine-free medium, including cycloheximide and chloramphenicol. They were then labeled with L-[methyl- 3 H] methionine for 3 hours. Cell lysates were immunoprecipitated with FLAG-M2 agarose, and methylated LSD1 was visualized by fluorography. The membrane was immunoblotted with an anti-FLAG (an internal control) antibody.

    Techniques Used: Methylation, In Vitro, In Vivo, Staining, Sequencing, Labeling, Transfection, Immunoprecipitation

    SUV39H2 stabilizes LSD1 protein A. FLAG-LSD1 was co-expressed with HA-Mock or HA-SUV39H2 into 293T cells. After treating cells with cycloheximide (CHX) (100 μg/ml) for indicated time intervals, expression of LSD1 was examined (left panel). The intensity of LSD1 protein for each time point was quantified by densitometry and plotted (right panel). Results are the mean ± SD of three independent experiments. B. A549 cells were transfected with control EGFP, LSD1 and two different SUV39H2 siRNAs. Expression of LSD1, SUV30H2 and β-Actin (internal control) was examined by western blot (left panel). The mRNA levels of LSD1 and SUV39H2 were quantified by real-time PCR. All error bars indicate SEM of two independent experiments. P -values were calculated using Student's t -test (* P
    Figure Legend Snippet: SUV39H2 stabilizes LSD1 protein A. FLAG-LSD1 was co-expressed with HA-Mock or HA-SUV39H2 into 293T cells. After treating cells with cycloheximide (CHX) (100 μg/ml) for indicated time intervals, expression of LSD1 was examined (left panel). The intensity of LSD1 protein for each time point was quantified by densitometry and plotted (right panel). Results are the mean ± SD of three independent experiments. B. A549 cells were transfected with control EGFP, LSD1 and two different SUV39H2 siRNAs. Expression of LSD1, SUV30H2 and β-Actin (internal control) was examined by western blot (left panel). The mRNA levels of LSD1 and SUV39H2 were quantified by real-time PCR. All error bars indicate SEM of two independent experiments. P -values were calculated using Student's t -test (* P

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

    SUV39H2-dependent LSD1 methylation inhibits LSD1 protein degradation mediated by polyubiquitination A. FLAG-LSD1 and HA-Ubiquitin were co-expressed in 293T cells with or without HA-SUV39H2. Cells were incubated with 5 μM MG115 and 10 μM MG132 for 6 hours before lysis. LSD1 protein was immunoprecipitated with anti-FLAG M2 agarose beads, and polyubiquitinated LSD1 proteins were detected by anti-HA antibody. B. After incubation with siEGFP or siSUV39H2 for 72 hours, A549 (left panel) or SBC5 (right panel) cells were treated with 5 μM MG115 and 10 μM MG132 for 6 hours. Cell lysates were immunoprecipitated with anti-LSD1 antibody. Polyubiquitinated LSD1 protein was detected using an anti-Ubiquitin antibody.
    Figure Legend Snippet: SUV39H2-dependent LSD1 methylation inhibits LSD1 protein degradation mediated by polyubiquitination A. FLAG-LSD1 and HA-Ubiquitin were co-expressed in 293T cells with or without HA-SUV39H2. Cells were incubated with 5 μM MG115 and 10 μM MG132 for 6 hours before lysis. LSD1 protein was immunoprecipitated with anti-FLAG M2 agarose beads, and polyubiquitinated LSD1 proteins were detected by anti-HA antibody. B. After incubation with siEGFP or siSUV39H2 for 72 hours, A549 (left panel) or SBC5 (right panel) cells were treated with 5 μM MG115 and 10 μM MG132 for 6 hours. Cell lysates were immunoprecipitated with anti-LSD1 antibody. Polyubiquitinated LSD1 protein was detected using an anti-Ubiquitin antibody.

    Techniques Used: Methylation, Incubation, Lysis, Immunoprecipitation

    15) Product Images from "GDI-1 preferably interacts with Rab10 in insulin-stimulated GLUT4 translocation"

    Article Title: GDI-1 preferably interacts with Rab10 in insulin-stimulated GLUT4 translocation

    Journal: Biochemical Journal

    doi: 10.1042/BJ20090624

    Rab10 preferably interacts with GDI-1 in vivo ( A ) Interaction of GDI-1 with Rab10 demonstrated by acceptor photobleaching FRET. HEK-293 cells were co-transfected with EGFP–GDI-1 and mKO–Rab10(T23N), and images were collected 36 h post-transfection. Pre-bleaching images of EGFP–GDI-1 and mKO–Rab10 were acquired first (left-hand column), and then cells were exposed to maximal intensity laser at 532 nm for 3 min to bleach mKO–Rab10 (acceptor). After mKO–Rab10 was bleached, post-bleaching images of EGFP–GDI-1 and mKO–Rab10 were acquired (right-hand column). ( B ) Quantification of the interaction of GDI-1 and GDI-2 with Rab10. Intensities of post-bleaching EGFP–GDI images are normalized with the corresponding pre-bleaching images, and mean intensity increments are displayed. An mKO–EGFP construction in which EGFP is directly fused to the C-terminal of mKO was employed as a positive control, and co-transfection of mKO–Rab10(T23N) with the EGFP empty vector was taken as a negative control. Results are represented as the means±S.E.M. (GDI-1, n =31 cells; GDI-2, n =35 cells).
    Figure Legend Snippet: Rab10 preferably interacts with GDI-1 in vivo ( A ) Interaction of GDI-1 with Rab10 demonstrated by acceptor photobleaching FRET. HEK-293 cells were co-transfected with EGFP–GDI-1 and mKO–Rab10(T23N), and images were collected 36 h post-transfection. Pre-bleaching images of EGFP–GDI-1 and mKO–Rab10 were acquired first (left-hand column), and then cells were exposed to maximal intensity laser at 532 nm for 3 min to bleach mKO–Rab10 (acceptor). After mKO–Rab10 was bleached, post-bleaching images of EGFP–GDI-1 and mKO–Rab10 were acquired (right-hand column). ( B ) Quantification of the interaction of GDI-1 and GDI-2 with Rab10. Intensities of post-bleaching EGFP–GDI images are normalized with the corresponding pre-bleaching images, and mean intensity increments are displayed. An mKO–EGFP construction in which EGFP is directly fused to the C-terminal of mKO was employed as a positive control, and co-transfection of mKO–Rab10(T23N) with the EGFP empty vector was taken as a negative control. Results are represented as the means±S.E.M. (GDI-1, n =31 cells; GDI-2, n =35 cells).

    Techniques Used: In Vivo, Transfection, Positive Control, Cotransfection, Plasmid Preparation, Negative Control

    16) Product Images from "C-Terminal Region of EBNA-2 Determines the Superior Transforming Ability of Type 1 Epstein-Barr Virus by Enhanced Gene Regulation of LMP-1 and CXCR7"

    Article Title: C-Terminal Region of EBNA-2 Determines the Superior Transforming Ability of Type 1 Epstein-Barr Virus by Enhanced Gene Regulation of LMP-1 and CXCR7

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002164

    Characterization of oestrogen-independent LCLs established from EREB2.5 cells expressing chimaeras 2 and 7. EREB2.5 cells transfected with OriP-p294 plasmids coding for type 1 EBNA-2 ( A ) or chimaeras 2 and 7 ( B ) and grown in the absence of oestrogen gave rise to continuously proliferating LCLs. Protein samples were harvested from these LCLs at each month after transfection over a period of 6 months ( A ) or after 1.5 and 3 months ( B ) and analyzed by western blotting. The anti-EBNA-2 antibody (PE2), detected the transfected type 1 (E2) and chimaeric (chim E2) EBNA-2 proteins as well as the endogenous ER-EBNA-2 (ER-E2). EBNA-LP immunoblotting was performed with the antibodies JF186 (type 1-specific) and 4D3 (recognizes both types). At any time-point analyzed, both antibodies detected a type 1 3-repeat EBNA-LP species (T1 3R), expressed from the endogenous p554-4 plasmid, indicating that the plasmid was not lost from the cells. The number of repeats was judged by comparison to 293 cells transfected with a type 1 3-repeat EBNA-LP-expressing vector (T1ELP3R). A type 2 Y1Y2-truncated isoform of around 52 kDa (T2 ΔY1Y2), expressed by the resident P3HR1 genome, was also detected by the 4D3 antibody. Numbers alongside the EBNA-LP immunoblot panel represent protein molecular weight (in kDa). β-actin immunoblots ensure equal loading of the proteins.
    Figure Legend Snippet: Characterization of oestrogen-independent LCLs established from EREB2.5 cells expressing chimaeras 2 and 7. EREB2.5 cells transfected with OriP-p294 plasmids coding for type 1 EBNA-2 ( A ) or chimaeras 2 and 7 ( B ) and grown in the absence of oestrogen gave rise to continuously proliferating LCLs. Protein samples were harvested from these LCLs at each month after transfection over a period of 6 months ( A ) or after 1.5 and 3 months ( B ) and analyzed by western blotting. The anti-EBNA-2 antibody (PE2), detected the transfected type 1 (E2) and chimaeric (chim E2) EBNA-2 proteins as well as the endogenous ER-EBNA-2 (ER-E2). EBNA-LP immunoblotting was performed with the antibodies JF186 (type 1-specific) and 4D3 (recognizes both types). At any time-point analyzed, both antibodies detected a type 1 3-repeat EBNA-LP species (T1 3R), expressed from the endogenous p554-4 plasmid, indicating that the plasmid was not lost from the cells. The number of repeats was judged by comparison to 293 cells transfected with a type 1 3-repeat EBNA-LP-expressing vector (T1ELP3R). A type 2 Y1Y2-truncated isoform of around 52 kDa (T2 ΔY1Y2), expressed by the resident P3HR1 genome, was also detected by the 4D3 antibody. Numbers alongside the EBNA-LP immunoblot panel represent protein molecular weight (in kDa). β-actin immunoblots ensure equal loading of the proteins.

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

    In a transient transfection assay in Daudi cells, the weaker induction of LMP-1 by type 2 EBNA-2, compared to the type 1 EBNA-2, is not affected by the EBNA-LP type. 2×10 6 Daudi cells were transiently transfected with an array of combinations of EBNA-2-expressing plasmids (OriP-p294) and EBNA-LP-expressing plasmids (pSNOC) of type 1 (T1) and type 2 (T2). The Neon transfection system (Invitrogen) was used. 24 hours after transfection, cells were lysed in RIPA lysis buffer and analyzed by SDS-PAGE followed by immunoblotting for LMP-1 (CS 1-4 antibody), EBNA-2 (PE2 antibody), EBNA-LP (4D3 antibody) and β-actin (to ensure equal loading of the proteins). The pSNOC plasmids express EBNA-LP proteins with 3 W repeats (3R). Daudi cells already express an endogenous species of EBNA-LP which bears 4 repeats and lacks the Y1Y2 domains (4R ΔY1Y2). Numbers next to the EBNA-LP blot indicate protein molecular weight in kDa. 1 representative experiment of 2 is shown.
    Figure Legend Snippet: In a transient transfection assay in Daudi cells, the weaker induction of LMP-1 by type 2 EBNA-2, compared to the type 1 EBNA-2, is not affected by the EBNA-LP type. 2×10 6 Daudi cells were transiently transfected with an array of combinations of EBNA-2-expressing plasmids (OriP-p294) and EBNA-LP-expressing plasmids (pSNOC) of type 1 (T1) and type 2 (T2). The Neon transfection system (Invitrogen) was used. 24 hours after transfection, cells were lysed in RIPA lysis buffer and analyzed by SDS-PAGE followed by immunoblotting for LMP-1 (CS 1-4 antibody), EBNA-2 (PE2 antibody), EBNA-LP (4D3 antibody) and β-actin (to ensure equal loading of the proteins). The pSNOC plasmids express EBNA-LP proteins with 3 W repeats (3R). Daudi cells already express an endogenous species of EBNA-LP which bears 4 repeats and lacks the Y1Y2 domains (4R ΔY1Y2). Numbers next to the EBNA-LP blot indicate protein molecular weight in kDa. 1 representative experiment of 2 is shown.

    Techniques Used: Transient Transfection Assay, Transfection, Expressing, Lysis, SDS Page, Molecular Weight

    Type 1/type 2 EBNA-2 chimaeras tested in the EREB2.5 growth assay. The carboxyl-terminal region of type 1 EBNA-2 complements the deficiency of type 2 EBNA-2 in the EREB2.5 growth assay. RG, CR7 and TAD domains are the minimum type 1 sequences required. ( A ) Structure of EBNA-2 protein. Type 1 EBNA-2 protein of the prototype B95-8 strain is 487 amino acids long, whereas the type 2 protein from the strain AG876 comprises 455 amino acids. The two types of EBNA-2 proteins share around 50% of sequence identity, which consists mainly of 9 short stretches of homology, named conserved regions (CR1 - CR9). Characteristic parts of the EBNA-2 protein are: two N-terminal self-association domains (SAD1 and SAD2); a poly-proline region (PPR); a diversity region, a sequence with low similarity between EBV types; a region interacting with the cell DNA-binding protein RBP-Jk (RBP-Jk); a short sequence rich in arginine and glycine residues (RG); a transactivation domain (TAD); a carboxyl-terminal nuclear localisation signal (NLS). EBNA-LP indicates regions involved in cooperation between EBNA-2 and EBNA-LP in transcriptional activation. ( B ) Panel of chimaeric proteins (C1 - C7) tested for their ability to rescue proliferation of oestrogen-starved EREB2.5 cells. The growth phenotype was scored as “++” (type 1 and chimaera 2) and “+” (chimaera 7), when cell proliferation was maintained over time after transfection and oestrogen withdrawal and LCLs were successfully established, or as “–” (type 2, chimaeras 1, 3, 4, 5 and 6), when proliferation ceased after transfection. ( C ) Live cell counts of EREB2.5 cells expressing wild-type type 1/type 2 or chimaeric EBNA-2 proteins. EREB2.5 cells were transfected with OriP-p294 plasmids expressing wild-type type 1 (T1), type 2 (T2) EBNA-2, chimaeras 1 to 7 (C1 - C7) or with empty vector (e.v.). Oestrogen was removed from the culture medium and accumulation of proliferating cells was assessed at 1, 2, 3 and 4 weeks after transfection by counting cells that exclude Trypan Blue on a haemocytometer. Data from 1 representative experiment of at least 4 is shown. Transfections were performed in duplicate and for each replica, triplicate wells of cells were counted. Error bars indicate standard deviations. ( D ) Western blot analysis of protein extracts from EREB2.5 cells transfected with wild-type type 1 (E2T1) and type 2 (E2T2) and chimaeric (C1 - C7) EBNA-2 proteins. Cells were harvested 4 days after transfection. EBNA-2 was detected using the PE2 antibody, which recognizes both types. All the chimaeric EBNA-2 proteins were expressed at levels comparable to wild-type type 1 and type 2 and displayed the expected size, which corresponds to 85 kDa (similar to type 1) for chimaera 1 and to 75 kDa (similar to type 2) for all the other chimaeras. ER-EBNA-2 fusion protein (120 kDa) is also detected in every transfection. Triplicate samples were analyzed for each transfection. B95-8 and AG876: positive controls for expression of EBNA-2 type 1 (85 kDa) and type 2 (75 kDa), respectively; E2.5 + est: non-transfected EREB2.5 cells normally grown in medium supplemented with oestrogen. Immunoblotting for β-actin was performed as a loading control.
    Figure Legend Snippet: Type 1/type 2 EBNA-2 chimaeras tested in the EREB2.5 growth assay. The carboxyl-terminal region of type 1 EBNA-2 complements the deficiency of type 2 EBNA-2 in the EREB2.5 growth assay. RG, CR7 and TAD domains are the minimum type 1 sequences required. ( A ) Structure of EBNA-2 protein. Type 1 EBNA-2 protein of the prototype B95-8 strain is 487 amino acids long, whereas the type 2 protein from the strain AG876 comprises 455 amino acids. The two types of EBNA-2 proteins share around 50% of sequence identity, which consists mainly of 9 short stretches of homology, named conserved regions (CR1 - CR9). Characteristic parts of the EBNA-2 protein are: two N-terminal self-association domains (SAD1 and SAD2); a poly-proline region (PPR); a diversity region, a sequence with low similarity between EBV types; a region interacting with the cell DNA-binding protein RBP-Jk (RBP-Jk); a short sequence rich in arginine and glycine residues (RG); a transactivation domain (TAD); a carboxyl-terminal nuclear localisation signal (NLS). EBNA-LP indicates regions involved in cooperation between EBNA-2 and EBNA-LP in transcriptional activation. ( B ) Panel of chimaeric proteins (C1 - C7) tested for their ability to rescue proliferation of oestrogen-starved EREB2.5 cells. The growth phenotype was scored as “++” (type 1 and chimaera 2) and “+” (chimaera 7), when cell proliferation was maintained over time after transfection and oestrogen withdrawal and LCLs were successfully established, or as “–” (type 2, chimaeras 1, 3, 4, 5 and 6), when proliferation ceased after transfection. ( C ) Live cell counts of EREB2.5 cells expressing wild-type type 1/type 2 or chimaeric EBNA-2 proteins. EREB2.5 cells were transfected with OriP-p294 plasmids expressing wild-type type 1 (T1), type 2 (T2) EBNA-2, chimaeras 1 to 7 (C1 - C7) or with empty vector (e.v.). Oestrogen was removed from the culture medium and accumulation of proliferating cells was assessed at 1, 2, 3 and 4 weeks after transfection by counting cells that exclude Trypan Blue on a haemocytometer. Data from 1 representative experiment of at least 4 is shown. Transfections were performed in duplicate and for each replica, triplicate wells of cells were counted. Error bars indicate standard deviations. ( D ) Western blot analysis of protein extracts from EREB2.5 cells transfected with wild-type type 1 (E2T1) and type 2 (E2T2) and chimaeric (C1 - C7) EBNA-2 proteins. Cells were harvested 4 days after transfection. EBNA-2 was detected using the PE2 antibody, which recognizes both types. All the chimaeric EBNA-2 proteins were expressed at levels comparable to wild-type type 1 and type 2 and displayed the expected size, which corresponds to 85 kDa (similar to type 1) for chimaera 1 and to 75 kDa (similar to type 2) for all the other chimaeras. ER-EBNA-2 fusion protein (120 kDa) is also detected in every transfection. Triplicate samples were analyzed for each transfection. B95-8 and AG876: positive controls for expression of EBNA-2 type 1 (85 kDa) and type 2 (75 kDa), respectively; E2.5 + est: non-transfected EREB2.5 cells normally grown in medium supplemented with oestrogen. Immunoblotting for β-actin was performed as a loading control.

    Techniques Used: Growth Assay, Sequencing, Binding Assay, Activation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot

    17) Product Images from "Myriocin-mediated up-regulation of hepatocyte apoA-I synthesis is associated with ERK inhibition"

    Article Title: Myriocin-mediated up-regulation of hepatocyte apoA-I synthesis is associated with ERK inhibition

    Journal: Clinical Science (London, England : 1979)

    doi: 10.1042/CS20090452

    Myriocin-mediated up-regulation of HepG2 cell apoA-I production is associated with inhibition of ERK phosphorylation (A) HepG2 cells were treated with 200 μM myriocin for 24 h, and apoA-I, ERK, phospho-ERK (pERK) and β-actin levels were analysed by Western blotting. (B) Signal intensity of the bands quantified by Image-J software. Analysis of apoA-I mRNA expression via qPCR was performed under identical conditions. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. ** P
    Figure Legend Snippet: Myriocin-mediated up-regulation of HepG2 cell apoA-I production is associated with inhibition of ERK phosphorylation (A) HepG2 cells were treated with 200 μM myriocin for 24 h, and apoA-I, ERK, phospho-ERK (pERK) and β-actin levels were analysed by Western blotting. (B) Signal intensity of the bands quantified by Image-J software. Analysis of apoA-I mRNA expression via qPCR was performed under identical conditions. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. ** P

    Techniques Used: Inhibition, Western Blot, Software, Expressing, Real-time Polymerase Chain Reaction

    Myriocin decreases SM levels in mouse hepatocytes and HepG2 cells Cell lysate (5 μl) was added to a 95-μl reaction buffer (see Materials and methods section for details) and, after 45 min of incubation at 37 °C, the absorbance was measured at 595 nm using a spectrophotometric plate reader. The absolute value for total SM, represented by the 100% relative SM, amount for hepatocytes, was 1.20±0.02 nmol/mg of cell protein. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. ** P
    Figure Legend Snippet: Myriocin decreases SM levels in mouse hepatocytes and HepG2 cells Cell lysate (5 μl) was added to a 95-μl reaction buffer (see Materials and methods section for details) and, after 45 min of incubation at 37 °C, the absorbance was measured at 595 nm using a spectrophotometric plate reader. The absolute value for total SM, represented by the 100% relative SM, amount for hepatocytes, was 1.20±0.02 nmol/mg of cell protein. Grey bars, control; black bars, myriocin-treated. Results are means±S.E.M. ** P

    Techniques Used: Incubation

    Myriocin up-regulates HepG2 cell apoA-I production (A) HepG2 cells were treated with 100 or 200 μM myriocin for 24 or 48 h, and mRNA expression was assessed by qPCR. (B and C) HepG2 cells were treated with 100 or 200 μM myriocin for 24 h. The cell medium was collected after 24 h, and apoA-I expression and secretion were analysed by Western blotting. Signal intensity of the apoA-I bands was quantified using Image-J software and the results are expressed relative to controls. Results are means±S.E.M. * P
    Figure Legend Snippet: Myriocin up-regulates HepG2 cell apoA-I production (A) HepG2 cells were treated with 100 or 200 μM myriocin for 24 or 48 h, and mRNA expression was assessed by qPCR. (B and C) HepG2 cells were treated with 100 or 200 μM myriocin for 24 h. The cell medium was collected after 24 h, and apoA-I expression and secretion were analysed by Western blotting. Signal intensity of the apoA-I bands was quantified using Image-J software and the results are expressed relative to controls. Results are means±S.E.M. * P

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

    18) Product Images from "Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown"

    Article Title: Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013568

    Soluble JAM-A serum levels do not indicate BBB disturbance in multiple sclerosis and ischemic stroke. A Levels of sJAM-A in serum samples from 45 patients with stable relapsing-remitting MS not receiving secondary prophylactic therapy were measured in duplicates by ELISA and compared to those in 14 untreated MS patients during an acute relapse. B Levels of sJAM-A were measured in serum samples from 13 patients with non-small vessel complete ischemic stroke where a first sample was obtained within the first 3 h of symptom onset and a second sample 24 h after clinical onset. Serum samples were not concentrated before ELISA measurement. Dots represent single values, lines median values. Serum levels in stable and inflammatory-active MS patients were statistically compared by the Mann-Whitney U test, serum levels in stroke patients over time by the Wilcoxon matched pairs test. n.s., not significant.
    Figure Legend Snippet: Soluble JAM-A serum levels do not indicate BBB disturbance in multiple sclerosis and ischemic stroke. A Levels of sJAM-A in serum samples from 45 patients with stable relapsing-remitting MS not receiving secondary prophylactic therapy were measured in duplicates by ELISA and compared to those in 14 untreated MS patients during an acute relapse. B Levels of sJAM-A were measured in serum samples from 13 patients with non-small vessel complete ischemic stroke where a first sample was obtained within the first 3 h of symptom onset and a second sample 24 h after clinical onset. Serum samples were not concentrated before ELISA measurement. Dots represent single values, lines median values. Serum levels in stable and inflammatory-active MS patients were statistically compared by the Mann-Whitney U test, serum levels in stroke patients over time by the Wilcoxon matched pairs test. n.s., not significant.

    Techniques Used: Mass Spectrometry, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    19) Product Images from "TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production"

    Article Title: TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq264

    Stabilization of cellular Rrn3p levels attenuates the reduction in initiation competent Pol I–Rrn3p complexes observed upon nutrient depletion. ( A ) Gelfiltration analysis. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were grown in YPD at 30°C to mid-log phase. Cells were either starved for 2 h in SDC-Trp (-Trp) or further cultured in YPD and collected by centrifugation. After lysis, same amounts of WCE (900 µg) were separated on a Superose-6® column in a buffer containing 1.5 M potassium acetate. An amount of 250 µl of the collected 500 µl fractions were TCA precipitated and analysed by western blotting together with the ‘Load’ (30 µg). Antibodies used were directed against the Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively. The gel filtration fractions containing the initiation competent Pol I–Rrn3p complexes are labelled in red. ( B ) Co-immunoprecipitations. Yeast strains TOY 684 (WT) and TOY 685 (ΔN), both expressing chromosomally HA 3 -tagged Pol I subunit A43 and either full length or truncated Prot.A-tagged Rrn3p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested and lysed ( t = 0 min). The remainder of the cells was starved in SDC-Trp (-Trp) for 2 h and treated as described above ( t = 120 min). The HA 3 -tagged Pol I subunit A43 was immunoprecipitated (α-HA-IPs) from 250 µl of extracts (Inputs) with anti-HA antibody. Fifty percent of the α-HA-IPs as well as 1% of the inputs were analysed by western blotting using antibodies directed against the Prot.A-tag of the Rrn3p versions and the HA-tag of the Pol I subunit, respectively. As a control an identical co-immunoprecipitation experiment was performed using extracts from yeast strain pNOP1-RRN3-Prot.A and pNOP1-Rrn3-ΔN-Prot.A, which do not express the HA-tagged Pol I subunit A43 (ctr.). Western blot signal intensities were measured, and quantified using the LAS 3000 imaging system and the AIDA software. Rrn3p/A43 ratios were calculated, and the ratio of the 120 min samples was normalized to the ratio of the respective 0 min samples which was set to 100%. Numbers calculated are given below each lane. ( C ) Chromatin-IP (ChIP) experiments. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN), both expressing either chromosomally HA 3 -tagged Pol I subunit A43 or the core-factor subunit Rrn6p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested, lysed and sonified. The remainder of the cells was starved in SDC-Trp for 2 h and treated as described above (-TRP). Rrn3p-Prot.A, the HA 3 -tagged Pol I subunit A43 or Rrn6p were immunoprecipitated from the chromatin extracts. After DNA isolation the relative amounts of specific DNA fragments co-purifying with the proteins were measured in triplicate real-time PCR reactions using primers specific for the rDNA promoter (P) and the 25S rRNA coding region (25S) as well as for the 5S rRNA gene (5S) which served as an internal control. Data were normalized to the promoter occupancy in growing wild-type cells and represent the mean of at least three independent ChIP experiments. ( D ) Reduction of 35S pre-rRNA synthesis is attenuated in the ΔN-mutant after TOR inactivation. (Upper panel) Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were cultured to mid-log phase ( t = 0 min), before the cells were treated with 200 ng/ml of rapamycin. At the time points indicated 5 ml of the cultures were pulse labelled for 15 min with 20 µCi of [ 5 , 6- 3 H] uracil, and total RNA was isolated. Equal amounts of total RNA were separated in a denaturing agarose gel and blotted onto a nylon membrane. 3 H-labelled RNAs were visualized and quantified using the BAS 1000 imaging system and the Image Gauge software. To determine the total RNA load per lane the membrane was hybridized with a 32 P-labelled oligonucleotide probe directed against the mature 25S rRNA (northern blot). Radioactive signals were visualized and quantified as described above. (Lower panel) The ratio of nascent 35S precursor rRNA to total 25S rRNA in the different rapamycin treated samples was determined and normalized to the 35S rRNA to 25S rRNA ratio of the untreated sample, which was arbitrarily set to 100.
    Figure Legend Snippet: Stabilization of cellular Rrn3p levels attenuates the reduction in initiation competent Pol I–Rrn3p complexes observed upon nutrient depletion. ( A ) Gelfiltration analysis. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were grown in YPD at 30°C to mid-log phase. Cells were either starved for 2 h in SDC-Trp (-Trp) or further cultured in YPD and collected by centrifugation. After lysis, same amounts of WCE (900 µg) were separated on a Superose-6® column in a buffer containing 1.5 M potassium acetate. An amount of 250 µl of the collected 500 µl fractions were TCA precipitated and analysed by western blotting together with the ‘Load’ (30 µg). Antibodies used were directed against the Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively. The gel filtration fractions containing the initiation competent Pol I–Rrn3p complexes are labelled in red. ( B ) Co-immunoprecipitations. Yeast strains TOY 684 (WT) and TOY 685 (ΔN), both expressing chromosomally HA 3 -tagged Pol I subunit A43 and either full length or truncated Prot.A-tagged Rrn3p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested and lysed ( t = 0 min). The remainder of the cells was starved in SDC-Trp (-Trp) for 2 h and treated as described above ( t = 120 min). The HA 3 -tagged Pol I subunit A43 was immunoprecipitated (α-HA-IPs) from 250 µl of extracts (Inputs) with anti-HA antibody. Fifty percent of the α-HA-IPs as well as 1% of the inputs were analysed by western blotting using antibodies directed against the Prot.A-tag of the Rrn3p versions and the HA-tag of the Pol I subunit, respectively. As a control an identical co-immunoprecipitation experiment was performed using extracts from yeast strain pNOP1-RRN3-Prot.A and pNOP1-Rrn3-ΔN-Prot.A, which do not express the HA-tagged Pol I subunit A43 (ctr.). Western blot signal intensities were measured, and quantified using the LAS 3000 imaging system and the AIDA software. Rrn3p/A43 ratios were calculated, and the ratio of the 120 min samples was normalized to the ratio of the respective 0 min samples which was set to 100%. Numbers calculated are given below each lane. ( C ) Chromatin-IP (ChIP) experiments. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN), both expressing either chromosomally HA 3 -tagged Pol I subunit A43 or the core-factor subunit Rrn6p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested, lysed and sonified. The remainder of the cells was starved in SDC-Trp for 2 h and treated as described above (-TRP). Rrn3p-Prot.A, the HA 3 -tagged Pol I subunit A43 or Rrn6p were immunoprecipitated from the chromatin extracts. After DNA isolation the relative amounts of specific DNA fragments co-purifying with the proteins were measured in triplicate real-time PCR reactions using primers specific for the rDNA promoter (P) and the 25S rRNA coding region (25S) as well as for the 5S rRNA gene (5S) which served as an internal control. Data were normalized to the promoter occupancy in growing wild-type cells and represent the mean of at least three independent ChIP experiments. ( D ) Reduction of 35S pre-rRNA synthesis is attenuated in the ΔN-mutant after TOR inactivation. (Upper panel) Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were cultured to mid-log phase ( t = 0 min), before the cells were treated with 200 ng/ml of rapamycin. At the time points indicated 5 ml of the cultures were pulse labelled for 15 min with 20 µCi of [ 5 , 6- 3 H] uracil, and total RNA was isolated. Equal amounts of total RNA were separated in a denaturing agarose gel and blotted onto a nylon membrane. 3 H-labelled RNAs were visualized and quantified using the BAS 1000 imaging system and the Image Gauge software. To determine the total RNA load per lane the membrane was hybridized with a 32 P-labelled oligonucleotide probe directed against the mature 25S rRNA (northern blot). Radioactive signals were visualized and quantified as described above. (Lower panel) The ratio of nascent 35S precursor rRNA to total 25S rRNA in the different rapamycin treated samples was determined and normalized to the 35S rRNA to 25S rRNA ratio of the untreated sample, which was arbitrarily set to 100.

    Techniques Used: Cell Culture, Centrifugation, Lysis, Western Blot, Filtration, Expressing, Immunoprecipitation, Imaging, Software, Chromatin Immunoprecipitation, DNA Extraction, Real-time Polymerase Chain Reaction, Mutagenesis, Isolation, Agarose Gel Electrophoresis, Northern Blot

    20) Product Images from "EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling"

    Article Title: EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-13-3724

    EGF mediated tyrosine phosphorylation of the Notch3 receptor. A B, HCC2429 cells were stimulated with EGF (25 ng/ml) for the indicated times to detect the ligand induced tyrosine phosphorylation of Notch3 receptor. A Cell lysates were immunoprecipitated with Notch3 antibody followed by western analysis using a phosphotyrosine antibody. The blot was stripped and re-probed with anti-Notch3 (bottom). B, A reciprocal immunoprecipitation was done with a phosphotyrosine antibody with a subsequent western blot probed for Notch3 (top). Equal amount of cell lysate was analyzed for Notch3 expression (middle) and tubulin (bottom).
    Figure Legend Snippet: EGF mediated tyrosine phosphorylation of the Notch3 receptor. A B, HCC2429 cells were stimulated with EGF (25 ng/ml) for the indicated times to detect the ligand induced tyrosine phosphorylation of Notch3 receptor. A Cell lysates were immunoprecipitated with Notch3 antibody followed by western analysis using a phosphotyrosine antibody. The blot was stripped and re-probed with anti-Notch3 (bottom). B, A reciprocal immunoprecipitation was done with a phosphotyrosine antibody with a subsequent western blot probed for Notch3 (top). Equal amount of cell lysate was analyzed for Notch3 expression (middle) and tubulin (bottom).

    Techniques Used: Immunoprecipitation, Western Blot, Expressing

    EGF-mediated association between EGFR and Notch 3 receptors in HCC2429 cells is dependent on EGFR kinase activity. A. HCC2429 cells were stimulated with EGF (25 ng/ml) for the indicated times. Cell lysate was prepared from each time point and immunoprecipitated with EGFR antibody. Western analysis of precipitated proteins was performedand probed with a Notch3 antibody (top) and an EGFR antibody (middle). Whole cell lysate was subjected to western analyses using Notch3, and β-tubulin antibodies to detect the total expression of these proteins. B, HCC4006 C, HCC827cells were treated with DMSO or 0.1 μM erlotinib for 24 hours. Cell lysate was prepared from each treatment and immunoprecipitated with EGFR antibody and blotted for Notch3, or reciprocal immunoprecipitation was performed by precipitating Notch3 and blotting for EGFR. Whole cell lysate was subjected to western analyses using Notch3, EGFR and actin antibodies to detect the total expression of these proteins. IP= Immunoprecipitation, WCL = Whole Cell Lysate.
    Figure Legend Snippet: EGF-mediated association between EGFR and Notch 3 receptors in HCC2429 cells is dependent on EGFR kinase activity. A. HCC2429 cells were stimulated with EGF (25 ng/ml) for the indicated times. Cell lysate was prepared from each time point and immunoprecipitated with EGFR antibody. Western analysis of precipitated proteins was performedand probed with a Notch3 antibody (top) and an EGFR antibody (middle). Whole cell lysate was subjected to western analyses using Notch3, and β-tubulin antibodies to detect the total expression of these proteins. B, HCC4006 C, HCC827cells were treated with DMSO or 0.1 μM erlotinib for 24 hours. Cell lysate was prepared from each treatment and immunoprecipitated with EGFR antibody and blotted for Notch3, or reciprocal immunoprecipitation was performed by precipitating Notch3 and blotting for EGFR. Whole cell lysate was subjected to western analyses using Notch3, EGFR and actin antibodies to detect the total expression of these proteins. IP= Immunoprecipitation, WCL = Whole Cell Lysate.

    Techniques Used: Activity Assay, Immunoprecipitation, Western Blot, Expressing

    21) Product Images from "Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair"

    Article Title: Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

    Journal: Respiratory Research

    doi: 10.1186/s12931-015-0263-x

    Cellular co-distribution, co-immunoprecipitation and membrane expression of β1-integrin and KCa3.1 channels. a . Representative immunofluorescence images of KCa3.1 and β1-integrin staining performed on ATII cells using anti-KCa3.1, anti-β1-integrin, anti-rabbit 633 (for KCa3.1 detection) and anti-mouse 488 (for β1-integrin detection) antibodies. Color superposition shows similar cellular distribution of KCa3.1 and β1-integrin in ATII cells (merge panel, Scale bars, 10 μm). No or diffuse signal was detected with the Alexa fluor 488 and Alexa fluor 633 coupled secondary antibodies in control experiments (negative controls). b . Representative immunoblots showing β1-integrin and KCa3.1 co-immunoprecipitations. β1-integrin (upper panels, IB: β1-integrin) and KCa3.1 (lower panels, IB: KCa3.1) proteins were revealed with specific antibodies after β1-integrin and KCa3.1 immunoprecipitation with anti-β1-integrin (lane 2 « β1-integrin IP ») or anti-KCa3.1 (lane 3 « KCa3.1 IP ») antibodies in ATII cell extracts. Endogenous expression of β1-integrin and KCa3.1 proteins in ATII cell lysate is also shown in lane 1, « Total Lysate ». Lanes 4 and 5 are negative control assays showing an absence of band in IB (IB β1-integrin and IB KCa3.1) after IP in the absence of lysate (lane 4, « Negative IP Control (no lysate) ») and in the absence of β-integrin and KCa3.1 antibodies (lane 5, « Negative IP control (no antibody) »). c . The level of β1-integrin and KCa3.1 channel expression in membrane fractions were determined by immunoblotting using anti-β1-integrin and anti-KCa3.1 antibodies. A representative immunoblot is shown in the left panel. The band intensities were compared in control condition (no coating, −) and in the presence of a fibronectin (+) matrix ( right panel , n = 11). * p
    Figure Legend Snippet: Cellular co-distribution, co-immunoprecipitation and membrane expression of β1-integrin and KCa3.1 channels. a . Representative immunofluorescence images of KCa3.1 and β1-integrin staining performed on ATII cells using anti-KCa3.1, anti-β1-integrin, anti-rabbit 633 (for KCa3.1 detection) and anti-mouse 488 (for β1-integrin detection) antibodies. Color superposition shows similar cellular distribution of KCa3.1 and β1-integrin in ATII cells (merge panel, Scale bars, 10 μm). No or diffuse signal was detected with the Alexa fluor 488 and Alexa fluor 633 coupled secondary antibodies in control experiments (negative controls). b . Representative immunoblots showing β1-integrin and KCa3.1 co-immunoprecipitations. β1-integrin (upper panels, IB: β1-integrin) and KCa3.1 (lower panels, IB: KCa3.1) proteins were revealed with specific antibodies after β1-integrin and KCa3.1 immunoprecipitation with anti-β1-integrin (lane 2 « β1-integrin IP ») or anti-KCa3.1 (lane 3 « KCa3.1 IP ») antibodies in ATII cell extracts. Endogenous expression of β1-integrin and KCa3.1 proteins in ATII cell lysate is also shown in lane 1, « Total Lysate ». Lanes 4 and 5 are negative control assays showing an absence of band in IB (IB β1-integrin and IB KCa3.1) after IP in the absence of lysate (lane 4, « Negative IP Control (no lysate) ») and in the absence of β-integrin and KCa3.1 antibodies (lane 5, « Negative IP control (no antibody) »). c . The level of β1-integrin and KCa3.1 channel expression in membrane fractions were determined by immunoblotting using anti-β1-integrin and anti-KCa3.1 antibodies. A representative immunoblot is shown in the left panel. The band intensities were compared in control condition (no coating, −) and in the presence of a fibronectin (+) matrix ( right panel , n = 11). * p

    Techniques Used: Immunoprecipitation, Expressing, Immunofluorescence, Staining, Western Blot, Negative Control

    22) Product Images from "IKKα negatively regulates ASC-dependent inflammasome activation"

    Article Title: IKKα negatively regulates ASC-dependent inflammasome activation

    Journal: Nature communications

    doi: 10.1038/ncomms5977

    IKKα regulates the inflammasome via interaction with ASC ( a ) HEK 293 cells were transfected with ASC and either GFP- IKKα WT or GFP-IKKα K44A using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with the indicated antibodies. ( b , c ) BMDMs from WT and IKKα K44A mice ( b ) or WT mice only ( c ) were either left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min) ( b ), or either left untreated or treated with LPS (1 μg/ml) for 4 hours, LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), or LPS (1 μg/ml) 4 hours + 1 uM gramicidin (30 min) ( c ). Cell lysates were then immunoprecipitated using anti-ASC antibody or control IgG antibody, and were immunoblotted with the indicated antibodies. ( d ) BMDMs from WT mice were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours ( left panel ), or were infected with 50 MOI of S. typhimurium for indicated times ( right panel ). Cell lysates were then immunoprecipitated using anti-ASC antibody or control IgG antibody, and were immunoblotted with the indicated antibodies. ( e ) WT, IKKα K44A and IKKα AA BMDMs were either left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), then washed, fixed, and stained with anti-ASC antibody and caspase-1 FLICA (FAM-YVAD-FMK). Images were then acquired using confocal microscopy with a 60X objective and 4X enlargement; scale bars are 5 μm. Images were quantified by counting number of cells with ASC specks co-staining with caspase-1 FLICA as a percentage of total cells counted in three fields. ( far right panel ). “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P
    Figure Legend Snippet: IKKα regulates the inflammasome via interaction with ASC ( a ) HEK 293 cells were transfected with ASC and either GFP- IKKα WT or GFP-IKKα K44A using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with the indicated antibodies. ( b , c ) BMDMs from WT and IKKα K44A mice ( b ) or WT mice only ( c ) were either left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min) ( b ), or either left untreated or treated with LPS (1 μg/ml) for 4 hours, LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), or LPS (1 μg/ml) 4 hours + 1 uM gramicidin (30 min) ( c ). Cell lysates were then immunoprecipitated using anti-ASC antibody or control IgG antibody, and were immunoblotted with the indicated antibodies. ( d ) BMDMs from WT mice were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours ( left panel ), or were infected with 50 MOI of S. typhimurium for indicated times ( right panel ). Cell lysates were then immunoprecipitated using anti-ASC antibody or control IgG antibody, and were immunoblotted with the indicated antibodies. ( e ) WT, IKKα K44A and IKKα AA BMDMs were either left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), then washed, fixed, and stained with anti-ASC antibody and caspase-1 FLICA (FAM-YVAD-FMK). Images were then acquired using confocal microscopy with a 60X objective and 4X enlargement; scale bars are 5 μm. Images were quantified by counting number of cells with ASC specks co-staining with caspase-1 FLICA as a percentage of total cells counted in three fields. ( far right panel ). “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Mouse Assay, Infection, Staining, Confocal Microscopy

    S193 and S16 are critical residues on ASC for interaction with IKKα ( a ) HEK 293 cells were transfected with HA-IKKα and either WT ASC, S16A ASC, or S193A ASC using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with the indicated antibodies. ( b ) HEK 293 cells were transfected with HA-IKKα or HA-IKKα K44A along with either WT ASC, S16A ASC, or S193A ASC using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and in vitro kinase assay. ( c,d ) Immortalized ASC −/− macrophages retrovirally reconstituted with empty vector, WT ASC, S193A ASC, or S16A ASC were either left untreated or treated with LPS (1 μg/mL) for 4 hours, followed by 30 min 5 mM ATP when indicated. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with indicated antibodies ( c ), or cells were then washed, fixed, and stained with anti-ASC antibody and caspase-1 FLICA (FAM-YVAD-FMK). Images were then acquired using confocal microscopy with 60X magnification and 4X enlargement; scale bars are 5 μm ( d ). Images from were quantified by counting the number of cells with ASC specks co-staining with caspase-1 FLICA as a percentage of total cells counted in three fields. ( e–g ) Retrovirally reconstituted ASC −/− macrophages from ( c ) were either left untreated or treated with LPS (1 μg/ml) for 4 hours, followed by 30 minutes of 5 mM ATP when indicated ( e,f ), or were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours ( g ). Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies ( e,g ), or were analyzed by ELISA ( f ). N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P
    Figure Legend Snippet: S193 and S16 are critical residues on ASC for interaction with IKKα ( a ) HEK 293 cells were transfected with HA-IKKα and either WT ASC, S16A ASC, or S193A ASC using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with the indicated antibodies. ( b ) HEK 293 cells were transfected with HA-IKKα or HA-IKKα K44A along with either WT ASC, S16A ASC, or S193A ASC using lipofectamine 2000. Empty vector was used to normalize total plasmid transfected. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and in vitro kinase assay. ( c,d ) Immortalized ASC −/− macrophages retrovirally reconstituted with empty vector, WT ASC, S193A ASC, or S16A ASC were either left untreated or treated with LPS (1 μg/mL) for 4 hours, followed by 30 min 5 mM ATP when indicated. Cells were then lysed, followed by immunoprecipitation with anti-ASC antibody and immunoblotting with indicated antibodies ( c ), or cells were then washed, fixed, and stained with anti-ASC antibody and caspase-1 FLICA (FAM-YVAD-FMK). Images were then acquired using confocal microscopy with 60X magnification and 4X enlargement; scale bars are 5 μm ( d ). Images from were quantified by counting the number of cells with ASC specks co-staining with caspase-1 FLICA as a percentage of total cells counted in three fields. ( e–g ) Retrovirally reconstituted ASC −/− macrophages from ( c ) were either left untreated or treated with LPS (1 μg/ml) for 4 hours, followed by 30 minutes of 5 mM ATP when indicated ( e,f ), or were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000 for 4 hours ( g ). Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies ( e,g ), or were analyzed by ELISA ( f ). N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least four independent experiments. Error bars represent s.e.m. of technical replicates. * P

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, In Vitro, Kinase Assay, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

    IKKα is a negative regulator of the inflammasome ( a ) BMDMs from WT and IKKα K44A mice were either left untreated or treated with LPS (1 μg/ml) for 4 hours, 5 mM ATP (30 min), or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min). Cell lysates and supernatants were collected together and were immunoblotted with the indicated antibodies. ( b ) Cell-free supernatants were collected from BMDMs that were either left untreated, or treated with LPS (1 μg/ml) for 4 hours, Pam 3 CSK 4 (1 μg/ml) for 4 hours, LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), or Pam 3 CSK 4 (1 μg/ml) 4 hours + 5 mM ATP (30 min). Levels of IL-1β and TNF-α were measured by ELISA. ( c ) BMDMs from WT and IKKα K44A mice were left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min). Cell lysates were collected and immunoprecipitated with anti-ASC antibody or control IgG antibody, followed by immunoblotting with the indicated antibodies. ( d,e ) BMDMs from WT and either IKKα K44A ( d ) or IKKα AA ( e ) mice were treated with LPS (1 μg/mL) for 0, 10, 30, or 60 minutes, followed by the addition of 5 mM ATP for 30 minutes. ( f ) BMDMs from WT and IKKα K44A mice were infected with Salmonella typhimurium at a multiplicity of infection (MOI) of 50 for 0, 0.5, 1, or 2 hours. Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies. ( g ) BMDMs from WT, IKKα K44A ( left panel ) and IKKα AA mice ( right panel ) were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000. After indicated times, cell lysates and supernatants were collected together and immunoblotted with the indicated antibodies. N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least five independent experiments. Error bars represent s.e.m. of technical replicates. * P
    Figure Legend Snippet: IKKα is a negative regulator of the inflammasome ( a ) BMDMs from WT and IKKα K44A mice were either left untreated or treated with LPS (1 μg/ml) for 4 hours, 5 mM ATP (30 min), or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min). Cell lysates and supernatants were collected together and were immunoblotted with the indicated antibodies. ( b ) Cell-free supernatants were collected from BMDMs that were either left untreated, or treated with LPS (1 μg/ml) for 4 hours, Pam 3 CSK 4 (1 μg/ml) for 4 hours, LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min), or Pam 3 CSK 4 (1 μg/ml) 4 hours + 5 mM ATP (30 min). Levels of IL-1β and TNF-α were measured by ELISA. ( c ) BMDMs from WT and IKKα K44A mice were left untreated or treated with LPS (1 μg/ml) for 4 hours, or LPS (1 μg/ml) 4 hours + 5 mM ATP (30 min). Cell lysates were collected and immunoprecipitated with anti-ASC antibody or control IgG antibody, followed by immunoblotting with the indicated antibodies. ( d,e ) BMDMs from WT and either IKKα K44A ( d ) or IKKα AA ( e ) mice were treated with LPS (1 μg/mL) for 0, 10, 30, or 60 minutes, followed by the addition of 5 mM ATP for 30 minutes. ( f ) BMDMs from WT and IKKα K44A mice were infected with Salmonella typhimurium at a multiplicity of infection (MOI) of 50 for 0, 0.5, 1, or 2 hours. Cell lysates and supernatants were then collected together and immunoblotted with the indicated antibodies. ( g ) BMDMs from WT, IKKα K44A ( left panel ) and IKKα AA mice ( right panel ) were transfected with poly(dA:dT) (1.5 μg/ml) using lipofectamine 2000. After indicated times, cell lysates and supernatants were collected together and immunoblotted with the indicated antibodies. N.D. indicates not detectable, and “UN” indicates left untreated. Data are representative of at least five independent experiments. Error bars represent s.e.m. of technical replicates. * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Infection, Transfection

    23) Product Images from "Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes"

    Article Title: Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057018

    A combined single molecule – bulk FRET approach to study nucleosome stability. A ) Theoretical diagram of nucleosome stability as a function of salt and nucleosome concentration (adapted from ref. [22] ). The solid line represents the amount of salt needed to destabilize nucleosomes at a given nucleosome concentration. Nucleosomes generally remain stable at higher concentrations and lower ionic strength, dissociation occurs at elevated ionic strength and nucleosome concentrations in the sub-nM range. The dashed line represents changes in nucleosome stability from altered nucleosome composition. B) DNA labeling for nucleosome FRET experiments. 170 bp long DNA fragments were labeled at positions -42 and +52 from the dyad axis. In the intact nucleosome both dyes are located ≈ 6 nm apart, allowing for FRET, while in a fully dissociated nucleosome or free DNA fragment both dyes are too far apart to undergo FRET. C ) (i) Schematic of confocal single molecule detection of nucleosomes in solution. A detailed description of the setup is given in Section S1 in File S1 . (ii) The passage of individual nucleosomes through the focus generates bursts of fluorescence. (iii) For each burst a proximity ratio is calculated and data binned for histogram analysis. The position of relevant subpopulations in the histogram is indicated. D ) (i) Schematic setup for microplate-scanning FRET (μpsFRET). Samples are loaded into a 384-well multiplate and imaged in three spectral channels using a commercial Typhoon™ multimode scanner with confocal optics (i). Grey scale images and intensity profiles of samples with different bulk FRET efficiencies (ii). Higher FRET leads to a decrease of signal in the donor channel and a corresponding increase of signal in the transfer channel. The signal in the acceptor channel remains unaffected. From these intensities P-values are calculated for each well. Abbreviations: DM: dichroic mirror, F: emission filter, APD: avalanche photodiode, PMT, photomultiplier tube, PH: pinhole.
    Figure Legend Snippet: A combined single molecule – bulk FRET approach to study nucleosome stability. A ) Theoretical diagram of nucleosome stability as a function of salt and nucleosome concentration (adapted from ref. [22] ). The solid line represents the amount of salt needed to destabilize nucleosomes at a given nucleosome concentration. Nucleosomes generally remain stable at higher concentrations and lower ionic strength, dissociation occurs at elevated ionic strength and nucleosome concentrations in the sub-nM range. The dashed line represents changes in nucleosome stability from altered nucleosome composition. B) DNA labeling for nucleosome FRET experiments. 170 bp long DNA fragments were labeled at positions -42 and +52 from the dyad axis. In the intact nucleosome both dyes are located ≈ 6 nm apart, allowing for FRET, while in a fully dissociated nucleosome or free DNA fragment both dyes are too far apart to undergo FRET. C ) (i) Schematic of confocal single molecule detection of nucleosomes in solution. A detailed description of the setup is given in Section S1 in File S1 . (ii) The passage of individual nucleosomes through the focus generates bursts of fluorescence. (iii) For each burst a proximity ratio is calculated and data binned for histogram analysis. The position of relevant subpopulations in the histogram is indicated. D ) (i) Schematic setup for microplate-scanning FRET (μpsFRET). Samples are loaded into a 384-well multiplate and imaged in three spectral channels using a commercial Typhoon™ multimode scanner with confocal optics (i). Grey scale images and intensity profiles of samples with different bulk FRET efficiencies (ii). Higher FRET leads to a decrease of signal in the donor channel and a corresponding increase of signal in the transfer channel. The signal in the acceptor channel remains unaffected. From these intensities P-values are calculated for each well. Abbreviations: DM: dichroic mirror, F: emission filter, APD: avalanche photodiode, PMT, photomultiplier tube, PH: pinhole.

    Techniques Used: Concentration Assay, DNA Labeling, Labeling, Fluorescence

    smFRET results on nucleosome stability are consistent to μpsFRET data. ( A ) Average proximity ratio calculated from all photons from double-labeled nucleosomes as a function of salt concentration. Photons from the donor and transfer channel were summed for all detected molecules, except donor-only and acceptor-only species. ( B ) Salt dependence of the fraction of intact nucleosomes in smFRET histograms from Fig. 5. For each histogram, the donor-only and acceptor-only population was excluded from the analysis. The relative fraction of FRET-active molecules (0.25
    Figure Legend Snippet: smFRET results on nucleosome stability are consistent to μpsFRET data. ( A ) Average proximity ratio calculated from all photons from double-labeled nucleosomes as a function of salt concentration. Photons from the donor and transfer channel were summed for all detected molecules, except donor-only and acceptor-only species. ( B ) Salt dependence of the fraction of intact nucleosomes in smFRET histograms from Fig. 5. For each histogram, the donor-only and acceptor-only population was excluded from the analysis. The relative fraction of FRET-active molecules (0.25

    Techniques Used: Labeling, Concentration Assay

    Characterization of microplate-scanning FRET spectroscopy (μpsFRET). A) μpsFRET grey scale images of a nucleosome sample (nuc) and a DNA fragment (DNA) at different sample concentrations (donor channel: excitation at 488 nm, detection at 500–540 nm; transfer channel: excitation at 488 nm, detection at 595–625 nm; acceptor channel: excitation at 532 nm, detection at 595–625 nm). Due to the absence of FRET, the DNA sample has a lower signal in the transfer channel. Concentrations are (from left to right): 2.5 nM, 1.7 nM, 1.1 nM, 600 pM, 350 pM, 180 pM, 120 pM, 70 p M, 40 pM, 20 pM, The last row to the right contained pure buffer solution. B ) A plot showing the integrated fluorescence signal (donor channel + transfer channel) as a function of sample concentration. The measured intensity is linear throughout the dilution series. Concentrations below 50 pM can still be distinguished from background. C ) A plot showing calculated P-values of nucleosomes and DNA as a function of sample concentration. For both samples P-values were consistent at larger concentrations, while for DNA P deviated at concentrations lower than 200 pM. Nucleosomal P-values were consistent to slightly lower concentrations (100 pM). D ) Noise analysis of P-values from a donor-only sample under sub-nanomolar concentrations. Black circles are experimental standard deviations from 25 wells, white circles show estimated shot noise values. The low signal to noise level at lowest concentrations results in a large well-to-well variation in P. Shot noise accounts for
    Figure Legend Snippet: Characterization of microplate-scanning FRET spectroscopy (μpsFRET). A) μpsFRET grey scale images of a nucleosome sample (nuc) and a DNA fragment (DNA) at different sample concentrations (donor channel: excitation at 488 nm, detection at 500–540 nm; transfer channel: excitation at 488 nm, detection at 595–625 nm; acceptor channel: excitation at 532 nm, detection at 595–625 nm). Due to the absence of FRET, the DNA sample has a lower signal in the transfer channel. Concentrations are (from left to right): 2.5 nM, 1.7 nM, 1.1 nM, 600 pM, 350 pM, 180 pM, 120 pM, 70 p M, 40 pM, 20 pM, The last row to the right contained pure buffer solution. B ) A plot showing the integrated fluorescence signal (donor channel + transfer channel) as a function of sample concentration. The measured intensity is linear throughout the dilution series. Concentrations below 50 pM can still be distinguished from background. C ) A plot showing calculated P-values of nucleosomes and DNA as a function of sample concentration. For both samples P-values were consistent at larger concentrations, while for DNA P deviated at concentrations lower than 200 pM. Nucleosomal P-values were consistent to slightly lower concentrations (100 pM). D ) Noise analysis of P-values from a donor-only sample under sub-nanomolar concentrations. Black circles are experimental standard deviations from 25 wells, white circles show estimated shot noise values. The low signal to noise level at lowest concentrations results in a large well-to-well variation in P. Shot noise accounts for

    Techniques Used: Spectroscopy, Fluorescence, Concentration Assay

    smFRET analysis reveals a conformational transition prior to nucleosome unwrapping. A,B ) smFRET histograms of non-acetylated and H3-acetylated nucleosomes at various salt concentrations and 300 pM total nucleosome concentration. Above 300 mM NaCl, a fraction of H3-acetylated nucleosomes populates a second conformation with slightly increased proximity ratio compared to non-acetylated nucleosomes, which appear to retain their initial structure. C, D ) Overlay of histograms for salt concentrations between 150 mM and 600 mM NaCl for non-acetylated (C) and H3-acetylated nucleosomes (D). Data were smoothed once to better visualize the gradual transition of nucleosomes into the high FRET state.
    Figure Legend Snippet: smFRET analysis reveals a conformational transition prior to nucleosome unwrapping. A,B ) smFRET histograms of non-acetylated and H3-acetylated nucleosomes at various salt concentrations and 300 pM total nucleosome concentration. Above 300 mM NaCl, a fraction of H3-acetylated nucleosomes populates a second conformation with slightly increased proximity ratio compared to non-acetylated nucleosomes, which appear to retain their initial structure. C, D ) Overlay of histograms for salt concentrations between 150 mM and 600 mM NaCl for non-acetylated (C) and H3-acetylated nucleosomes (D). Data were smoothed once to better visualize the gradual transition of nucleosomes into the high FRET state.

    Techniques Used: Concentration Assay

    Working range of conventional and quasi-bulk single particle FRET. A – C ) smFRET histograms and burst size to burst duration distributions for a binary DNA mixture (noFRET and FRET-active) at 60 pM (A), 150 pM (B), and 330 pM (C) sample concentrations. While at 60 pM both subpopulations are clearly separated, coincident detection of both species occurs at 150 pM and above. The presence of multi-particle events is evident from the burst size to burst duration distribution. While at 50 pM burst duration and burst size strongly correlate, additional populations appear outside the ellipsoidal point cloud at higher sample concentrations. D, E ) Principle of quasi-bulk smFRET of nucleosomes. Nucleosomes were reconstituted on 5S rDNA (D) or the high affinity Widom 601 sequence (E). Histograms are shown for 5 mM or 150 mM salt concentrations and in the presence or absence of 10 nM unlabeled nucleosomes. At 5 mM NaCl (left panels) most nucleosomes were intact as expected from Figure 1A . At 150 mM NaCl (right panels) and in the absence of unlabeled nucleosomes, diluted nucleosomes dissociated, whereas under quasi-bulk conditions, nucleosomes on both 5S and 601 DNA remained intact.
    Figure Legend Snippet: Working range of conventional and quasi-bulk single particle FRET. A – C ) smFRET histograms and burst size to burst duration distributions for a binary DNA mixture (noFRET and FRET-active) at 60 pM (A), 150 pM (B), and 330 pM (C) sample concentrations. While at 60 pM both subpopulations are clearly separated, coincident detection of both species occurs at 150 pM and above. The presence of multi-particle events is evident from the burst size to burst duration distribution. While at 50 pM burst duration and burst size strongly correlate, additional populations appear outside the ellipsoidal point cloud at higher sample concentrations. D, E ) Principle of quasi-bulk smFRET of nucleosomes. Nucleosomes were reconstituted on 5S rDNA (D) or the high affinity Widom 601 sequence (E). Histograms are shown for 5 mM or 150 mM salt concentrations and in the presence or absence of 10 nM unlabeled nucleosomes. At 5 mM NaCl (left panels) most nucleosomes were intact as expected from Figure 1A . At 150 mM NaCl (right panels) and in the absence of unlabeled nucleosomes, diluted nucleosomes dissociated, whereas under quasi-bulk conditions, nucleosomes on both 5S and 601 DNA remained intact.

    Techniques Used: Sequencing

    Acetylation of histone H3 decreases nucleosome stability. Salt-dependent proximity ratio at 1.5 nM and 300 pM nucleosome concentration measured with μpsFRET. A loss in P is interpreted as nucleosome dissociation. Salt titration curves were approximated by a sigmoidal function and nucleosome stability was quantified in terms of the c 1/2 value, the salt concentration at which P is half the maximum observed around 500–600 mM NaCl. Measured c 1/2 values were (995±20) mM and (980±15) mM for 1.5 nM and 300 pM non-acetylated nucleosomes, while measured c 1/2 -values were 120−130 mM lower for H3-acetylated nucleosomes ((875±10) mM and (850±20) mM for 1.5 nM and 300 pM).
    Figure Legend Snippet: Acetylation of histone H3 decreases nucleosome stability. Salt-dependent proximity ratio at 1.5 nM and 300 pM nucleosome concentration measured with μpsFRET. A loss in P is interpreted as nucleosome dissociation. Salt titration curves were approximated by a sigmoidal function and nucleosome stability was quantified in terms of the c 1/2 value, the salt concentration at which P is half the maximum observed around 500–600 mM NaCl. Measured c 1/2 values were (995±20) mM and (980±15) mM for 1.5 nM and 300 pM non-acetylated nucleosomes, while measured c 1/2 -values were 120−130 mM lower for H3-acetylated nucleosomes ((875±10) mM and (850±20) mM for 1.5 nM and 300 pM).

    Techniques Used: Concentration Assay, Titration

    24) Product Images from "In Vitro Phenotypic, Genomic and Proteomic Characterization of a Cytokine-Resistant Murine ?-TC3 Cell Line"

    Article Title: In Vitro Phenotypic, Genomic and Proteomic Characterization of a Cytokine-Resistant Murine ?-TC3 Cell Line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032109

    SUMO4 hyperexpression blocks NF- k B and JAK/STAT signalling pathways in β-TC3R cells. ( A ) Analysis of SUMO4 gene expression by qRT-PCR in β-TC3 and β-TC3R cells after cytokines exposure compared with untreated cells. SUMO4 is undetectable in β-TC3 cells, while it is strongly upregulated after cytokine exposure in β-TC3R cells. Cytokine treatment is 100 IU/ml IL-1β, 100 IU/ml IFN-γ or their combination, for 72 hours. ( B ) Effects of cytokine treatment on unphosphorilated and phosphorylated forms of Mek, p65 and Stat-1α. Cytokine treatment upregulates p-Mek protein levels in β-TC3R cells in comparison to β-TC3; whereas it does not activate p-65 and Stat-1α. U: untreated; Cyt: 100 IU/ml IL-1β + IFN-γ for 20 min. Data shown are representative of 5 independent experiments. ( C ) Quantification of proteic bands performed by densitometric analysis. OD: optical density units. Each bar represents average ± SD of 5 independent experiments. ( D ) NF- k B and JAK/STAT signalling pathways are blocked in β-TC3R cells. Cytokine treatment induces expression of NF- k B and the JAK/STAT-dependent genes iNOS, MnSOD, FAS and TRAIL, in β-TC3 cells compared to untreated cells. By contrast, these genes are not expressed in both untreated and cytokine-treated β-TC3R cells. U: untreated; cytokine treatment is 100 IU/ml IL-1β, 100 IU/ml IFN-γ or their combination, after 72 hours. bp = base pairs.
    Figure Legend Snippet: SUMO4 hyperexpression blocks NF- k B and JAK/STAT signalling pathways in β-TC3R cells. ( A ) Analysis of SUMO4 gene expression by qRT-PCR in β-TC3 and β-TC3R cells after cytokines exposure compared with untreated cells. SUMO4 is undetectable in β-TC3 cells, while it is strongly upregulated after cytokine exposure in β-TC3R cells. Cytokine treatment is 100 IU/ml IL-1β, 100 IU/ml IFN-γ or their combination, for 72 hours. ( B ) Effects of cytokine treatment on unphosphorilated and phosphorylated forms of Mek, p65 and Stat-1α. Cytokine treatment upregulates p-Mek protein levels in β-TC3R cells in comparison to β-TC3; whereas it does not activate p-65 and Stat-1α. U: untreated; Cyt: 100 IU/ml IL-1β + IFN-γ for 20 min. Data shown are representative of 5 independent experiments. ( C ) Quantification of proteic bands performed by densitometric analysis. OD: optical density units. Each bar represents average ± SD of 5 independent experiments. ( D ) NF- k B and JAK/STAT signalling pathways are blocked in β-TC3R cells. Cytokine treatment induces expression of NF- k B and the JAK/STAT-dependent genes iNOS, MnSOD, FAS and TRAIL, in β-TC3 cells compared to untreated cells. By contrast, these genes are not expressed in both untreated and cytokine-treated β-TC3R cells. U: untreated; cytokine treatment is 100 IU/ml IL-1β, 100 IU/ml IFN-γ or their combination, after 72 hours. bp = base pairs.

    Techniques Used: Expressing, Quantitative RT-PCR

    Evaluation of β-cell specific markers and secretory function in β-TC3R cells. ( A ) Immunofluorescence analysis shows persistence of β-cell markers in β-TC3R cells (right panel) despite the acquisition of cytokine resistance. Left panel: sensitive β-TC3. ( B–D ) Stimulation with 20 mM glucose (GLUC), 100 µM of the secretagogue tolbutamide (TOL) or direct depolarization with 30 mM potassium chloride (KCl) during 2-h static incubation of both β-TC3 and β-TC3R cells. ( E ) C-peptide secretion after 1-hr static incubation in β-TC3 and β-TC3R cells. ( F ) western blot analysis of glucokinase (GCK) in β-TC3 and β-TC3R cells. Graph shows quantification of proteic bands performed by densitometric analysis.
    Figure Legend Snippet: Evaluation of β-cell specific markers and secretory function in β-TC3R cells. ( A ) Immunofluorescence analysis shows persistence of β-cell markers in β-TC3R cells (right panel) despite the acquisition of cytokine resistance. Left panel: sensitive β-TC3. ( B–D ) Stimulation with 20 mM glucose (GLUC), 100 µM of the secretagogue tolbutamide (TOL) or direct depolarization with 30 mM potassium chloride (KCl) during 2-h static incubation of both β-TC3 and β-TC3R cells. ( E ) C-peptide secretion after 1-hr static incubation in β-TC3 and β-TC3R cells. ( F ) western blot analysis of glucokinase (GCK) in β-TC3 and β-TC3R cells. Graph shows quantification of proteic bands performed by densitometric analysis.

    Techniques Used: Immunofluorescence, Incubation, Western Blot

    Proteomic analysis of β-TC3 and β-TC3R cells. ( A–B ) Proteomic maps. β-TC3R gel ( B ) displays the locations of the proteins that are differentially expressed in comparison to β-TC3; see Table S3 for further information relative to single detected protein. Gels are representative of three experiments. ( C ) Comparative proteomic profile. Graph represents protein expression levels in β-TC3 (black peaks) and β-TC3R (grey peaks). Relative volumes of the spots (V%) were calculated by ImageMaster 2D Platinum software, the same software used for gel analysis. ( D ) Proteomic groups. Proteins characterized by significant variation are grouped into four different classes. ( E ) Proteomic network. Protein interaction network reveals that 28 out of 99 proteins are differentially expressed, which are distributed into groups of different biological functions (mainly belonging to cell death pathway, negative regulators of apoptosis and protein folding).
    Figure Legend Snippet: Proteomic analysis of β-TC3 and β-TC3R cells. ( A–B ) Proteomic maps. β-TC3R gel ( B ) displays the locations of the proteins that are differentially expressed in comparison to β-TC3; see Table S3 for further information relative to single detected protein. Gels are representative of three experiments. ( C ) Comparative proteomic profile. Graph represents protein expression levels in β-TC3 (black peaks) and β-TC3R (grey peaks). Relative volumes of the spots (V%) were calculated by ImageMaster 2D Platinum software, the same software used for gel analysis. ( D ) Proteomic groups. Proteins characterized by significant variation are grouped into four different classes. ( E ) Proteomic network. Protein interaction network reveals that 28 out of 99 proteins are differentially expressed, which are distributed into groups of different biological functions (mainly belonging to cell death pathway, negative regulators of apoptosis and protein folding).

    Techniques Used: Expressing, Software

    Evaluation of cytokine-mediated apoptosis in β-TC3 and β-TC3R cells. ( A ) Flow cytometry analysis of cell-cycle in β-TC3 and β-TC3R cells. Green line indicates cycle profile in untreated cells, while red line indicates cell cycle after treatment with cytokines. ( B ) Effects of cytokines on DNA fragmentation in β-TC3 and β-TC3R cells. M = DNA laddering Marker (100 bp). ( C ) Flow cytometry analysis of Caspase 3 in β-TC3 and β-TC3R cells after treatment with cytokines. No significant change was observed in β-TC3R cells. Cytokine treatment is 100 IU/ml of IL-1β + IFN-γ for 72 hours. U = untreated.
    Figure Legend Snippet: Evaluation of cytokine-mediated apoptosis in β-TC3 and β-TC3R cells. ( A ) Flow cytometry analysis of cell-cycle in β-TC3 and β-TC3R cells. Green line indicates cycle profile in untreated cells, while red line indicates cell cycle after treatment with cytokines. ( B ) Effects of cytokines on DNA fragmentation in β-TC3 and β-TC3R cells. M = DNA laddering Marker (100 bp). ( C ) Flow cytometry analysis of Caspase 3 in β-TC3 and β-TC3R cells after treatment with cytokines. No significant change was observed in β-TC3R cells. Cytokine treatment is 100 IU/ml of IL-1β + IFN-γ for 72 hours. U = untreated.

    Techniques Used: Flow Cytometry, Cytometry, DNA Laddering, Marker

    Molecular characterization of β-TC3R cells. qRT-PCR analysis of ( A ) IL-1RI, ( B ) IFN-γR, and ( C ) SOCS-3 in β-TC3 and β-TC3R cells after cytokine treatment compared to respective untreated cells. IL-1RI and IFN-γR are markedly downregulated, while SOCS-3 shows a 65-fold increase in β-TC3R compared to β-TC3 cells. Cytokine treatment is 100 IU/ml IL-1β, 100 IU/ml IFN-γ or their combination, for 72 hours.
    Figure Legend Snippet: Molecular characterization of β-TC3R cells. qRT-PCR analysis of ( A ) IL-1RI, ( B ) IFN-γR, and ( C ) SOCS-3 in β-TC3 and β-TC3R cells after cytokine treatment compared to respective untreated cells. IL-1RI and IFN-γR are markedly downregulated, while SOCS-3 shows a 65-fold increase in β-TC3R compared to β-TC3 cells. Cytokine treatment is 100 IU/ml IL-1β, 100 IU/ml IFN-γ or their combination, for 72 hours.

    Techniques Used: Quantitative RT-PCR

    Selection of a β-TC3 cell population resistant to cytokine-mediated cytotoxicity. ( A ) Cell phenotypes: β-TC3R cells (right) show a more elongated (fibroblastic-like) appearance in comparison to parental β-TC3 cells (left). ( B ) Proliferation assay (MTT) at 0, 24, 48, 72 and 96 hours for β-TC3 (continuous line) and resistant β-TC3R (dotted line). ( C ) Cytotoxicity assay (MTT) in β-TC3 (left) and β-TC3R (right) after cytokine treatment (100 IU/ml IL-1β, 100 IU/ml IFN-γ or their combination, for 72 hours).
    Figure Legend Snippet: Selection of a β-TC3 cell population resistant to cytokine-mediated cytotoxicity. ( A ) Cell phenotypes: β-TC3R cells (right) show a more elongated (fibroblastic-like) appearance in comparison to parental β-TC3 cells (left). ( B ) Proliferation assay (MTT) at 0, 24, 48, 72 and 96 hours for β-TC3 (continuous line) and resistant β-TC3R (dotted line). ( C ) Cytotoxicity assay (MTT) in β-TC3 (left) and β-TC3R (right) after cytokine treatment (100 IU/ml IL-1β, 100 IU/ml IFN-γ or their combination, for 72 hours).

    Techniques Used: Selection, Proliferation Assay, MTT Assay, Cytotoxicity Assay

    25) Product Images from "Inorganic Polyphosphate Modulates TRPM8 Channels"

    Article Title: Inorganic Polyphosphate Modulates TRPM8 Channels

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005404

    Activation of TRPM8 channels in Planar Lipid Bilayers by cold. Representative current traces of TRPM8 activated by lowering the temperature from 23 to 16°C in planar lipid bilayers: Channels were incorporated in planar lipid bilayers of synthetic POPC, POPE (3∶1) in presence of diC 16 PtdIns(4,5)P 2 . Experimental conditions are the same as described in the legend to Fig. 6 . Channels were inserted cis at 23°C and the temperature was then lowered to 16°C at ∼1 degree per min. Upper trace: TRPM8 activity at 23°C; lower trace: TRPM8 channel activity at 16°C (representative of 12 independent experiments). The temperature of the chambers was controlled by pyroelectric controller (see Experimental Procedures). The temperature in the cis bath (ground) was read directly using a thermoelectric junction thermometer, which also served as a point of reference for the pyroelectric controller. Data were filtered at 50 Hz. Clamping potential was −60 mV.
    Figure Legend Snippet: Activation of TRPM8 channels in Planar Lipid Bilayers by cold. Representative current traces of TRPM8 activated by lowering the temperature from 23 to 16°C in planar lipid bilayers: Channels were incorporated in planar lipid bilayers of synthetic POPC, POPE (3∶1) in presence of diC 16 PtdIns(4,5)P 2 . Experimental conditions are the same as described in the legend to Fig. 6 . Channels were inserted cis at 23°C and the temperature was then lowered to 16°C at ∼1 degree per min. Upper trace: TRPM8 activity at 23°C; lower trace: TRPM8 channel activity at 16°C (representative of 12 independent experiments). The temperature of the chambers was controlled by pyroelectric controller (see Experimental Procedures). The temperature in the cis bath (ground) was read directly using a thermoelectric junction thermometer, which also served as a point of reference for the pyroelectric controller. Data were filtered at 50 Hz. Clamping potential was −60 mV.

    Techniques Used: Activation Assay, Activity Assay

    Inhibition of TRPM8 currents by scPPX1 in whole-cell patch clamp. Upper panels: Whole-cell patch clamp measurements of menthol-induced currents were performed at −60 mV in the whole-cell configuration on HEK cells expressing TRPM8, in nominally Ca 2+ -free solution (NCF), to avoid desensitization. Menthol pulses (500 µM) were applied in the first 3–5 min after establishment of whole-cell configuration: HEK-293 cells were transiently transfected with TRPM8 (0.4 µg) and co-transfected with GFP clone (0.2 µg) to allow detection of transfected cells. Panel A: the control. Panel B: the pipette solution was supplemented with 2.3 µg/ml scPPX1. Midle panels: Whole-cell patch clamp was performed on HEK-293 TRPM8 stable cell line, which was transiently transfected with GFP (0.2 µg) alone (panel D) or with scPPX1 clone (0.4 µg) and GFP (0.2 µg) (panel E). The summaries are shown in panel F. The protocol of experiment is the same as for the measurements in the upper panel. Lower panels: Current/Voltage relationships of TRPM8 channels obtained in whole-cell patch clamp performed at −100 +100 mV voltage ramps for HEK-293 TRPM8 stable cell line, which was transiently transfected with GFP (0.2 µg) alone (panel G) or with scPPX1 clone (0.4 µg) and GFP (0.2 µg) (panel H). The summaries are shown in panel I at −100 and +100 mV.
    Figure Legend Snippet: Inhibition of TRPM8 currents by scPPX1 in whole-cell patch clamp. Upper panels: Whole-cell patch clamp measurements of menthol-induced currents were performed at −60 mV in the whole-cell configuration on HEK cells expressing TRPM8, in nominally Ca 2+ -free solution (NCF), to avoid desensitization. Menthol pulses (500 µM) were applied in the first 3–5 min after establishment of whole-cell configuration: HEK-293 cells were transiently transfected with TRPM8 (0.4 µg) and co-transfected with GFP clone (0.2 µg) to allow detection of transfected cells. Panel A: the control. Panel B: the pipette solution was supplemented with 2.3 µg/ml scPPX1. Midle panels: Whole-cell patch clamp was performed on HEK-293 TRPM8 stable cell line, which was transiently transfected with GFP (0.2 µg) alone (panel D) or with scPPX1 clone (0.4 µg) and GFP (0.2 µg) (panel E). The summaries are shown in panel F. The protocol of experiment is the same as for the measurements in the upper panel. Lower panels: Current/Voltage relationships of TRPM8 channels obtained in whole-cell patch clamp performed at −100 +100 mV voltage ramps for HEK-293 TRPM8 stable cell line, which was transiently transfected with GFP (0.2 µg) alone (panel G) or with scPPX1 clone (0.4 µg) and GFP (0.2 µg) (panel H). The summaries are shown in panel I at −100 and +100 mV.

    Techniques Used: Inhibition, Patch Clamp, Expressing, Transfection, Transferring, Stable Transfection

    A. Representative current/voltage relationship of TRPM8: Channels were incorporated in planar lipid bilayers of synthetic POPC, POPE (3∶1) in the presence of diC 16 PtdIns(4,5)P 2 . Experimental conditions are the same as described in the legend to Fig. 5 . TRPM8 channels were stimulated with the application of 500 µM of menthol. The dashed line corresponds to the mean conductance of fully open channels, working in inward direction, this state is rarely observed due to the low open probability of this subconductance level. B: Representative current traces and all points' histograms of outward (upper) and inward (lower) currents of TRPM8 channels with clamping potentials were +60 mV and −60 mV, respectively. Experimental conditions are the same as in the legend to figure 6A . C: Open probability of TRPM8 channels operating in inward and outward directions measured at +100 mV and −100 mV. Data were analyzed from a total of 9 experiments. D: Menthol dose response of the open probability of TRPM8. Demonstrated P o values were obtained at 100 mV. Data were analyzed from a total of 36 experimens.
    Figure Legend Snippet: A. Representative current/voltage relationship of TRPM8: Channels were incorporated in planar lipid bilayers of synthetic POPC, POPE (3∶1) in the presence of diC 16 PtdIns(4,5)P 2 . Experimental conditions are the same as described in the legend to Fig. 5 . TRPM8 channels were stimulated with the application of 500 µM of menthol. The dashed line corresponds to the mean conductance of fully open channels, working in inward direction, this state is rarely observed due to the low open probability of this subconductance level. B: Representative current traces and all points' histograms of outward (upper) and inward (lower) currents of TRPM8 channels with clamping potentials were +60 mV and −60 mV, respectively. Experimental conditions are the same as in the legend to figure 6A . C: Open probability of TRPM8 channels operating in inward and outward directions measured at +100 mV and −100 mV. Data were analyzed from a total of 9 experiments. D: Menthol dose response of the open probability of TRPM8. Demonstrated P o values were obtained at 100 mV. Data were analyzed from a total of 36 experimens.

    Techniques Used:

    Western blots of TRPM8 protein derived from expression in HEK-293 cell lines. TRPM8 protein samples were separated on a 10% SDS-PAGE and blotted on nitrocellulose membranes overnight in the presence of CAPS buffer (pH 11.1). Immunodetection was revealed by chemiluminescence. Lanes 1–3 probed with anti-Myc-IgG: Lane 1 – plasma membrane fractions of HEK-293 cells not expressing TRPM8; Lane 2 – plasma membrane extracts of cells stably expressing TRPM8; Lane 3 – TRPM8 protein purified on Sephacryl-300 gel-filtration chromatography. Lane 4 – Coomassie blue staining of purified TRPM8. Samples were heated for 5 min. at 70°C before loading.
    Figure Legend Snippet: Western blots of TRPM8 protein derived from expression in HEK-293 cell lines. TRPM8 protein samples were separated on a 10% SDS-PAGE and blotted on nitrocellulose membranes overnight in the presence of CAPS buffer (pH 11.1). Immunodetection was revealed by chemiluminescence. Lanes 1–3 probed with anti-Myc-IgG: Lane 1 – plasma membrane fractions of HEK-293 cells not expressing TRPM8; Lane 2 – plasma membrane extracts of cells stably expressing TRPM8; Lane 3 – TRPM8 protein purified on Sephacryl-300 gel-filtration chromatography. Lane 4 – Coomassie blue staining of purified TRPM8. Samples were heated for 5 min. at 70°C before loading.

    Techniques Used: Western Blot, Derivative Assay, Expressing, SDS Page, Immunodetection, Stable Transfection, Purification, Filtration, Chromatography, Staining

    Voltage-dependence of TRPM8 before and after the treatment with scPPX1. A: Representative current traces recordings obtained at −150 +150 mV voltage ramps before and after the treatment with polyphosphatase in a time course at the beginning of 3 rd , 10 th , 18 th , 28 th and 33 rd minutes. B: The changes in open probability obtained at different voltages in gap free recordings for TRPM8 alone (▪) or after the treatment with scPPX1 for the following intervals of time: 5–7 min (♦), 9–11 min (Δ), 14–16 min (▾), 20–23 min (◊), and 28–32 min (•). Data were analyzed from overall of 16 experiments.
    Figure Legend Snippet: Voltage-dependence of TRPM8 before and after the treatment with scPPX1. A: Representative current traces recordings obtained at −150 +150 mV voltage ramps before and after the treatment with polyphosphatase in a time course at the beginning of 3 rd , 10 th , 18 th , 28 th and 33 rd minutes. B: The changes in open probability obtained at different voltages in gap free recordings for TRPM8 alone (▪) or after the treatment with scPPX1 for the following intervals of time: 5–7 min (♦), 9–11 min (Δ), 14–16 min (▾), 20–23 min (◊), and 28–32 min (•). Data were analyzed from overall of 16 experiments.

    Techniques Used:

    Activation of TRPM8 channels in Planar Lipid Bilayers by menthol and PtdIns(4,5)P 2 . Representative single-channel current recordings of TRPM8 channels incorporated in planar lipid bilayers formed from POPC/POPE (3∶1) in n -decane, between symmetric bathing solutions of 150 mM KCl, 0.2 mM MgCl 2 in 20 mM Hepes buffer, pH 7.4 at 22°C. 0.2–0.5 µl of 0.2 µg/ml TRPM8 protein (isolated from the plasma membrane of HEK-293 cells stably expressing TRPM8) was incorporated in POPC/POPE micelles, which were added to the cis compartment (ground). Clamping potential was +60 mV. Data were filtered at 50 Hz. Upper and lower traces consist of three segments with additions of components as indicated in the figure: 2 µM of diC 8 PtdIns(4,5)P 2 and 500 µM of menthol were added to both compartments. The current recordings are representative of a total of 22 independent experiments for the upper traces and 12 independent experiments for the lower traces.
    Figure Legend Snippet: Activation of TRPM8 channels in Planar Lipid Bilayers by menthol and PtdIns(4,5)P 2 . Representative single-channel current recordings of TRPM8 channels incorporated in planar lipid bilayers formed from POPC/POPE (3∶1) in n -decane, between symmetric bathing solutions of 150 mM KCl, 0.2 mM MgCl 2 in 20 mM Hepes buffer, pH 7.4 at 22°C. 0.2–0.5 µl of 0.2 µg/ml TRPM8 protein (isolated from the plasma membrane of HEK-293 cells stably expressing TRPM8) was incorporated in POPC/POPE micelles, which were added to the cis compartment (ground). Clamping potential was +60 mV. Data were filtered at 50 Hz. Upper and lower traces consist of three segments with additions of components as indicated in the figure: 2 µM of diC 8 PtdIns(4,5)P 2 and 500 µM of menthol were added to both compartments. The current recordings are representative of a total of 22 independent experiments for the upper traces and 12 independent experiments for the lower traces.

    Techniques Used: Activation Assay, Isolation, Stable Transfection, Expressing

    Reduction of TRPM8 channel conductance by exopolyphosphatase scPPX1. A: Representative single-channel current recordings of TRPM8 channels: upper traces – TRPM8 channels recordings before treatment with scPPX1; middle traces – TRPM8 channel recording 15 minutes later after the addition of scPPX1 (2 µg); lower traces – TRPM8 channel recordings after 30 minutes of addition of scPPX1. Clamping potential was +100 mV. Data were filtered at 50 Hz. B: Symbols (▪) (n = 5) correspond to the mean conductance values of scPPX1 treated TRPM8 channels, where 2 µM of scPPX1 were added to the internal side of the channel; (○) (n = 8) mean conductance of control, untreated channels. Experimental conditions are the same as described in the legend to Fig. 6 .
    Figure Legend Snippet: Reduction of TRPM8 channel conductance by exopolyphosphatase scPPX1. A: Representative single-channel current recordings of TRPM8 channels: upper traces – TRPM8 channels recordings before treatment with scPPX1; middle traces – TRPM8 channel recording 15 minutes later after the addition of scPPX1 (2 µg); lower traces – TRPM8 channel recordings after 30 minutes of addition of scPPX1. Clamping potential was +100 mV. Data were filtered at 50 Hz. B: Symbols (▪) (n = 5) correspond to the mean conductance values of scPPX1 treated TRPM8 channels, where 2 µM of scPPX1 were added to the internal side of the channel; (○) (n = 8) mean conductance of control, untreated channels. Experimental conditions are the same as described in the legend to Fig. 6 .

    Techniques Used:

    Inhibition of TRPM8 activity by scPPX1 in intracellular Ca 2+ measurements. Upper panels: Fluorescence measurements of intracellular Ca 2+ concentration were performed on HEK-293 TRPM8 stable cell lines with transiently transfected GFP (0.2 µg) alone (panel A) or together with the scPPX1 clone (0.4 µg) (panel B). The summaries of averaged menthol responses are represented in panel C. Lower panels: Fluorescence measurements of intracellular Ca 2+ signals were performed on F-11 neuronal cells with transiently transfected TRPM8 (0.4 µg) and GFP (0.2 µg) (panel D) or together with the scPPX1 clone (0.4 µg) (panel E). The summaries of averaged menthol responses are represented in panel F.
    Figure Legend Snippet: Inhibition of TRPM8 activity by scPPX1 in intracellular Ca 2+ measurements. Upper panels: Fluorescence measurements of intracellular Ca 2+ concentration were performed on HEK-293 TRPM8 stable cell lines with transiently transfected GFP (0.2 µg) alone (panel A) or together with the scPPX1 clone (0.4 µg) (panel B). The summaries of averaged menthol responses are represented in panel C. Lower panels: Fluorescence measurements of intracellular Ca 2+ signals were performed on F-11 neuronal cells with transiently transfected TRPM8 (0.4 µg) and GFP (0.2 µg) (panel D) or together with the scPPX1 clone (0.4 µg) (panel E). The summaries of averaged menthol responses are represented in panel F.

    Techniques Used: Inhibition, Activity Assay, Fluorescence, Concentration Assay, Stable Transfection, Transfection

    A. Detection of polyP associated with the TRPM8 protein. TRPM8 was separated on native PAGE to preserve its migration in the tetrameric form. Lane 1 – standards ladder (The High-Mark Pre-stained High Molecular Weight Protein Standards, Invitrogen); Lane 2 – purified TRPM8 sample with o -toluidine blue stain of native PAGE gel; Lane 3 – o -toluidine blue stain of native PAGE gel of the same TRPM8 sample treated with 1 µl scPPX1 (2 µg/ml) for 3 h. before loading: Lane 4 and 5 are lanes 2 and 3 re-stained with Coomassie blue. B. Detection of PHB in TRPM8 in Western blot. Lane 1 – purified TRPM8 protein detected with antiMyc_IgG; Lane 2 – Western blot of purified TRPM8 probed with anti-PHB-IgG. Samples were heated for 5 min. at 70°C before loading.
    Figure Legend Snippet: A. Detection of polyP associated with the TRPM8 protein. TRPM8 was separated on native PAGE to preserve its migration in the tetrameric form. Lane 1 – standards ladder (The High-Mark Pre-stained High Molecular Weight Protein Standards, Invitrogen); Lane 2 – purified TRPM8 sample with o -toluidine blue stain of native PAGE gel; Lane 3 – o -toluidine blue stain of native PAGE gel of the same TRPM8 sample treated with 1 µl scPPX1 (2 µg/ml) for 3 h. before loading: Lane 4 and 5 are lanes 2 and 3 re-stained with Coomassie blue. B. Detection of PHB in TRPM8 in Western blot. Lane 1 – purified TRPM8 protein detected with antiMyc_IgG; Lane 2 – Western blot of purified TRPM8 probed with anti-PHB-IgG. Samples were heated for 5 min. at 70°C before loading.

    Techniques Used: Clear Native PAGE, Migration, Staining, Molecular Weight, Purification, Western Blot

    26) Product Images from "Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses"

    Article Title: Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24905-y

    Interaction of chIFIT5 with RNA carrying modifications in their 5′ termini using RNA-protein immunoprecipitation. ( A ) The genomes of negative sense single stranded RNA viruses carry triphosphate linkage (5′ppp) in the first transcribed base of the RNA and removal of 5′ppp structure will leave non-IFN stimulatory hydroxyl group (OH). ( B ) RNA carrying both 5′ppp and OH termini were biotinylated and coated on streptavidin beads before interaction with V5-tagged chIFIT5. Total ribonucleoproteins were isolated and stained for the V5 tag. ( C ) Pull down of biotinylated RNA interacting with chIFIT5 indicated that both human and chicken IFIT5 interacted with RNA carrying 5′ppp structures. EV = empty vector, M = marker.
    Figure Legend Snippet: Interaction of chIFIT5 with RNA carrying modifications in their 5′ termini using RNA-protein immunoprecipitation. ( A ) The genomes of negative sense single stranded RNA viruses carry triphosphate linkage (5′ppp) in the first transcribed base of the RNA and removal of 5′ppp structure will leave non-IFN stimulatory hydroxyl group (OH). ( B ) RNA carrying both 5′ppp and OH termini were biotinylated and coated on streptavidin beads before interaction with V5-tagged chIFIT5. Total ribonucleoproteins were isolated and stained for the V5 tag. ( C ) Pull down of biotinylated RNA interacting with chIFIT5 indicated that both human and chicken IFIT5 interacted with RNA carrying 5′ppp structures. EV = empty vector, M = marker.

    Techniques Used: Immunoprecipitation, Isolation, Staining, Plasmid Preparation, Marker

    27) Product Images from "Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses"

    Article Title: Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24905-y

    Interaction of chIFIT5 with RNA carrying modifications in their 5′ termini using RNA-protein immunoprecipitation. ( A ) The genomes of negative sense single stranded RNA viruses carry triphosphate linkage (5′ppp) in the first transcribed base of the RNA and removal of 5′ppp structure will leave non-IFN stimulatory hydroxyl group (OH). ( B ) RNA carrying both 5′ppp and OH termini were biotinylated and coated on streptavidin beads before interaction with V5-tagged chIFIT5. Total ribonucleoproteins were isolated and stained for the V5 tag. ( C ) Pull down of biotinylated RNA interacting with chIFIT5 indicated that both human and chicken IFIT5 interacted with RNA carrying 5′ppp structures. EV = empty vector, M = marker.
    Figure Legend Snippet: Interaction of chIFIT5 with RNA carrying modifications in their 5′ termini using RNA-protein immunoprecipitation. ( A ) The genomes of negative sense single stranded RNA viruses carry triphosphate linkage (5′ppp) in the first transcribed base of the RNA and removal of 5′ppp structure will leave non-IFN stimulatory hydroxyl group (OH). ( B ) RNA carrying both 5′ppp and OH termini were biotinylated and coated on streptavidin beads before interaction with V5-tagged chIFIT5. Total ribonucleoproteins were isolated and stained for the V5 tag. ( C ) Pull down of biotinylated RNA interacting with chIFIT5 indicated that both human and chicken IFIT5 interacted with RNA carrying 5′ppp structures. EV = empty vector, M = marker.

    Techniques Used: Immunoprecipitation, Isolation, Staining, Plasmid Preparation, Marker

    28) Product Images from "Placental Growth Factor Contributes to Micro-Vascular Abnormalization and Blood-Retinal Barrier Breakdown in Diabetic Retinopathy"

    Article Title: Placental Growth Factor Contributes to Micro-Vascular Abnormalization and Blood-Retinal Barrier Breakdown in Diabetic Retinopathy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017462

    Angiogenic response on retinal vascular cells. (a-d) Confocal microscopy of vascular abnormalities within the neural retina, one month after pVAX2-rPGF-1 ET. Confocal microscopy of flat-mounted neuroretinas from eyes in which rPGF-1 was over-expressed shows lectin-positive blood vessels and cells at different depth levels, from the superficial (1) to the deep vascular plexus (4). Scale bars = 50 µm. ( a, b ) The abnormalities noticed in vivo on SLO angiograms from two eyes were found on retinal flat-mounts by comparison of the vascular architectures. ( c, d ) For illustration, retinal flat-mounts have been turned into grey-scale pictures. Red asterisks indicate the presence of infiltrating cells around vascular abnormalization. (e) Optic microscopy of vascular abnormalities within flat-mounted retinas, two months after ET. Vascular abnormalities ( e 1 ), sprouts ( e 2 ) and microaneurysmal-like structures ( e 3 ) were observed between the inner and the middle vascular beds. (f) Q PCR analysis of VEGF, IL-1beta, TNF-alpha and IL-6, two months after ET, in retina of control (in white, n = 3 in duplicate) and pVAX2-rPGF-1 ET treated (in black, n = 5 in duplicate) retina from BN rats. ***, p
    Figure Legend Snippet: Angiogenic response on retinal vascular cells. (a-d) Confocal microscopy of vascular abnormalities within the neural retina, one month after pVAX2-rPGF-1 ET. Confocal microscopy of flat-mounted neuroretinas from eyes in which rPGF-1 was over-expressed shows lectin-positive blood vessels and cells at different depth levels, from the superficial (1) to the deep vascular plexus (4). Scale bars = 50 µm. ( a, b ) The abnormalities noticed in vivo on SLO angiograms from two eyes were found on retinal flat-mounts by comparison of the vascular architectures. ( c, d ) For illustration, retinal flat-mounts have been turned into grey-scale pictures. Red asterisks indicate the presence of infiltrating cells around vascular abnormalization. (e) Optic microscopy of vascular abnormalities within flat-mounted retinas, two months after ET. Vascular abnormalities ( e 1 ), sprouts ( e 2 ) and microaneurysmal-like structures ( e 3 ) were observed between the inner and the middle vascular beds. (f) Q PCR analysis of VEGF, IL-1beta, TNF-alpha and IL-6, two months after ET, in retina of control (in white, n = 3 in duplicate) and pVAX2-rPGF-1 ET treated (in black, n = 5 in duplicate) retina from BN rats. ***, p

    Techniques Used: Confocal Microscopy, In Vivo, Microscopy, Polymerase Chain Reaction

    29) Product Images from "Prion Disease in Dromedary Camels, Algeria"

    Article Title: Prion Disease in Dromedary Camels, Algeria

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2406.172007

    Western blot analysis of protein-resistant core (PrP res ) of pathological dromedary prion protein. A) Western blot analysis of proteinase K (PK)–treated PrP Sc in brain homogenates from dromedary camels with neurologic symptoms (nos. 4 and 8), Algeria. A sample of sheep scrapie was loaded as control (indicated as C+). Membranes were probed with L42 (left) and 12B2 monoclonal antibody (mAb) (right). Molecular weights (kDa) are indicated on the left. Tissue equivalents loaded per lane were 2 mg for camel samples and 0.1 mg for sheep scrapie. B) Samples after deglycosylation. Membrane was probed with L42 mAb. C) Comparison of dromedary PrP res (from camel no. 4) with sheep bovine spongiform encephalopathy (BSE), bovine BSE, and sheep scrapie samples by ISS (Istituto Superiore di Sanità) discriminatory Western blot ( 17 ). Tissue equivalents loaded per lane were 2 mg for dromedary camel and bovine samples and 0.1 mg for sheep samples. In each blot, samples were loaded as follows: lane 1, ovine BSE; lane 2, bovine BSE; lane 3, dromedary camel no. 4; lane 4, ovine scrapie. Membranes were probed with L42, 12B2, and SAF32 mAbs, as indicated. For the analyses in panels B and C, protein standards were loaded and are indicated as M.
    Figure Legend Snippet: Western blot analysis of protein-resistant core (PrP res ) of pathological dromedary prion protein. A) Western blot analysis of proteinase K (PK)–treated PrP Sc in brain homogenates from dromedary camels with neurologic symptoms (nos. 4 and 8), Algeria. A sample of sheep scrapie was loaded as control (indicated as C+). Membranes were probed with L42 (left) and 12B2 monoclonal antibody (mAb) (right). Molecular weights (kDa) are indicated on the left. Tissue equivalents loaded per lane were 2 mg for camel samples and 0.1 mg for sheep scrapie. B) Samples after deglycosylation. Membrane was probed with L42 mAb. C) Comparison of dromedary PrP res (from camel no. 4) with sheep bovine spongiform encephalopathy (BSE), bovine BSE, and sheep scrapie samples by ISS (Istituto Superiore di Sanità) discriminatory Western blot ( 17 ). Tissue equivalents loaded per lane were 2 mg for dromedary camel and bovine samples and 0.1 mg for sheep samples. In each blot, samples were loaded as follows: lane 1, ovine BSE; lane 2, bovine BSE; lane 3, dromedary camel no. 4; lane 4, ovine scrapie. Membranes were probed with L42, 12B2, and SAF32 mAbs, as indicated. For the analyses in panels B and C, protein standards were loaded and are indicated as M.

    Techniques Used: Western Blot

    30) Product Images from "MT1-MMP deficiency leads to defective ependymal cell maturation, impaired ciliogenesis, and hydrocephalus"

    Article Title: MT1-MMP deficiency leads to defective ependymal cell maturation, impaired ciliogenesis, and hydrocephalus

    Journal: JCI Insight

    doi: 10.1172/jci.insight.132782

    Polarity analyses of basal body patches in ependymal cells. ( A ) Representative confocal images of whole-mount staining of γ-tubulin (green) and β-catenin (red) in the walls of LVs from WT and Mmp14 –/– brains at P10. Scale bar: 20 μm. ( B ) Distribution of the percentage of cells with different length/width ratios of the basal body patches marked by γ-tubulin staining in A . Contingency table test, P
    Figure Legend Snippet: Polarity analyses of basal body patches in ependymal cells. ( A ) Representative confocal images of whole-mount staining of γ-tubulin (green) and β-catenin (red) in the walls of LVs from WT and Mmp14 –/– brains at P10. Scale bar: 20 μm. ( B ) Distribution of the percentage of cells with different length/width ratios of the basal body patches marked by γ-tubulin staining in A . Contingency table test, P

    Techniques Used: Staining

    Examination of Notch signaling in lateral ventricles and inhibition of Notch signaling in cultured ECs isolated from mouse brains. (A ) Representative Western blotting shows the NICD, DLL1, and β-actin expression of LVs from WT and Mmp14 –/– brains at P7 and P10. ( B ) Fold change in mRNA levels of Hes5 and Hey1 in LVs of WT and Mmp14 –/– brains at P7 and P10. Gene expression levels in WT samples were designated as 1. Two-tailed Student’s t test, * P
    Figure Legend Snippet: Examination of Notch signaling in lateral ventricles and inhibition of Notch signaling in cultured ECs isolated from mouse brains. (A ) Representative Western blotting shows the NICD, DLL1, and β-actin expression of LVs from WT and Mmp14 –/– brains at P7 and P10. ( B ) Fold change in mRNA levels of Hes5 and Hey1 in LVs of WT and Mmp14 –/– brains at P7 and P10. Gene expression levels in WT samples were designated as 1. Two-tailed Student’s t test, * P

    Techniques Used: Inhibition, Cell Culture, Isolation, Western Blot, Expressing, Two Tailed Test

    MT1-MMP expression in the wall of the lateral ventricle in Mmp14 +/LacZ –transgenic mice. ( A ) Representative β-galactosidase staining in the developing brain at P3. ( B ) Diagram of a coronal section in the dorsal region of a mouse brain showing the LV. High-magnification images of coronal sections with different staining are shown in C and D . ( C ) β-Galactosidase staining (blue) in coronal sections of Mmp14 +/LacZ mouse brains at P3, P6, and P10. ( D ) Representative staining of LacZ, BLBP, Sox2, vimentin, and S100 in coronal sections of LVs from P0 and P10 mouse brains. Scale bar: 50 μm.
    Figure Legend Snippet: MT1-MMP expression in the wall of the lateral ventricle in Mmp14 +/LacZ –transgenic mice. ( A ) Representative β-galactosidase staining in the developing brain at P3. ( B ) Diagram of a coronal section in the dorsal region of a mouse brain showing the LV. High-magnification images of coronal sections with different staining are shown in C and D . ( C ) β-Galactosidase staining (blue) in coronal sections of Mmp14 +/LacZ mouse brains at P3, P6, and P10. ( D ) Representative staining of LacZ, BLBP, Sox2, vimentin, and S100 in coronal sections of LVs from P0 and P10 mouse brains. Scale bar: 50 μm.

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Staining

    Examination of ependymal cilia in WT and mutant LV. ( A ) Immunofluorescence staining of acetylated α-tubulin in LVs of P15 brains shows evenly distributed cilium bundles in a WT animal (arrows) and flattened and discontinued cilium bundles in a Mmp14 –/– mouse (arrowheads). Scale bar: 50 μm. ( B ) Scanning electron microscopy on the surface of LV walls from WT and Mmp14 –/– mice at different postnatal stages. Note the evenly distributed cilium bundles with a uniformed direction of cilia tufts across the LV wall in a WT mouse (arrows) and the sparsely distributed cilium bundles with shorter and fewer cilia in disrupted orientation in a MT1-MMP–deficient mouse (arrowheads). Scale bar: 20 μm. ( C ) Quantification of the number of cilia tufts at the surface of the LV in B . Two-tailed Student’s t test, ** P
    Figure Legend Snippet: Examination of ependymal cilia in WT and mutant LV. ( A ) Immunofluorescence staining of acetylated α-tubulin in LVs of P15 brains shows evenly distributed cilium bundles in a WT animal (arrows) and flattened and discontinued cilium bundles in a Mmp14 –/– mouse (arrowheads). Scale bar: 50 μm. ( B ) Scanning electron microscopy on the surface of LV walls from WT and Mmp14 –/– mice at different postnatal stages. Note the evenly distributed cilium bundles with a uniformed direction of cilia tufts across the LV wall in a WT mouse (arrows) and the sparsely distributed cilium bundles with shorter and fewer cilia in disrupted orientation in a MT1-MMP–deficient mouse (arrowheads). Scale bar: 20 μm. ( C ) Quantification of the number of cilia tufts at the surface of the LV in B . Two-tailed Student’s t test, ** P

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

    31) Product Images from "An OBSL1-Cul7Fbxw8 Ubiquitin Ligase Signaling Mechanism Regulates Golgi Morphology and Dendrite Patterning"

    Article Title: An OBSL1-Cul7Fbxw8 Ubiquitin Ligase Signaling Mechanism Regulates Golgi Morphology and Dendrite Patterning

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001060

    The cytoskeletal adaptor protein OBSL1 forms a physical complex with the scaffold protein Cul7. (A) Lysates of 293T cells harboring an inducible HA-Fbxw8 lentivirus were immunoprecipitated with the HA antibody and subjected to proteomic analysis using LC-MS/MS. CompPASS was utilized to interrogate datasets and assign the D N and Z scoring metrics. Proteins with a D N score greater than 1 and a Z score greater than 3.5 were considered HCIPs. We confirmed that endogenous Fbxw8 and endogenous Cul7 form a complex in cells ( Figure S6G ). (B) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and Cul7-HA or the control vectors were immunoprecipitated (IP) with the V5 or HA antibodies. Immunoprecipitates and lysates were immunoblotted (IB) with the V5 and HA antibodies. (C) Domain map of full-length (FL) Cul7 protein and deletion mutant proteins. Cul7 consists of a large N-terminal domain unique among the cullin family that contains a CPH domain, a DOC domain, a cullin domain, and an extreme C-terminal region that contains a neddylation motif. (D) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and full-length Cul7-HA, deletion mutants, or the control vector were immunoprecipitated with the HA antibody. Immunoprecipitates and lysates were immmunoblotted with the HA and V5 antibodies. (E) Granule neurons were transfected with the expression plasmids encoding V5-OBSL1 and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the V5 and GFP antibodies. Arrow denotes co-localization of OBSL1 and GFP-GT. Scale bar = 5 µm. (F) Granule neurons were transfected with the expression plasmids encoding Cul7-HA or deletion mutants and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the HA and GFP antibodies. Arrows denote the Golgi apparatus as labeled by GFP-GT. Scale bar = 5 µm.
    Figure Legend Snippet: The cytoskeletal adaptor protein OBSL1 forms a physical complex with the scaffold protein Cul7. (A) Lysates of 293T cells harboring an inducible HA-Fbxw8 lentivirus were immunoprecipitated with the HA antibody and subjected to proteomic analysis using LC-MS/MS. CompPASS was utilized to interrogate datasets and assign the D N and Z scoring metrics. Proteins with a D N score greater than 1 and a Z score greater than 3.5 were considered HCIPs. We confirmed that endogenous Fbxw8 and endogenous Cul7 form a complex in cells ( Figure S6G ). (B) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and Cul7-HA or the control vectors were immunoprecipitated (IP) with the V5 or HA antibodies. Immunoprecipitates and lysates were immunoblotted (IB) with the V5 and HA antibodies. (C) Domain map of full-length (FL) Cul7 protein and deletion mutant proteins. Cul7 consists of a large N-terminal domain unique among the cullin family that contains a CPH domain, a DOC domain, a cullin domain, and an extreme C-terminal region that contains a neddylation motif. (D) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and full-length Cul7-HA, deletion mutants, or the control vector were immunoprecipitated with the HA antibody. Immunoprecipitates and lysates were immmunoblotted with the HA and V5 antibodies. (E) Granule neurons were transfected with the expression plasmids encoding V5-OBSL1 and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the V5 and GFP antibodies. Arrow denotes co-localization of OBSL1 and GFP-GT. Scale bar = 5 µm. (F) Granule neurons were transfected with the expression plasmids encoding Cul7-HA or deletion mutants and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the HA and GFP antibodies. Arrows denote the Golgi apparatus as labeled by GFP-GT. Scale bar = 5 µm.

    Techniques Used: Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Marker, Immunocytochemistry, Labeling

    Fbxw8 is localized at the Golgi apparatus in neurons. (A) Lysates of embryonic brain and placenta were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (B) Lysates of cerebellum from rat pups from P6 to adult and of primary P6 granule neurons cultured DIV1 to DIV9 were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (C) Lysates of granule neurons were subjected to subcellular fractionation and immunoblotted with the Fbxw8, 14-3-3β, and SnoN antibodies, the latter two to mark cytoplasmic (C) and nuclear (N) fractions, respectively. (D) Granule neurons were subjected to immunocytochemistry with the Fbxw8 antibody together with the TGN38, GM130, Hsp60, or PDI antibody. DNA dye bisbenzimide (Hoechst 33258) was used to stain the nucleus. Arrows indicate localization of Fbxw8 at the Golgi apparatus. Scale bar = 10 µm. (E) Lysates of 293T cells transfected with the expression plasmids encoding Flag-Fbxw8 and GFP together with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Flag and GFP antibodies. (F) Lysates of granule neurons transfected with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (G) Granule neurons transfected at DIV2 with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid together with an expression plasmid encoding farnesylated GFP to label membranes were fixed at DIV5 and were subjected to immunocytochemistry using the GFP and Fbxw8 antibodies. Dotted lines represent tracing of transfected cells. Arrows indicate Golgi-localized Fbxw8 in transfected neurons. Scale bar = 10 µm. Fbxw8 knockdown reduced almost completely Fbxw8 immunofluorescence in neurons.
    Figure Legend Snippet: Fbxw8 is localized at the Golgi apparatus in neurons. (A) Lysates of embryonic brain and placenta were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (B) Lysates of cerebellum from rat pups from P6 to adult and of primary P6 granule neurons cultured DIV1 to DIV9 were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (C) Lysates of granule neurons were subjected to subcellular fractionation and immunoblotted with the Fbxw8, 14-3-3β, and SnoN antibodies, the latter two to mark cytoplasmic (C) and nuclear (N) fractions, respectively. (D) Granule neurons were subjected to immunocytochemistry with the Fbxw8 antibody together with the TGN38, GM130, Hsp60, or PDI antibody. DNA dye bisbenzimide (Hoechst 33258) was used to stain the nucleus. Arrows indicate localization of Fbxw8 at the Golgi apparatus. Scale bar = 10 µm. (E) Lysates of 293T cells transfected with the expression plasmids encoding Flag-Fbxw8 and GFP together with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Flag and GFP antibodies. (F) Lysates of granule neurons transfected with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (G) Granule neurons transfected at DIV2 with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid together with an expression plasmid encoding farnesylated GFP to label membranes were fixed at DIV5 and were subjected to immunocytochemistry using the GFP and Fbxw8 antibodies. Dotted lines represent tracing of transfected cells. Arrows indicate Golgi-localized Fbxw8 in transfected neurons. Scale bar = 10 µm. Fbxw8 knockdown reduced almost completely Fbxw8 immunofluorescence in neurons.

    Techniques Used: Cell Culture, Fractionation, Immunocytochemistry, Staining, Transfection, Expressing, Plasmid Preparation, Immunofluorescence

    32) Product Images from "A Tumor-Associated Mutation of FYVE-CENT Prevents Its Interaction with Beclin 1 and Interferes with Cytokinesis"

    Article Title: A Tumor-Associated Mutation of FYVE-CENT Prevents Its Interaction with Beclin 1 and Interferes with Cytokinesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017086

    The average expression of FYVE-CENT and associated genes is significantly lower in high vs. low grade breast cancers. (A)–(D) The gene expression data were derived from the Gene Expression Omnibus GSE1456 and GSE4922 datasets. The expression values were median centred within each of the series separately. The p-values were derived from comparing means by independent samples t-test (SPSS, v.16.0). (E) Proposed model: FYVE-CENT- Beclin 1 interplay in a positive feedback loop manner during cytokinesis.
    Figure Legend Snippet: The average expression of FYVE-CENT and associated genes is significantly lower in high vs. low grade breast cancers. (A)–(D) The gene expression data were derived from the Gene Expression Omnibus GSE1456 and GSE4922 datasets. The expression values were median centred within each of the series separately. The p-values were derived from comparing means by independent samples t-test (SPSS, v.16.0). (E) Proposed model: FYVE-CENT- Beclin 1 interplay in a positive feedback loop manner during cytokinesis.

    Techniques Used: Expressing, Derivative Assay

    Two-hybrid interactions of Beclin 1 with FYVE-CENT. The figure shows schematically the domain of Beclin 1 that interacts with FYVE-CENT.
    Figure Legend Snippet: Two-hybrid interactions of Beclin 1 with FYVE-CENT. The figure shows schematically the domain of Beclin 1 that interacts with FYVE-CENT.

    Techniques Used:

    Beclin 1 interacts with FYVE-CENT. (A) GST pull-down from HeLa cell lysates transiently over-expressing myc-Beclin 1 using recombinant GST-FYVE-CENT C-terminal fusion (2120–2539) protein or GST protein immobilized on glutathione-Sepharose beads. Proteins eluted from the beads were analyzed by SDS-PAGE and immuno-blotting using an anti-myc antibody. Equal amounts of GST-FYVE-CENT C-terminal fusion protein and GST protein were loaded. (B) HeLa cell lysates were subjected to immunoprecipitation (IP) with an antibody against FYVE-CENT. Immunoprecipitated proteins were detected by Western blotting, using anti-Beclin 1 and anti-FYVE-CEΝT antibodies. (C) HeLa cells transiently over-expressing myc-Beclin 1 were pulled down with recombinant GST-FYVE-CENT C-terminal fusion (1807–2539 or 1807–2539 R1945Q) protein or GST protein immobilized on glutathione-Sepharose beads. (D) HCC-1395 control cells and HCC-1954 FYVE-CENT R1945Q mutant cells were lysed and subjected to immunoprecipitation (IP) with an antibody against FYVE-CENT. Immunoprecipitated proteins were detected by Western blotting, using anti-Beclin 1 and anti-FYVE-CEΝT antibodies. (E) HeLa cells transiently over-expressing myc-TTC19 or myc-KIF13A were pulled down with recombinant GST-FYVE-CENT C-terminal fusion protein and GST-FYVE-CENT C-terminal R1945Q fusion protein or GST protein immobilized on glutathione-Sepharose beads.
    Figure Legend Snippet: Beclin 1 interacts with FYVE-CENT. (A) GST pull-down from HeLa cell lysates transiently over-expressing myc-Beclin 1 using recombinant GST-FYVE-CENT C-terminal fusion (2120–2539) protein or GST protein immobilized on glutathione-Sepharose beads. Proteins eluted from the beads were analyzed by SDS-PAGE and immuno-blotting using an anti-myc antibody. Equal amounts of GST-FYVE-CENT C-terminal fusion protein and GST protein were loaded. (B) HeLa cell lysates were subjected to immunoprecipitation (IP) with an antibody against FYVE-CENT. Immunoprecipitated proteins were detected by Western blotting, using anti-Beclin 1 and anti-FYVE-CEΝT antibodies. (C) HeLa cells transiently over-expressing myc-Beclin 1 were pulled down with recombinant GST-FYVE-CENT C-terminal fusion (1807–2539 or 1807–2539 R1945Q) protein or GST protein immobilized on glutathione-Sepharose beads. (D) HCC-1395 control cells and HCC-1954 FYVE-CENT R1945Q mutant cells were lysed and subjected to immunoprecipitation (IP) with an antibody against FYVE-CENT. Immunoprecipitated proteins were detected by Western blotting, using anti-Beclin 1 and anti-FYVE-CEΝT antibodies. (E) HeLa cells transiently over-expressing myc-TTC19 or myc-KIF13A were pulled down with recombinant GST-FYVE-CENT C-terminal fusion protein and GST-FYVE-CENT C-terminal R1945Q fusion protein or GST protein immobilized on glutathione-Sepharose beads.

    Techniques Used: Expressing, Recombinant, SDS Page, Immunoprecipitation, Western Blot, Mutagenesis

    The localization of Beclin 1 to the intercellular bridge during cytokinesis is abolished in FYVE-CENT R1945Q mutant breast cancer cells. (A) and (B) Confocal micrographs of HeLa, HCC-1395 and HCC-1954 cells stained with antibodies against Aurora B and Beclin 1 (A) or FYVE-CENT (B), and with Hoechst. Magnifications of the intercellular bridges are shown in the insets. Scale bars: 10 µm. (C) Graphic presentation of quantification of control cells (HCC-1395) and mutant cells (HCC-1954) labeled on the midbody with anti-FYVE-CENT or anti-Beclin 1 antibodies. Error bars show mean ± s.d. Control cells stained with anti-FYVE-CENT: 4 independent experiments, n = 1769 cells. Mutant cells stained with anti-FYVE-CENT: 4 independent experiments, n = 1781 cells. Control cells stained with anti-Beclin 1: 4 independent experiments, n = 1340. Mutant cells stained with anti-Beclin 1: 4 independent experiments, n = 1521. p value for cells labeled with anti-FYVE-CENT on the midbody: 0.01. p value for cells labeled with anti-Beclin 1 on the midbody: 0.01.
    Figure Legend Snippet: The localization of Beclin 1 to the intercellular bridge during cytokinesis is abolished in FYVE-CENT R1945Q mutant breast cancer cells. (A) and (B) Confocal micrographs of HeLa, HCC-1395 and HCC-1954 cells stained with antibodies against Aurora B and Beclin 1 (A) or FYVE-CENT (B), and with Hoechst. Magnifications of the intercellular bridges are shown in the insets. Scale bars: 10 µm. (C) Graphic presentation of quantification of control cells (HCC-1395) and mutant cells (HCC-1954) labeled on the midbody with anti-FYVE-CENT or anti-Beclin 1 antibodies. Error bars show mean ± s.d. Control cells stained with anti-FYVE-CENT: 4 independent experiments, n = 1769 cells. Mutant cells stained with anti-FYVE-CENT: 4 independent experiments, n = 1781 cells. Control cells stained with anti-Beclin 1: 4 independent experiments, n = 1340. Mutant cells stained with anti-Beclin 1: 4 independent experiments, n = 1521. p value for cells labeled with anti-FYVE-CENT on the midbody: 0.01. p value for cells labeled with anti-Beclin 1 on the midbody: 0.01.

    Techniques Used: Mutagenesis, Staining, Labeling

    A FYVE-CENT R1945Q mutant breast cancer cell line exhibits an increased number of cells arrested in cytokinesis as well as bi- and multinuclear cells. (A) Sequencing of cDNA for exons 31 to 33 of FYVE-CENT from the HCC-1395 and HCC1954 breast cancer cell lines revealed a G to A substitution at base position 5834 in the HCC1954 cell line. (B). Confocal micrographs of HCC-1395 and HCC-1954 breast cancer cell lines cells stained with α-tubulin, Alexa Fluor® 594 phalloidin and Hoechst. In FYVE-CENT mutant cells (HCC-1954) there is a significant increase in cells arrested in cytokinesis (arrows) compared to the control as well as increase in binuclear-multinuclear cells (asterisk). Scale bars: 20 µm. ( C ) Graphic presentation of quantification of cells arrested at the midbody stage and bi-multinuclear cells in control cells (HCC-1395) and FYVE-CENT R1945Q mutant cell line (HCC-1954). Error bars show mean ± s.d. Control: 3 independent experiments, n = 1142 cells. Mutant cells: 3 independent experiments, n = 1225 cells. p value for cells arrested at the midbody stage
    Figure Legend Snippet: A FYVE-CENT R1945Q mutant breast cancer cell line exhibits an increased number of cells arrested in cytokinesis as well as bi- and multinuclear cells. (A) Sequencing of cDNA for exons 31 to 33 of FYVE-CENT from the HCC-1395 and HCC1954 breast cancer cell lines revealed a G to A substitution at base position 5834 in the HCC1954 cell line. (B). Confocal micrographs of HCC-1395 and HCC-1954 breast cancer cell lines cells stained with α-tubulin, Alexa Fluor® 594 phalloidin and Hoechst. In FYVE-CENT mutant cells (HCC-1954) there is a significant increase in cells arrested in cytokinesis (arrows) compared to the control as well as increase in binuclear-multinuclear cells (asterisk). Scale bars: 20 µm. ( C ) Graphic presentation of quantification of cells arrested at the midbody stage and bi-multinuclear cells in control cells (HCC-1395) and FYVE-CENT R1945Q mutant cell line (HCC-1954). Error bars show mean ± s.d. Control: 3 independent experiments, n = 1142 cells. Mutant cells: 3 independent experiments, n = 1225 cells. p value for cells arrested at the midbody stage

    Techniques Used: Mutagenesis, Sequencing, Staining

    33) Product Images from "Sigma-2 receptor ligand as a novel method for delivering a SMAC mimetic drug for treating ovarian cancer"

    Article Title: Sigma-2 receptor ligand as a novel method for delivering a SMAC mimetic drug for treating ovarian cancer

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2013.593

    SW IV-52s and SW III-123 induced NF- κ B activation. ( A ) SKOV-3 cells were treated with 0, 1, 3 and 10 μ M SW 43 , SW IV-52s or SW III-123 for 24 h. The whole-cell lysates were analysed by western blot. ( B ) SKOV-3 cells were treated with 3 μ M of SW 43 , SW IV-52s or SW III-123 for indicated time. The whole-cell lysates were analysed by western blot.
    Figure Legend Snippet: SW IV-52s and SW III-123 induced NF- κ B activation. ( A ) SKOV-3 cells were treated with 0, 1, 3 and 10 μ M SW 43 , SW IV-52s or SW III-123 for 24 h. The whole-cell lysates were analysed by western blot. ( B ) SKOV-3 cells were treated with 3 μ M of SW 43 , SW IV-52s or SW III-123 for indicated time. The whole-cell lysates were analysed by western blot.

    Techniques Used: Activation Assay, Western Blot

    SW IV-52s and SW III-123 induced TNF α -dependent apoptosis. ( A ) SKOV-3 cells were pre-treated with or without 2 μ g ml –1 TNF α antibody for 1 h, and then treated with 3 μ M SW IV-52s, SW 43 or SW III-123 for 24 h. The cells were assayed for caspase 3 activity. ( B – D ) SKOV-3 cells were pre-treated with or without 2 μ g ml –1 TNF α antibody for 1 h, and then treated with 3 or 10 μ M SW 43 ( B ), SW IV-52s ( C ) or SW III-123 ( D ) for 48 h. Viability of cells was determined by MTS assay. The representative data from three independent experiments are shown. * P
    Figure Legend Snippet: SW IV-52s and SW III-123 induced TNF α -dependent apoptosis. ( A ) SKOV-3 cells were pre-treated with or without 2 μ g ml –1 TNF α antibody for 1 h, and then treated with 3 μ M SW IV-52s, SW 43 or SW III-123 for 24 h. The cells were assayed for caspase 3 activity. ( B – D ) SKOV-3 cells were pre-treated with or without 2 μ g ml –1 TNF α antibody for 1 h, and then treated with 3 or 10 μ M SW 43 ( B ), SW IV-52s ( C ) or SW III-123 ( D ) for 48 h. Viability of cells was determined by MTS assay. The representative data from three independent experiments are shown. * P

    Techniques Used: Activity Assay, MTS Assay

    SW III-123 potently induced cell death in ovarian cancer cells. SKOV-3 ( A ), CaOV-3 ( B ) or BG-1 ( C ) cells were treated with increasing concentrations of SW 43 (○), SW IV-52s (●), the combination of SW 43 and SW IV-52s (□), or SW III-123 (▪) for 24 h. Cell viability was determined by MTS assay. ( D ) Flow cytometric determination of the internalisation of SW120 in SKOV-3 cells with the blocking compound of SW 43 (▪), SW IV-52s (▴) or SW III-123 (●). The bars represent mean±s.e.m. in at least three independent experiments.
    Figure Legend Snippet: SW III-123 potently induced cell death in ovarian cancer cells. SKOV-3 ( A ), CaOV-3 ( B ) or BG-1 ( C ) cells were treated with increasing concentrations of SW 43 (○), SW IV-52s (●), the combination of SW 43 and SW IV-52s (□), or SW III-123 (▪) for 24 h. Cell viability was determined by MTS assay. ( D ) Flow cytometric determination of the internalisation of SW120 in SKOV-3 cells with the blocking compound of SW 43 (▪), SW IV-52s (▴) or SW III-123 (●). The bars represent mean±s.e.m. in at least three independent experiments.

    Techniques Used: MTS Assay, Flow Cytometry, Blocking Assay

    SW IV-52s and SW III-123 rapidly degraded cIAP1 and cIAP2 and cleaved pro-caspase-8, -9, and -3. (A) SKOV-3 cells were treated with 0, 1, 3 and 10 μ M SW 43, SW IV-52s or SW III-123 for 24 h. The whole-cell lysates were analysed by western blot. ( B ) SKOV-3 cells were treated with 3 μ M of SW 43 , SW IV-52s or SW III-123 for indicated time. The whole-cell lysates were analysed by western blot.
    Figure Legend Snippet: SW IV-52s and SW III-123 rapidly degraded cIAP1 and cIAP2 and cleaved pro-caspase-8, -9, and -3. (A) SKOV-3 cells were treated with 0, 1, 3 and 10 μ M SW 43, SW IV-52s or SW III-123 for 24 h. The whole-cell lysates were analysed by western blot. ( B ) SKOV-3 cells were treated with 3 μ M of SW 43 , SW IV-52s or SW III-123 for indicated time. The whole-cell lysates were analysed by western blot.

    Techniques Used: Western Blot

    The synthetic schemes for generating SW III-123 (A) and SW IV-52s (B).
    Figure Legend Snippet: The synthetic schemes for generating SW III-123 (A) and SW IV-52s (B).

    Techniques Used:

    34) Product Images from "Wnt10b Activates the Wnt, Notch and NF?B Pathways in U2OS Osteosarcoma Cells"

    Article Title: Wnt10b Activates the Wnt, Notch and NF?B Pathways in U2OS Osteosarcoma Cells

    Journal: Journal of cellular biochemistry

    doi: 10.1002/jcb.23048

    The Wnt pathway is activated in U2OS-Wnt10b cells. QPCR analysis of genes in the Wnt pathway was performed in U2OS and U2OS-Wnt10b cells using primers specific for the indicated genes. The bars represent the fold change between U2OS and U2OS-Wnt10b cells
    Figure Legend Snippet: The Wnt pathway is activated in U2OS-Wnt10b cells. QPCR analysis of genes in the Wnt pathway was performed in U2OS and U2OS-Wnt10b cells using primers specific for the indicated genes. The bars represent the fold change between U2OS and U2OS-Wnt10b cells

    Techniques Used: Real-time Polymerase Chain Reaction

    The Notch pathway is activated in U2OS-Wnt10b cells. (A) QPCR analysis of genes in the Notch pathway was performed in U2OS and U2OS-Wnt10b cells using primers specific for the indicated genes. (B) U2OS and U2OS-Wnt10b cells were transiently transfected
    Figure Legend Snippet: The Notch pathway is activated in U2OS-Wnt10b cells. (A) QPCR analysis of genes in the Notch pathway was performed in U2OS and U2OS-Wnt10b cells using primers specific for the indicated genes. (B) U2OS and U2OS-Wnt10b cells were transiently transfected

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection

    Production and characterization of U2OS cells expressing Wnt10b. (A) U2OS cells were transiently transfected with either TOP-Flash or FOP-Flash luciferase constructs, treated with control media or 100 ng/mL purified Wnt3a and luciferase assays performed.
    Figure Legend Snippet: Production and characterization of U2OS cells expressing Wnt10b. (A) U2OS cells were transiently transfected with either TOP-Flash or FOP-Flash luciferase constructs, treated with control media or 100 ng/mL purified Wnt3a and luciferase assays performed.

    Techniques Used: Expressing, Transfection, Luciferase, Construct, Purification

    Statistical categorization of differentially regulated genes in the U2OS-Wnt10b cell line. All 3387 differentially expressed genes regulated p
    Figure Legend Snippet: Statistical categorization of differentially regulated genes in the U2OS-Wnt10b cell line. All 3387 differentially expressed genes regulated p

    Techniques Used:

    Pathway analysis of differentially regulated genes in the U2OS-Wnt10b cell line. The 1003 genes identified as differentially regulated from the 2 SD and 3 SD statistical categories were subjected to pathway analysis. The 28 regulated pathways at the p
    Figure Legend Snippet: Pathway analysis of differentially regulated genes in the U2OS-Wnt10b cell line. The 1003 genes identified as differentially regulated from the 2 SD and 3 SD statistical categories were subjected to pathway analysis. The 28 regulated pathways at the p

    Techniques Used:

    Lack of activation of the Notch and NFκB pathways in U2OS cells by Wnt3a. U2OS cells were treated with Wnt3a (100 ng/mL) for 48 hr and QPCR was performed using primers specific for the indicated genes for A) Wnt targets, B) Notch targets and C)
    Figure Legend Snippet: Lack of activation of the Notch and NFκB pathways in U2OS cells by Wnt3a. U2OS cells were treated with Wnt3a (100 ng/mL) for 48 hr and QPCR was performed using primers specific for the indicated genes for A) Wnt targets, B) Notch targets and C)

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

    The NFκB pathway is activated in U2OS-Wnt10b cells. (A) U2OS and U2OS-Wnt10b cells were transduced with an NFκB-dependent luciferase reporter adenovirus. In a subset of U2OS cells, Wnt3a (100 ng/mL) was added (hatched bars). Forty-eight
    Figure Legend Snippet: The NFκB pathway is activated in U2OS-Wnt10b cells. (A) U2OS and U2OS-Wnt10b cells were transduced with an NFκB-dependent luciferase reporter adenovirus. In a subset of U2OS cells, Wnt3a (100 ng/mL) was added (hatched bars). Forty-eight

    Techniques Used: Transduction, Luciferase

    35) Product Images from "Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage"

    Article Title: Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158966

    Heterogeneous expression of BrABI5a and BrABI5b reverse the insensitivity of Arabidopsis abi5-1 to ABA during seed germination. (A, B) Sensitivity of seed germination to ABA. The seeds of Ws-2, abi5-1 , and transgenic abi5-1 lines carrying Myc-tagged BrABI5a or BrABI5b ( abi5-1 :: Myc-BrABI5a or abi5-1 :: Myc-BrABI5b ) were germinated on MS medium (A) and MS medium supplemented with 3μM ABA (B) for the indicated days. The emergence rate of green cotyledons (C) and radicle (D) from Ws-2, abi5-1 and abi5-1 :: Myc-BrABI5a or abi5-1 :: Myc-BrABI5b transgenic seeds plated on MS supplemented with ABA. Approximately 150 seeds were used in each experiment. Error bars represent SD (seed number > 100). (E) Immunoblots of Myc-BrABI5a or Myc-BrABI5b protein levels in the transgenic abi5-1 lines ( abi5-1 :: Myc-BrABI5a or abi5-1 :: Myc-BrABI5b ). N, transgenic abi5-1 lines carrying the empty Myc-tagged vector; CBB (Coomassie Brilliant Blue) R250-stained RLS (Rubisco large subunit) served as a loading control.
    Figure Legend Snippet: Heterogeneous expression of BrABI5a and BrABI5b reverse the insensitivity of Arabidopsis abi5-1 to ABA during seed germination. (A, B) Sensitivity of seed germination to ABA. The seeds of Ws-2, abi5-1 , and transgenic abi5-1 lines carrying Myc-tagged BrABI5a or BrABI5b ( abi5-1 :: Myc-BrABI5a or abi5-1 :: Myc-BrABI5b ) were germinated on MS medium (A) and MS medium supplemented with 3μM ABA (B) for the indicated days. The emergence rate of green cotyledons (C) and radicle (D) from Ws-2, abi5-1 and abi5-1 :: Myc-BrABI5a or abi5-1 :: Myc-BrABI5b transgenic seeds plated on MS supplemented with ABA. Approximately 150 seeds were used in each experiment. Error bars represent SD (seed number > 100). (E) Immunoblots of Myc-BrABI5a or Myc-BrABI5b protein levels in the transgenic abi5-1 lines ( abi5-1 :: Myc-BrABI5a or abi5-1 :: Myc-BrABI5b ). N, transgenic abi5-1 lines carrying the empty Myc-tagged vector; CBB (Coomassie Brilliant Blue) R250-stained RLS (Rubisco large subunit) served as a loading control.

    Techniques Used: Expressing, Transgenic Assay, Mass Spectrometry, Western Blot, Plasmid Preparation, Staining

    36) Product Images from "Arl2- and Msps-dependent microtubule growth governs asymmetric division"

    Article Title: Arl2- and Msps-dependent microtubule growth governs asymmetric division

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201503047

    Arl2 functions together with TBCD to regulate microtubule growth and D-TACC/Msps localization. (A) Silver staining of Elution fractions E1 and E2 after TAP. E2 sample contains most of the proteins associated with Arl2-CTAP. Asterisk indicates the bait protein. (B) Coimmunoprecipitation (IP) of Arl2 WT -Venus and FLAG-TBCD, HA-TBCC or HA-TBCE. (C) Coimmunoprecipitation of different forms of Arl2-Venus and HA-TBCC or HA-TBCE. (D) Coimmunoprecipitation of different forms of Arl2-Venus and Myc–β-tubulin or Myc–α-tubulin. (E) NBs of control and TBCD overexpression labeled with α-tubulin and DNA. (F) NBs of control and TBCD overexpression labeled for D-TACC, γ-tubulin, and DNA. Arrows indicate the centrosomes. (G) Metaphase NBs of control, Arl2 T30N , TBCD, and Arl2 T30N , TBCD coexpression labeled with α-tubulin and DNA. (H) Metaphase NBs of control, TBCD overexpression, and TBCD overexpression in heterozygous arl2 Δ156 background ( arl2 Δ156 /+ ) labeled for α-tubulin, PH3 and DNA. (E–H) The white dotted circles label the cell outlines. (I) Quantification of spindle length (with SD) in H. Control: 9.48 ± 0.91 µm ( n = 16); TBCD: 6.61 ± 1.23 µm ( n = 15); TBCD, arl2 Δ156 /+ : 4.41 ± 0.82 µm ( n = 15). ***, P
    Figure Legend Snippet: Arl2 functions together with TBCD to regulate microtubule growth and D-TACC/Msps localization. (A) Silver staining of Elution fractions E1 and E2 after TAP. E2 sample contains most of the proteins associated with Arl2-CTAP. Asterisk indicates the bait protein. (B) Coimmunoprecipitation (IP) of Arl2 WT -Venus and FLAG-TBCD, HA-TBCC or HA-TBCE. (C) Coimmunoprecipitation of different forms of Arl2-Venus and HA-TBCC or HA-TBCE. (D) Coimmunoprecipitation of different forms of Arl2-Venus and Myc–β-tubulin or Myc–α-tubulin. (E) NBs of control and TBCD overexpression labeled with α-tubulin and DNA. (F) NBs of control and TBCD overexpression labeled for D-TACC, γ-tubulin, and DNA. Arrows indicate the centrosomes. (G) Metaphase NBs of control, Arl2 T30N , TBCD, and Arl2 T30N , TBCD coexpression labeled with α-tubulin and DNA. (H) Metaphase NBs of control, TBCD overexpression, and TBCD overexpression in heterozygous arl2 Δ156 background ( arl2 Δ156 /+ ) labeled for α-tubulin, PH3 and DNA. (E–H) The white dotted circles label the cell outlines. (I) Quantification of spindle length (with SD) in H. Control: 9.48 ± 0.91 µm ( n = 16); TBCD: 6.61 ± 1.23 µm ( n = 15); TBCD, arl2 Δ156 /+ : 4.41 ± 0.82 µm ( n = 15). ***, P

    Techniques Used: Silver Staining, Over Expression, Labeling

    37) Product Images from "Movements of HIV-1 Genomic RNA-APOBEC3F Complexes and PKR Reveal Cytoplasmic and Nuclear PKR Defenses and HIV-1 Evasion Strategies"

    Article Title: Movements of HIV-1 Genomic RNA-APOBEC3F Complexes and PKR Reveal Cytoplasmic and Nuclear PKR Defenses and HIV-1 Evasion Strategies

    Journal: Virus research

    doi: 10.1016/j.virusres.2015.11.023

    Cytoplasmic gRNA in cultures transfected with pHIV gpt coalesces into DDX6-positive stress granules
    Figure Legend Snippet: Cytoplasmic gRNA in cultures transfected with pHIV gpt coalesces into DDX6-positive stress granules

    Techniques Used: Transfection

    38) Product Images from "Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes"

    Article Title: Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057018

    smFRET results on nucleosome stability are consistent to μpsFRET data. ( A ) Average proximity ratio calculated from all photons from double-labeled nucleosomes as a function of salt concentration. Photons from the donor and transfer channel were summed for all detected molecules, except donor-only and acceptor-only species. ( B ) Salt dependence of the fraction of intact nucleosomes in smFRET histograms from Fig. 5. For each histogram, the donor-only and acceptor-only population was excluded from the analysis. The relative fraction of FRET-active molecules (0.25
    Figure Legend Snippet: smFRET results on nucleosome stability are consistent to μpsFRET data. ( A ) Average proximity ratio calculated from all photons from double-labeled nucleosomes as a function of salt concentration. Photons from the donor and transfer channel were summed for all detected molecules, except donor-only and acceptor-only species. ( B ) Salt dependence of the fraction of intact nucleosomes in smFRET histograms from Fig. 5. For each histogram, the donor-only and acceptor-only population was excluded from the analysis. The relative fraction of FRET-active molecules (0.25

    Techniques Used: Labeling, Concentration Assay

    smFRET analysis reveals a conformational transition prior to nucleosome unwrapping. A,B ) smFRET histograms of non-acetylated and H3-acetylated nucleosomes at various salt concentrations and 300 pM total nucleosome concentration. Above 300 mM NaCl, a fraction of H3-acetylated nucleosomes populates a second conformation with slightly increased proximity ratio compared to non-acetylated nucleosomes, which appear to retain their initial structure. C, D ) Overlay of histograms for salt concentrations between 150 mM and 600 mM NaCl for non-acetylated (C) and H3-acetylated nucleosomes (D). Data were smoothed once to better visualize the gradual transition of nucleosomes into the high FRET state.
    Figure Legend Snippet: smFRET analysis reveals a conformational transition prior to nucleosome unwrapping. A,B ) smFRET histograms of non-acetylated and H3-acetylated nucleosomes at various salt concentrations and 300 pM total nucleosome concentration. Above 300 mM NaCl, a fraction of H3-acetylated nucleosomes populates a second conformation with slightly increased proximity ratio compared to non-acetylated nucleosomes, which appear to retain their initial structure. C, D ) Overlay of histograms for salt concentrations between 150 mM and 600 mM NaCl for non-acetylated (C) and H3-acetylated nucleosomes (D). Data were smoothed once to better visualize the gradual transition of nucleosomes into the high FRET state.

    Techniques Used: Concentration Assay

    Working range of conventional and quasi-bulk single particle FRET. A – C ) smFRET histograms and burst size to burst duration distributions for a binary DNA mixture (noFRET and FRET-active) at 60 pM (A), 150 pM (B), and 330 pM (C) sample concentrations. While at 60 pM both subpopulations are clearly separated, coincident detection of both species occurs at 150 pM and above. The presence of multi-particle events is evident from the burst size to burst duration distribution. While at 50 pM burst duration and burst size strongly correlate, additional populations appear outside the ellipsoidal point cloud at higher sample concentrations. D, E ) Principle of quasi-bulk smFRET of nucleosomes. Nucleosomes were reconstituted on 5S rDNA (D) or the high affinity Widom 601 sequence (E). Histograms are shown for 5 mM or 150 mM salt concentrations and in the presence or absence of 10 nM unlabeled nucleosomes. At 5 mM NaCl (left panels) most nucleosomes were intact as expected from Figure 1A . At 150 mM NaCl (right panels) and in the absence of unlabeled nucleosomes, diluted nucleosomes dissociated, whereas under quasi-bulk conditions, nucleosomes on both 5S and 601 DNA remained intact.
    Figure Legend Snippet: Working range of conventional and quasi-bulk single particle FRET. A – C ) smFRET histograms and burst size to burst duration distributions for a binary DNA mixture (noFRET and FRET-active) at 60 pM (A), 150 pM (B), and 330 pM (C) sample concentrations. While at 60 pM both subpopulations are clearly separated, coincident detection of both species occurs at 150 pM and above. The presence of multi-particle events is evident from the burst size to burst duration distribution. While at 50 pM burst duration and burst size strongly correlate, additional populations appear outside the ellipsoidal point cloud at higher sample concentrations. D, E ) Principle of quasi-bulk smFRET of nucleosomes. Nucleosomes were reconstituted on 5S rDNA (D) or the high affinity Widom 601 sequence (E). Histograms are shown for 5 mM or 150 mM salt concentrations and in the presence or absence of 10 nM unlabeled nucleosomes. At 5 mM NaCl (left panels) most nucleosomes were intact as expected from Figure 1A . At 150 mM NaCl (right panels) and in the absence of unlabeled nucleosomes, diluted nucleosomes dissociated, whereas under quasi-bulk conditions, nucleosomes on both 5S and 601 DNA remained intact.

    Techniques Used: Sequencing

    39) Product Images from "Temporal Regulation of Distinct Internal Ribosome Entry Sites of the Dicistroviridae Cricket Paralysis Virus"

    Article Title: Temporal Regulation of Distinct Internal Ribosome Entry Sites of the Dicistroviridae Cricket Paralysis Virus

    Journal: Viruses

    doi: 10.3390/v8010025

    Viral non-structural and structural protein expression in CrPV-infected S2 cells. ( A ) Schematic of the genome arrangement of CrPV; ( B ) Autoradiography of pulse-labelled protein lysates from S2 cells either mock- or CrPV-infected (MOI 10) resolved on a 12% SDS-PAGE gel. The protein lysates were collected from S2 cells at the indicated times hours post-infection (h.p.i.) and metabolically labelled with [ 35 S]-Met/Cys for twenty minutes at the end of each time point (top). The identities of specific CrPV viral proteins, as shown on the right of the gel, were previously described [ 7 , 8 ] and determined by immunoblotting with α-RdRp and α-VP2 peptide antibodies or predicted by molecular weight (below); ( C ) Raw densitometric quantitation of [ 35 S]-Met/Cys pulse-labelled RdRp*, RdRp**, RdRp***, 2C, VP0, and VP1, 2, and 3 during CrPV infection from three independent experiments (±s.d.). RdRp*, RdRp**, and RdRp*** denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa respectively.
    Figure Legend Snippet: Viral non-structural and structural protein expression in CrPV-infected S2 cells. ( A ) Schematic of the genome arrangement of CrPV; ( B ) Autoradiography of pulse-labelled protein lysates from S2 cells either mock- or CrPV-infected (MOI 10) resolved on a 12% SDS-PAGE gel. The protein lysates were collected from S2 cells at the indicated times hours post-infection (h.p.i.) and metabolically labelled with [ 35 S]-Met/Cys for twenty minutes at the end of each time point (top). The identities of specific CrPV viral proteins, as shown on the right of the gel, were previously described [ 7 , 8 ] and determined by immunoblotting with α-RdRp and α-VP2 peptide antibodies or predicted by molecular weight (below); ( C ) Raw densitometric quantitation of [ 35 S]-Met/Cys pulse-labelled RdRp*, RdRp**, RdRp***, 2C, VP0, and VP1, 2, and 3 during CrPV infection from three independent experiments (±s.d.). RdRp*, RdRp**, and RdRp*** denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa respectively.

    Techniques Used: Expressing, Infection, Autoradiography, SDS Page, Metabolic Labelling, Molecular Weight, Quantitation Assay

    Role of dPERK and dGCN2 eIF2α kinases in CrPV-infected S2 cells. ( A ) Immunoblots (anti-phospho-eIF2α (P-eIF2α) and anti-Tubulin antibodies) of lysates from cells treated with control (GFP) dsRNAs or singly dPERK or dGCN2 dsRNAs or both dPERK/dGCN2 dsRNAs for 48 h followed by arsenite (500 μM) treatment or infection with CrPV (MOI 10); ( B ) [ 35 S]-Met/Cys pulse-labelled protein lysates from control or dPERK and dGCN2 dsRNA-treated S2 cells that were either mock or CrPV-infected (MOI 10) for the indicted times (h.p.i.) were resolved on a 12% SDS-PAGE gel. Shown are representative autoradiographs from at least three independent experiments. RdRp*, RdRp**, and RdRp*** denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa, respectively.
    Figure Legend Snippet: Role of dPERK and dGCN2 eIF2α kinases in CrPV-infected S2 cells. ( A ) Immunoblots (anti-phospho-eIF2α (P-eIF2α) and anti-Tubulin antibodies) of lysates from cells treated with control (GFP) dsRNAs or singly dPERK or dGCN2 dsRNAs or both dPERK/dGCN2 dsRNAs for 48 h followed by arsenite (500 μM) treatment or infection with CrPV (MOI 10); ( B ) [ 35 S]-Met/Cys pulse-labelled protein lysates from control or dPERK and dGCN2 dsRNA-treated S2 cells that were either mock or CrPV-infected (MOI 10) for the indicted times (h.p.i.) were resolved on a 12% SDS-PAGE gel. Shown are representative autoradiographs from at least three independent experiments. RdRp*, RdRp**, and RdRp*** denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa, respectively.

    Techniques Used: Infection, Western Blot, SDS Page

    Effects of pateamine A (PatA) and DTT on CrPV protein synthesis. Pulse-labelled protein lysates from mock- or CrPV-infected (MOI 10) S2 cells treated with either ( A ) DMSO; ( B ) 50 nM pateamine A; ( E ) 50 nM DTT; ( D ) or left untreated were resolved on a 12% SDS-PAGE gel. The cells were metabolically labelled with [ 35 S]-Met/Cys for twenty minutes at the end of each time point. Shown are representative autoradiographs from at least three independent experiments; ( C ) Viral titers from infected lysates treated with DMSO or PatA after 10 h.p.i. RdRp*, RdRp**, and RdRp*** denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa respectively.
    Figure Legend Snippet: Effects of pateamine A (PatA) and DTT on CrPV protein synthesis. Pulse-labelled protein lysates from mock- or CrPV-infected (MOI 10) S2 cells treated with either ( A ) DMSO; ( B ) 50 nM pateamine A; ( E ) 50 nM DTT; ( D ) or left untreated were resolved on a 12% SDS-PAGE gel. The cells were metabolically labelled with [ 35 S]-Met/Cys for twenty minutes at the end of each time point. Shown are representative autoradiographs from at least three independent experiments; ( C ) Viral titers from infected lysates treated with DMSO or PatA after 10 h.p.i. RdRp*, RdRp**, and RdRp*** denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa respectively.

    Techniques Used: Infection, SDS Page, Metabolic Labelling

    ( A ) Mock- and CrPV-infected S2 cells were pulse-labelled with [ 3 H]-uridine for ten minutes at the indicated hours post infection (h.p.i.) before harvesting. Where indicated, the cells were left untreated or treated with actinomycin D (Act D, 5 µg/mL) for fifteen minutes prior to pulse-labeling. RNA was extracted, loaded on a denaturing agarose gel, transferred to a nylon membrane, and analyzed by phosphorimager analysis. Representative autoradiogram and a corresponding methylene blue stain are shown in ( A , B ), respectively; ( C ) Quantitation of host and viral transcription rates in CrPV-infected cells. The rate of host transcription at each time point was calculated using the formula [(Total radioactivity in lane − Act D) − (Total radioactivity in lane + Act D)] / (Total radioactivity in lane − Act D). The rate of host transcription was normalized to that in mock-infected cells given as 100%. The rate of viral transcription was calculated at each h.p.i. by using the formula [(Total radioactivity in lane + Act D) / (Total radioactivity in lane – Act D)]. The rate of viral transcription was normalized to that in mock-infected cells given as 0%. Shown are representative gels and averages from at least 2 independent experiments; ( D ) Viral RNA levels were detected by Northern blot analysis; ( E ) Viral RNA levels were quantified using ImageQuant and plotted to show levels of viral RNA/GAPDH RNA at different time points post infection; ( F , G ) Autoradiography of pulse-labelled protein lysates resolved on a 12% SDS-PAGE gel from S2 cells mock- or CrPV-infected (MOI 10) and treated with DMSO ( F ) or 50 nM Act D ( G ) three hours prior to infection. RdRp*, RdRp**, and RdRp*** denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa, respectively. Averages are shown from at least three independent experiments (±s.d.).
    Figure Legend Snippet: ( A ) Mock- and CrPV-infected S2 cells were pulse-labelled with [ 3 H]-uridine for ten minutes at the indicated hours post infection (h.p.i.) before harvesting. Where indicated, the cells were left untreated or treated with actinomycin D (Act D, 5 µg/mL) for fifteen minutes prior to pulse-labeling. RNA was extracted, loaded on a denaturing agarose gel, transferred to a nylon membrane, and analyzed by phosphorimager analysis. Representative autoradiogram and a corresponding methylene blue stain are shown in ( A , B ), respectively; ( C ) Quantitation of host and viral transcription rates in CrPV-infected cells. The rate of host transcription at each time point was calculated using the formula [(Total radioactivity in lane − Act D) − (Total radioactivity in lane + Act D)] / (Total radioactivity in lane − Act D). The rate of host transcription was normalized to that in mock-infected cells given as 100%. The rate of viral transcription was calculated at each h.p.i. by using the formula [(Total radioactivity in lane + Act D) / (Total radioactivity in lane – Act D)]. The rate of viral transcription was normalized to that in mock-infected cells given as 0%. Shown are representative gels and averages from at least 2 independent experiments; ( D ) Viral RNA levels were detected by Northern blot analysis; ( E ) Viral RNA levels were quantified using ImageQuant and plotted to show levels of viral RNA/GAPDH RNA at different time points post infection; ( F , G ) Autoradiography of pulse-labelled protein lysates resolved on a 12% SDS-PAGE gel from S2 cells mock- or CrPV-infected (MOI 10) and treated with DMSO ( F ) or 50 nM Act D ( G ) three hours prior to infection. RdRp*, RdRp**, and RdRp*** denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa, respectively. Averages are shown from at least three independent experiments (±s.d.).

    Techniques Used: Infection, Activated Clotting Time Assay, Labeling, Agarose Gel Electrophoresis, Staining, Quantitation Assay, Radioactivity, Northern Blot, Autoradiography, SDS Page

    40) Product Images from "DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury"

    Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23978

    GAS6 triggered Schwann cell proliferation primarily through CIP2A and DAPK (A) RSC96 cells were transfected with either control or DAPK siRNA for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. Immunoblotting evaluations of pAxl, Axl, CIP2A, DAPK, pERK1/2, ERK1/2, pAKT, AKT, Myc and Survivin. (B) RSC96 cells were transfected with either control or CIP2A siRNA for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. Immunoblotting evaluations of pAxl, Axl, DAPK, pDAPK, pERK1/2, ERK1/2, pAKT, AKT, Myc and Survivin. (C) DAPK or CIP2A was knocked down for 48 h then RSC96 cells were incubated with GAS6 (100 ng/ml) at the indicated hours. Cell viability was analysed via the WST-1 assay. Data are the mean ± SD, and n = 3 for each time point. * p
    Figure Legend Snippet: GAS6 triggered Schwann cell proliferation primarily through CIP2A and DAPK (A) RSC96 cells were transfected with either control or DAPK siRNA for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. Immunoblotting evaluations of pAxl, Axl, CIP2A, DAPK, pERK1/2, ERK1/2, pAKT, AKT, Myc and Survivin. (B) RSC96 cells were transfected with either control or CIP2A siRNA for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. Immunoblotting evaluations of pAxl, Axl, DAPK, pDAPK, pERK1/2, ERK1/2, pAKT, AKT, Myc and Survivin. (C) DAPK or CIP2A was knocked down for 48 h then RSC96 cells were incubated with GAS6 (100 ng/ml) at the indicated hours. Cell viability was analysed via the WST-1 assay. Data are the mean ± SD, and n = 3 for each time point. * p

    Techniques Used: Transfection, Incubation, WST-1 Assay

    Schematic diagram of GAS6-induced Schwann cell proliferation GAS6 binds to its receptor, AXL, and p-AXL induce Schwann cell proliferation via MYC and Survivin signalling. GAS6 increases the expression level of CIP2A to form a p-AXL, p-DAPK and CIP2A protein complex. The presence of CIP2A inhibits the enzyme activity of PP2A and activates downstream ERK1/2 and AKT signals for proliferation.
    Figure Legend Snippet: Schematic diagram of GAS6-induced Schwann cell proliferation GAS6 binds to its receptor, AXL, and p-AXL induce Schwann cell proliferation via MYC and Survivin signalling. GAS6 increases the expression level of CIP2A to form a p-AXL, p-DAPK and CIP2A protein complex. The presence of CIP2A inhibits the enzyme activity of PP2A and activates downstream ERK1/2 and AKT signals for proliferation.

    Techniques Used: Expressing, Activity Assay

    GAS6 triggers p-Axl, p-DAPK and CIP2A to form a protein complex in RSC96 cells (A-B) Co-immunoprecipitation between p-Axl, p-DAPK and CIP2A with GAS6 stimulation for 30 min in RSC96 cells. Total lysate indicates 1/10 input in each experiment. The relative quantification of protein expression was normalized with respect to β-actin expression. (C) Immunofluorescence image demonstrating co-localized CIP2A and p-Axl and co-localized p-Axl and p-DAPK (D) after GAS6 stimulation for 30 min in RSC96 cells.
    Figure Legend Snippet: GAS6 triggers p-Axl, p-DAPK and CIP2A to form a protein complex in RSC96 cells (A-B) Co-immunoprecipitation between p-Axl, p-DAPK and CIP2A with GAS6 stimulation for 30 min in RSC96 cells. Total lysate indicates 1/10 input in each experiment. The relative quantification of protein expression was normalized with respect to β-actin expression. (C) Immunofluorescence image demonstrating co-localized CIP2A and p-Axl and co-localized p-Axl and p-DAPK (D) after GAS6 stimulation for 30 min in RSC96 cells.

    Techniques Used: Immunoprecipitation, Expressing, Immunofluorescence

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    Sequencing:

    Article Title: Hepatitis B virus X stimulates redox signaling through activation of ataxia telangiectasia mutated kinase
    Article Snippet: .. After the verification of DNA sequence, HepG2 cells were transfected with mock plasmid pCAG-IRES-Puro or pCAG-FLAG-HBx using FuGENE6 (Roche Diagnostics, Indianapolis, IN, USA) with MA lipofection enhancer (IBA, St. Louis, MO, USA). .. Stable transfected cells were established by transfection with the linearized pCAG-IRES-Puro or pCAG-FLAG-HBx, and selected using 2 μg/ml puromycin (Cayla, Toulouse, France).

    Lysis:

    Article Title: Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)
    Article Snippet: .. To monitor the association between SERCA and Bcl-2, 150 μg of SR in lysis buffer containing 10 mM Tris/HCl (pH 7.4), 10 mM EDTA, 1% Nonidet P40, 1 mM PMSF and protease inhibitors (Roche Diagnostics) was incubated with 3 μg of GST–Bcl-2 or GST alone for 2 h at 4 °C. .. After the incubation with 10 μl (packed volume) of glutathione–Sepharose beads for 2 h, the beads were separated by centrifugation at 7500 g for 10 min and washed six times with the lysis buffer.

    Article Title: SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells
    Article Snippet: .. Immunoblotting For Immunoblotting, cells were lysed in high salt lysis buffer, IPLB (50 mM Tris–HCl, 500 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM β-glycerol phosphate, 0.1 mM NaOrthovanadate, 0.2% Triton X-100, 0.3% NP-40, 10 U/ml of Benzonase nuclease, plus protease inhibitor cocktail (Roche, Basel, Switzerland) at 4°C. .. Total proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes.

    Article Title: The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain
    Article Snippet: .. Twenty-four hours post transfection (hpt), the cells were washed with ice-cold PBS, incubated for 20 min with ice-cold lysis buffer (20 mM Tris-HCl pH8, 150 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100, 2 mM EDTA, with complete Protease-Inhibitor Cocktail (Roche)). .. The lysates were pre-cleared by centrifugation (4°C, 12 000 rpm, 10 min) and incubation with Protein A Sepharose beads (GE Healthcare) for 10 min at 4°C.

    Article Title: The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7
    Article Snippet: .. IP For coimmunoprecipitations of tagged Treslin/TICRR and MTBP from 293T cells, a 10-cm plate of transiently transfected 293T cells were lysed in 5× pellet volume of lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, complete EDTA-free protease inhibitor cocktail [Roche, 05056489001], 0.1% Triton X-100, 2 mM 2-mercaptoethanol). .. For flag affinity purifications 50% of cell lysates was incubated with 1 μg anti-FLAG mouse monoclonal antibody or 1 μg mouse IgG coupled to 150 μg Protein G Dynabeads (Invitrogen, 100-04D).

    SDS Page:

    Article Title: Arabidopsis ROOT PHOTOTROPISM2 is a Light-Dependent Dynamic Modulator of Phototropin1
    Article Snippet: .. Immunoblotting Analysis For immunoblotting of Phos-tag SDS-PAGE gels, total proteins were extracted from etiolated seedlings in a buffer containing 50 mM Tris-MES, pH 7.5, 300 mM sucrose, 150 mM NaCl, 10 mM potassium acetate, 0.2% Triton X-100, and a protease inhibitor mixture (Complete Mini EDTA-free; Roche Diagnostics). .. The extracts were then centrifuged at 10,000 g at 4°C for 10 min to remove cell debris and the supernatants were collected and mixed with a half volume of 3× SDS gel loading buffer, and boiled at 95°C for 15 min.

    Plasmid Preparation:

    Article Title: Hepatitis B virus X stimulates redox signaling through activation of ataxia telangiectasia mutated kinase
    Article Snippet: .. After the verification of DNA sequence, HepG2 cells were transfected with mock plasmid pCAG-IRES-Puro or pCAG-FLAG-HBx using FuGENE6 (Roche Diagnostics, Indianapolis, IN, USA) with MA lipofection enhancer (IBA, St. Louis, MO, USA). .. Stable transfected cells were established by transfection with the linearized pCAG-IRES-Puro or pCAG-FLAG-HBx, and selected using 2 μg/ml puromycin (Cayla, Toulouse, France).

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  • 97
    Roche ripa buffer
    Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in <t>293T</t> cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with <t>RIPA</t> buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.
    Ripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 5816 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche nonidet p40
    Substitution of Lys-110 with an alanine residue results in the accumulation of the mutant PCNA in the heterochromatin region ( A ) The cell fractionation protocol. CHO cells transfected with WT or mutant constructs were fractionated at 12 h post-transfection. MNase, micrococcal nuclease; NP-40, <t>Nonidet</t> <t>P40;</t> Sup, supernatant. ( B ) Fractionated samples were analysed by SDS/PAGE and Western blotting with anti-GFP, -PCNA, -(histone H1) or -HP1α antibodies as indicated. The striking differences in chromatin localization of PCNA(K110A) and endogenous PCNA are shown with * and arrowheads. ( C ) DNA purified from each fraction was separated by agarose gel electrophoresis (2% gel). Di, di-nucleosome; M, DNA size makers; Mono, mononucleosome.
    Nonidet P40, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche nt rna immunoprecipitation buffer
    Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by <t>RNA</t> <t>immunoprecipitation</t> assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008
    Nt Rna Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.

    Journal: Oncotarget

    Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    doi: 10.18632/oncotarget.8072

    Figure Lengend Snippet: Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.

    Article Snippet: After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany).

    Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Staining, Plasmid Preparation, Mutagenesis, Transfection

    Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.

    Journal: Oncotarget

    Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    doi: 10.18632/oncotarget.8072

    Figure Lengend Snippet: Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.

    Article Snippet: After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany).

    Techniques: Incubation, Immunoprecipitation, Transfection, Western Blot

    Substitution of Lys-110 with an alanine residue results in the accumulation of the mutant PCNA in the heterochromatin region ( A ) The cell fractionation protocol. CHO cells transfected with WT or mutant constructs were fractionated at 12 h post-transfection. MNase, micrococcal nuclease; NP-40, Nonidet P40; Sup, supernatant. ( B ) Fractionated samples were analysed by SDS/PAGE and Western blotting with anti-GFP, -PCNA, -(histone H1) or -HP1α antibodies as indicated. The striking differences in chromatin localization of PCNA(K110A) and endogenous PCNA are shown with * and arrowheads. ( C ) DNA purified from each fraction was separated by agarose gel electrophoresis (2% gel). Di, di-nucleosome; M, DNA size makers; Mono, mononucleosome.

    Journal: Biology of the cell

    Article Title: Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis

    doi: 10.1042/BC20070158

    Figure Lengend Snippet: Substitution of Lys-110 with an alanine residue results in the accumulation of the mutant PCNA in the heterochromatin region ( A ) The cell fractionation protocol. CHO cells transfected with WT or mutant constructs were fractionated at 12 h post-transfection. MNase, micrococcal nuclease; NP-40, Nonidet P40; Sup, supernatant. ( B ) Fractionated samples were analysed by SDS/PAGE and Western blotting with anti-GFP, -PCNA, -(histone H1) or -HP1α antibodies as indicated. The striking differences in chromatin localization of PCNA(K110A) and endogenous PCNA are shown with * and arrowheads. ( C ) DNA purified from each fraction was separated by agarose gel electrophoresis (2% gel). Di, di-nucleosome; M, DNA size makers; Mono, mononucleosome.

    Article Snippet: Briefly, nuclei were lysed in lysis buffer [15 mM Tris/HCl (pH 7.4), 60 mM KCl, 15 mM MgCl2 , 15 mM NaCl, 1mM CaCl2 , 1 mM PMSF, 0.6% Nonidet P40 and 1×protease inhibitor cocktail (Roche)].

    Techniques: Mutagenesis, Cell Fractionation, Transfection, Construct, SDS Page, Western Blot, Purification, Agarose Gel Electrophoresis

    Detection of intracellular CD83 in monocytes, macrophages and imDCs ( A ) Monocytes (Mo), macrophages (MΦ) and imDCs were fixed with paraformaldehyde and permeabilized with saponin. Upon blocking with 20% (v/v) goat serum, the cells were stained with a PE-labelled CD83 antibody (clone HB15e; filled histograms) or, as a control, isotype IgG (open histograms). The monocytic THP-1 cells were used as controls. ( B ) Monocytes (lane 3), resting macrophages (lane 4), LPS-activated macrophages (lane 5), imDCs (lane 6) and mDCs (lane 7) were lysed with Nonidet P40, and the soluble fractions were analysed by Western blotting using the HB15a CD83 antibody (Santa Cruz Biotechnology). Aliquots (50 μg) of each sample were separated by SDS/PAGE. Also included in the blots was the soluble fraction of THP-1 cells (lane 8), and 293T cells transfected with the pcDNA3.1 plasmid (lane 1) or the phCD83 expression vector (lane 2). The transfected cells were cultured for 24 h. For monocytes, both the soluble (lane 9) and insoluble (lane 10) fractions were subjected to Western blot analysis. ( C ) Expression of CD83 by 293T cells: 293T cells were transfected with the phCD83 expression vector (lower panel) or, as a control, with the pcDNA3.1 plasmid (upper panel). The cells were stained with a PE-labelled CD83 antibody (HB15e; open histograms). The filled histograms represent signals detected with isotype IgG. All results shown are representative of at least three similar experiments.

    Journal: Biochemical Journal

    Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells

    doi: 10.1042/BJ20040741

    Figure Lengend Snippet: Detection of intracellular CD83 in monocytes, macrophages and imDCs ( A ) Monocytes (Mo), macrophages (MΦ) and imDCs were fixed with paraformaldehyde and permeabilized with saponin. Upon blocking with 20% (v/v) goat serum, the cells were stained with a PE-labelled CD83 antibody (clone HB15e; filled histograms) or, as a control, isotype IgG (open histograms). The monocytic THP-1 cells were used as controls. ( B ) Monocytes (lane 3), resting macrophages (lane 4), LPS-activated macrophages (lane 5), imDCs (lane 6) and mDCs (lane 7) were lysed with Nonidet P40, and the soluble fractions were analysed by Western blotting using the HB15a CD83 antibody (Santa Cruz Biotechnology). Aliquots (50 μg) of each sample were separated by SDS/PAGE. Also included in the blots was the soluble fraction of THP-1 cells (lane 8), and 293T cells transfected with the pcDNA3.1 plasmid (lane 1) or the phCD83 expression vector (lane 2). The transfected cells were cultured for 24 h. For monocytes, both the soluble (lane 9) and insoluble (lane 10) fractions were subjected to Western blot analysis. ( C ) Expression of CD83 by 293T cells: 293T cells were transfected with the phCD83 expression vector (lower panel) or, as a control, with the pcDNA3.1 plasmid (upper panel). The cells were stained with a PE-labelled CD83 antibody (HB15e; open histograms). The filled histograms represent signals detected with isotype IgG. All results shown are representative of at least three similar experiments.

    Article Snippet: Cells were washed in PBS and then lysed on ice in a lysis buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P40 and the Com plete™ protease-inhibitor cocktail (Roche).

    Techniques: Blocking Assay, Staining, Western Blot, SDS Page, Transfection, Plasmid Preparation, Expressing, Cell Culture

    Surface induction of CD83 on DCs requires Asn-linked glycosylation ( A ) Transfected 293T cells were lysed, and the soluble fraction was either untreated (lane 1) or treated with PNGase F (lane 2). The samples were then separated on SDS/PAGE gels and subjected to Western blotting using the HB15a CD83 antibody. The transfected 293T cells were also cultured in the presence of tunicamycin (lane 3) or DMSO (lane 4) for 24 h, and then lysed with Nonidet P40. The soluble fraction was subjected to SDS/PAGE, followed by Western blotting using the CD83 antibody. ImDCs were activated with LPS in the presence of tunicamycin (lane 5) or DMSO (lane 6) for 6 h, and were similarly analysed by Western blotting using the CD83 antibody. The molecular-mass standards are shown, and apply to all three blots. ( B ) Transfected 293T cells were cultured for 24 h in the presence of tunicamycin (solid line) or DMSO (dotted line), and then stained with the CD83 antibody and analysed by flow cytometry. The filled histogram represents signals on transfected 293T cells cultured in the absence of tunicamycin and DMSO. ( C ) ImDCs were activated with LPS in the presence of tunicamycin (solid line) or DMSO (dotted line), or were unactivated (filled histogram) and then stained with the CD83 antibody and analysed by flow cytometry. The results are representative of two or three independent experiments.

    Journal: Biochemical Journal

    Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells

    doi: 10.1042/BJ20040741

    Figure Lengend Snippet: Surface induction of CD83 on DCs requires Asn-linked glycosylation ( A ) Transfected 293T cells were lysed, and the soluble fraction was either untreated (lane 1) or treated with PNGase F (lane 2). The samples were then separated on SDS/PAGE gels and subjected to Western blotting using the HB15a CD83 antibody. The transfected 293T cells were also cultured in the presence of tunicamycin (lane 3) or DMSO (lane 4) for 24 h, and then lysed with Nonidet P40. The soluble fraction was subjected to SDS/PAGE, followed by Western blotting using the CD83 antibody. ImDCs were activated with LPS in the presence of tunicamycin (lane 5) or DMSO (lane 6) for 6 h, and were similarly analysed by Western blotting using the CD83 antibody. The molecular-mass standards are shown, and apply to all three blots. ( B ) Transfected 293T cells were cultured for 24 h in the presence of tunicamycin (solid line) or DMSO (dotted line), and then stained with the CD83 antibody and analysed by flow cytometry. The filled histogram represents signals on transfected 293T cells cultured in the absence of tunicamycin and DMSO. ( C ) ImDCs were activated with LPS in the presence of tunicamycin (solid line) or DMSO (dotted line), or were unactivated (filled histogram) and then stained with the CD83 antibody and analysed by flow cytometry. The results are representative of two or three independent experiments.

    Article Snippet: Cells were washed in PBS and then lysed on ice in a lysis buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P40 and the Com plete™ protease-inhibitor cocktail (Roche).

    Techniques: Transfection, SDS Page, Western Blot, Cell Culture, Staining, Flow Cytometry, Cytometry

    Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008

    Journal: eLife

    Article Title: RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response

    doi: 10.7554/eLife.09656

    Figure Lengend Snippet: Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008

    Article Snippet: After homogenization in 100 μl of NT- RNA immunoprecipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.5% Nonidet P40, 1 mM EDTA pH 8.0, 1 mM DTT), Complete protease inhibitor (Roche), 200 U/ml RNase Out (Invitrogen, USA) samples were incubated for 10 min on ice and pre-cleared with 25 μl of Protein G-coated Dynabeads (Life Technologies).

    Techniques: Binding Assay, Immunoprecipitation, Incubation, Quantitative RT-PCR