hela tet off cells  (TaKaRa)


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    TaKaRa hela tet off cells
    NMD is inhibited during doxorubicin treatment. ( a ) mRNA decay assays in MCF7 cells. MCF7 cells either were (red) or were not (black) pre-treated with 5 μM doxorubicin for 1 h before addition of 3 μg/ml actinomycin D to halt transcription. Cells were collected at the indicated times after actinomycin D addition. Levels of the indicated NMD-targeted mRNAs were assessed by RT-qPCR, normalized to 18s rRNA levels, and displayed as a percentage of the levels at t=0 h. Error bars=S.E.M., n=4 independent biological quadruplicates. ( b ) Human β-Gl mRNA half-life studies in <t>HeLa</t> <t>Tet-off</t> cells. HeLa Tet-off cells were transfected with plasmids encoding human β-Gl Norm mRNA and MUP mRNA or β-Gl Ter mRNA and MUP mRNA. β-Gl Norm and β-Gl 39 Ter mRNA transcription occurs under the agency of the non-stress-responsive Tet-off promoter. Cells were either pre-treated with nothing (top), 50 μM doxorubicin for 1 h (middle), or 50 μg/ml puromycin for 3 h (bottom) before transcriptional shut-off with 2 μg/ml doxycycline. Cell aliquots were removed at the indicated “chase” time points, and RT-qPCR was used to assess the remaining levels of β-Gl Norm and β-Gl Ter mRNAs, each after normalization to MUP mRNA. ( c ) Western blots of lysates of MCF7 cells from a (blots derive from and are representative of the three biological replicates in a ) that had been exposed to doxorubicin (5 μM) for the indicated times. CP, cleavage product. GAPDH levels serve as loading controls. Three-fold serial dilutions (wedge) reveal the dynamic range of analysis. ( d ) As in c , but cells were exposed to a 10-fold higher concentration of doxorubicin and analyzed at earlier time points. Representative of 2 biological replicates.
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    Images

    1) Product Images from "Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics"

    Article Title: Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics

    Journal: Nature communications

    doi: 10.1038/ncomms7632

    NMD is inhibited during doxorubicin treatment. ( a ) mRNA decay assays in MCF7 cells. MCF7 cells either were (red) or were not (black) pre-treated with 5 μM doxorubicin for 1 h before addition of 3 μg/ml actinomycin D to halt transcription. Cells were collected at the indicated times after actinomycin D addition. Levels of the indicated NMD-targeted mRNAs were assessed by RT-qPCR, normalized to 18s rRNA levels, and displayed as a percentage of the levels at t=0 h. Error bars=S.E.M., n=4 independent biological quadruplicates. ( b ) Human β-Gl mRNA half-life studies in HeLa Tet-off cells. HeLa Tet-off cells were transfected with plasmids encoding human β-Gl Norm mRNA and MUP mRNA or β-Gl Ter mRNA and MUP mRNA. β-Gl Norm and β-Gl 39 Ter mRNA transcription occurs under the agency of the non-stress-responsive Tet-off promoter. Cells were either pre-treated with nothing (top), 50 μM doxorubicin for 1 h (middle), or 50 μg/ml puromycin for 3 h (bottom) before transcriptional shut-off with 2 μg/ml doxycycline. Cell aliquots were removed at the indicated “chase” time points, and RT-qPCR was used to assess the remaining levels of β-Gl Norm and β-Gl Ter mRNAs, each after normalization to MUP mRNA. ( c ) Western blots of lysates of MCF7 cells from a (blots derive from and are representative of the three biological replicates in a ) that had been exposed to doxorubicin (5 μM) for the indicated times. CP, cleavage product. GAPDH levels serve as loading controls. Three-fold serial dilutions (wedge) reveal the dynamic range of analysis. ( d ) As in c , but cells were exposed to a 10-fold higher concentration of doxorubicin and analyzed at earlier time points. Representative of 2 biological replicates.
    Figure Legend Snippet: NMD is inhibited during doxorubicin treatment. ( a ) mRNA decay assays in MCF7 cells. MCF7 cells either were (red) or were not (black) pre-treated with 5 μM doxorubicin for 1 h before addition of 3 μg/ml actinomycin D to halt transcription. Cells were collected at the indicated times after actinomycin D addition. Levels of the indicated NMD-targeted mRNAs were assessed by RT-qPCR, normalized to 18s rRNA levels, and displayed as a percentage of the levels at t=0 h. Error bars=S.E.M., n=4 independent biological quadruplicates. ( b ) Human β-Gl mRNA half-life studies in HeLa Tet-off cells. HeLa Tet-off cells were transfected with plasmids encoding human β-Gl Norm mRNA and MUP mRNA or β-Gl Ter mRNA and MUP mRNA. β-Gl Norm and β-Gl 39 Ter mRNA transcription occurs under the agency of the non-stress-responsive Tet-off promoter. Cells were either pre-treated with nothing (top), 50 μM doxorubicin for 1 h (middle), or 50 μg/ml puromycin for 3 h (bottom) before transcriptional shut-off with 2 μg/ml doxycycline. Cell aliquots were removed at the indicated “chase” time points, and RT-qPCR was used to assess the remaining levels of β-Gl Norm and β-Gl Ter mRNAs, each after normalization to MUP mRNA. ( c ) Western blots of lysates of MCF7 cells from a (blots derive from and are representative of the three biological replicates in a ) that had been exposed to doxorubicin (5 μM) for the indicated times. CP, cleavage product. GAPDH levels serve as loading controls. Three-fold serial dilutions (wedge) reveal the dynamic range of analysis. ( d ) As in c , but cells were exposed to a 10-fold higher concentration of doxorubicin and analyzed at earlier time points. Representative of 2 biological replicates.

    Techniques Used: Quantitative RT-PCR, Transfection, Western Blot, Concentration Assay

    2) Product Images from "A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †"

    Article Title: A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01260-09

    Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.
    Figure Legend Snippet: Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection

    (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P
    Figure Legend Snippet: (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P

    Techniques Used: Western Blot, Expressing, Mouse Assay, Recombinant, Staining, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P
    Figure Legend Snippet: Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation

    3) Product Images from "Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿ †"

    Article Title: Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00624-10

    CUGBP1 is responsible for GU-repeat-mediated mRNA decay. (A) The decay of the GSN-GU beta-globin reporter was measured in cells that expressed a control siRNA (Ctrl-si) or two different siRNAs (siA and siB) directed against CUGBP1. HeLa Tet-Off cells were transfected with the GSN-GU-repeat-containing reporter and the indicated siRNAs. Knockdown efficiency was monitored for each experiment by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. (B) Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at the 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C) The experiment shown in panel B was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are given in minutes.
    Figure Legend Snippet: CUGBP1 is responsible for GU-repeat-mediated mRNA decay. (A) The decay of the GSN-GU beta-globin reporter was measured in cells that expressed a control siRNA (Ctrl-si) or two different siRNAs (siA and siB) directed against CUGBP1. HeLa Tet-Off cells were transfected with the GSN-GU-repeat-containing reporter and the indicated siRNAs. Knockdown efficiency was monitored for each experiment by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. (B) Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at the 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C) The experiment shown in panel B was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are given in minutes.

    Techniques Used: Transfection, Western Blot, Northern Blot

    The GU-repeat sequence mediates rapid decay of the beta-globin reporter transcript. (A) HeLa Tet-Off cells were transfected with the pTetBBB beta-globin reporter construct or with reporter constructs in which GRE-containing sequences from the 3′ UTR of the JUNB transcript (JUNB-GRE) or GU-repeat sequences from the 3′ UTRs of the NDUFS2 (NDUFS2-GU), GSN (GSN-GU), or PPIC (PPIC-GU) were inserted into the beta-globin 3′ UTR. Doxycycline was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (B) The experiment shown in panel A was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.
    Figure Legend Snippet: The GU-repeat sequence mediates rapid decay of the beta-globin reporter transcript. (A) HeLa Tet-Off cells were transfected with the pTetBBB beta-globin reporter construct or with reporter constructs in which GRE-containing sequences from the 3′ UTR of the JUNB transcript (JUNB-GRE) or GU-repeat sequences from the 3′ UTRs of the NDUFS2 (NDUFS2-GU), GSN (GSN-GU), or PPIC (PPIC-GU) were inserted into the beta-globin 3′ UTR. Doxycycline was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (B) The experiment shown in panel A was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.

    Techniques Used: Sequencing, Transfection, Construct, Northern Blot

    Mutation of the GU-repeat sequence abrogates mRNA decay. (A and B) The decay of the NDUFS2-GU (A) or the GSN-GU (B) beta-globin reporter was compared to the decay of these reporters in which the GU-repeats were deleted (NDUFS2-ΔGU and GSN-ΔGU) or mutated (NDUFS2-mGU and GSN-mGU). HeLa Tet-Off cells were transfected with the indicated beta-globin reporter constructs. Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C and D) The experiments shown in panels A and B were performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.
    Figure Legend Snippet: Mutation of the GU-repeat sequence abrogates mRNA decay. (A and B) The decay of the NDUFS2-GU (A) or the GSN-GU (B) beta-globin reporter was compared to the decay of these reporters in which the GU-repeats were deleted (NDUFS2-ΔGU and GSN-ΔGU) or mutated (NDUFS2-mGU and GSN-mGU). HeLa Tet-Off cells were transfected with the indicated beta-globin reporter constructs. Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C and D) The experiments shown in panels A and B were performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.

    Techniques Used: Mutagenesis, Sequencing, Transfection, Construct, Northern Blot

    Posttranscriptional regulation of the CUGBP1 target network. (A) The network diagram depicts the coordinate regulation of CUGBP1 target transcripts involved in apoptosis. Transcripts depicted in orange represent GRE-containing transcripts that were enriched in the CUGBP1 targets. These pathways were identi fied by Ingenuity Pathway Assistant software (Ingenuity Systems, CA). (B) HeLa Tet-Off cells were transfected twice within 24 h with either a control siRNA or two different siRNAs specific for CUGBP1 (siA and siB). Knockdown efficiency was monitored by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. Apoptosis was assessed with a specific anti-PARP antibody. (C) Cells were stained for FACS analysis with a PE-labeled anti-active caspase 3 antibody. A representative experiment is shown. Four independent experiments were analyzed. Average percentages of apoptosis, standard error, and P values are indicated.
    Figure Legend Snippet: Posttranscriptional regulation of the CUGBP1 target network. (A) The network diagram depicts the coordinate regulation of CUGBP1 target transcripts involved in apoptosis. Transcripts depicted in orange represent GRE-containing transcripts that were enriched in the CUGBP1 targets. These pathways were identi fied by Ingenuity Pathway Assistant software (Ingenuity Systems, CA). (B) HeLa Tet-Off cells were transfected twice within 24 h with either a control siRNA or two different siRNAs specific for CUGBP1 (siA and siB). Knockdown efficiency was monitored by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. Apoptosis was assessed with a specific anti-PARP antibody. (C) Cells were stained for FACS analysis with a PE-labeled anti-active caspase 3 antibody. A representative experiment is shown. Four independent experiments were analyzed. Average percentages of apoptosis, standard error, and P values are indicated.

    Techniques Used: Software, Transfection, Western Blot, Staining, FACS, Labeling

    4) Product Images from "Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila"

    Article Title: Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.397612

    Intracellular expression and production of VahC cause actin cytoskeleton disruption and induced caspase-mediated apoptosis in mammalian cells. A , episomal expression of the vahC gene in HeLa Tet-Off cells. Shown is a Western blot on HeLa Tet-Off cells
    Figure Legend Snippet: Intracellular expression and production of VahC cause actin cytoskeleton disruption and induced caspase-mediated apoptosis in mammalian cells. A , episomal expression of the vahC gene in HeLa Tet-Off cells. Shown is a Western blot on HeLa Tet-Off cells

    Techniques Used: Expressing, Western Blot

    5) Product Images from "A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †"

    Article Title: A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01260-09

    Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.
    Figure Legend Snippet: Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection

    (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P
    Figure Legend Snippet: (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P

    Techniques Used: Western Blot, Expressing, Mouse Assay, Recombinant, Staining, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P
    Figure Legend Snippet: Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation

    6) Product Images from "SGT1-HSP90 complex is required for CENP-A deposition at centromeres"

    Article Title: SGT1-HSP90 complex is required for CENP-A deposition at centromeres

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1325039

    The SGT1-HSP90 complex contributes to CENP-A monoubiquitylation and diubiquitylation in vivo. (A) Representative images of the in vivo ubiquitylation assay with the combination of SGT1 siRNA (#1 + #2), CUL4A siRNA (#1), or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods). HeLa Tet-Off cells were cotransfected with the indicated constructs and siRNAs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (**) Putative di-Ub-CENP-A-Flag, (*) Putative mono-Ub-CENP-A-Flag. (B) Representative images of the in vivo ubiquitylation assay performed as (A) with the combination of HSP90 siRNA (#1) or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods).
    Figure Legend Snippet: The SGT1-HSP90 complex contributes to CENP-A monoubiquitylation and diubiquitylation in vivo. (A) Representative images of the in vivo ubiquitylation assay with the combination of SGT1 siRNA (#1 + #2), CUL4A siRNA (#1), or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods). HeLa Tet-Off cells were cotransfected with the indicated constructs and siRNAs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (**) Putative di-Ub-CENP-A-Flag, (*) Putative mono-Ub-CENP-A-Flag. (B) Representative images of the in vivo ubiquitylation assay performed as (A) with the combination of HSP90 siRNA (#1) or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods).

    Techniques Used: In Vivo, Ubiquitin Assay, Construct, Western Blot

    SGT1-HSP90 complex is required for composition of the CUL4A complex and recognition of CENP-A by COPS8. (A) SGT1A interacts with CUL4A in vivo. Immunoblot analysis of CUL4A immunoprecipitates. Plasmids pTRM4-SGT1A-Flag and pcDNA3-myc3-CUL4A (Table S3) were cotransfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 1% of the total cell lysate (Input) and precipitate (IP) obtained by using anti-c-myc antibody (Table S1) were detected by Western blot analysis using anti-Flag antibody. (B) Interaction between CENP-A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of endogenous CENP-A immunoprecipitates. Indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using rabbit polyclonal anti-CENP-A antibody (Table S1) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (C) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of CUL4A immunoprecipitates. Plasmid pTRM4-CUL4A-Flag (Table S3) and indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using ANTI-FLAG M2 Affinity Gel (SIGMAALDRICH) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (D) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of COPS8 immunoprecipitates. The indicated plasmids (Table S3) and siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using Anti-HA.11 Epitope Tag Affinity Matrix (Covance) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input).
    Figure Legend Snippet: SGT1-HSP90 complex is required for composition of the CUL4A complex and recognition of CENP-A by COPS8. (A) SGT1A interacts with CUL4A in vivo. Immunoblot analysis of CUL4A immunoprecipitates. Plasmids pTRM4-SGT1A-Flag and pcDNA3-myc3-CUL4A (Table S3) were cotransfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 1% of the total cell lysate (Input) and precipitate (IP) obtained by using anti-c-myc antibody (Table S1) were detected by Western blot analysis using anti-Flag antibody. (B) Interaction between CENP-A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of endogenous CENP-A immunoprecipitates. Indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using rabbit polyclonal anti-CENP-A antibody (Table S1) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (C) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of CUL4A immunoprecipitates. Plasmid pTRM4-CUL4A-Flag (Table S3) and indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using ANTI-FLAG M2 Affinity Gel (SIGMAALDRICH) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (D) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of COPS8 immunoprecipitates. The indicated plasmids (Table S3) and siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using Anti-HA.11 Epitope Tag Affinity Matrix (Covance) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input).

    Techniques Used: In Vivo, Transfection, Western Blot, Plasmid Preparation

    7) Product Images from "ALTERATION IN THE ACTIVATION STATE OF NEW INFLAMMATION-ASSOCIATED TARGETS BY PHOSPHOLIPASE A2-ACTIVATING PROTEIN (PLAA)"

    Article Title: ALTERATION IN THE ACTIVATION STATE OF NEW INFLAMMATION-ASSOCIATED TARGETS BY PHOSPHOLIPASE A2-ACTIVATING PROTEIN (PLAA)

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2008.01.004

    Overexpression of the plaa gene increases COX-2 expression by TNF-α in HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene increases COX-2 expression by TNF-α in HeLa Tet-off cells

    Techniques Used: Over Expression, Expressing

    Overexpression of the plaa gene increases PGE 2 production and PLA 2 activation by TNF-α in HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene increases PGE 2 production and PLA 2 activation by TNF-α in HeLa Tet-off cells

    Techniques Used: Over Expression, Proximity Ligation Assay, Activation Assay

    Overexpression of the plaa gene increases IL-6 production in TNF-α-stimulated HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene increases IL-6 production in TNF-α-stimulated HeLa Tet-off cells

    Techniques Used: Over Expression

    Overexpression of the plaa gene enhances TNF-α-induced NF-κB activation in HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene enhances TNF-α-induced NF-κB activation in HeLa Tet-off cells

    Techniques Used: Over Expression, Activation Assay

    Overexpression of the plaa gene increases cPLA 2 membrane translocation by TNF-α in HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene increases cPLA 2 membrane translocation by TNF-α in HeLa Tet-off cells

    Techniques Used: Over Expression, Translocation Assay

    Overexpression of the plaa gene in HeLa Tet-off cells.
    Figure Legend Snippet: Overexpression of the plaa gene in HeLa Tet-off cells.

    Techniques Used: Over Expression

    8) Product Images from "Dimer‐specific immunoprecipitation of active caspase‐2 identifies TRAF proteins as novel activators"

    Article Title: Dimer‐specific immunoprecipitation of active caspase‐2 identifies TRAF proteins as novel activators

    Journal: The EMBO Journal

    doi: 10.15252/embj.201797072

    TRAF2 interacts directly with caspase‐2 to induce activation A diagram of the caspase‐2 protein showing the major domains and the location of a putative TRAF‐interacting motif (TIM). Casp2(C320A)‐Flag plasmids (wild type or TIM mutant) were expressed in HeLa cells for 24 h followed by anti‐Flag IP and IB. Stably transfected, catalytically active caspase‐2 (wild type or TIM mutant) was induced in HeLa Tet‐On cells with 0.1 μg/ml doxycycline for 24 h, followed by propidium iodide (PI) staining and flow cytometry. n = 3 independent experiments (means + s.e.m.). * P
    Figure Legend Snippet: TRAF2 interacts directly with caspase‐2 to induce activation A diagram of the caspase‐2 protein showing the major domains and the location of a putative TRAF‐interacting motif (TIM). Casp2(C320A)‐Flag plasmids (wild type or TIM mutant) were expressed in HeLa cells for 24 h followed by anti‐Flag IP and IB. Stably transfected, catalytically active caspase‐2 (wild type or TIM mutant) was induced in HeLa Tet‐On cells with 0.1 μg/ml doxycycline for 24 h, followed by propidium iodide (PI) staining and flow cytometry. n = 3 independent experiments (means + s.e.m.). * P

    Techniques Used: Activation Assay, Mutagenesis, Stable Transfection, Transfection, Staining, Flow Cytometry, Cytometry

    9) Product Images from "Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila"

    Article Title: Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.397612

    Intracellular expression and production of VahC cause actin cytoskeleton disruption and induced caspase-mediated apoptosis in mammalian cells. A , episomal expression of the vahC gene in HeLa Tet-Off cells. Shown is a Western blot on HeLa Tet-Off cells
    Figure Legend Snippet: Intracellular expression and production of VahC cause actin cytoskeleton disruption and induced caspase-mediated apoptosis in mammalian cells. A , episomal expression of the vahC gene in HeLa Tet-Off cells. Shown is a Western blot on HeLa Tet-Off cells

    Techniques Used: Expressing, Western Blot

    10) Product Images from "Ubiquitination and Proteasomal Degradation of Interferon Regulatory Factor-3 induced by Npro from a Cytopathic Bovine Viral Diarrhea Virus"

    Article Title: Ubiquitination and Proteasomal Degradation of Interferon Regulatory Factor-3 induced by Npro from a Cytopathic Bovine Viral Diarrhea Virus

    Journal: Virology

    doi: 10.1016/j.virol.2007.04.023

    Ectopic expression Npro reduces IRF-3 protein abundance and inhibits the induction of IRF-3 target genes. A. HeLa cell lines with tet-regulated expression of WT Npro (Npro-25 and Npro-29 cells, left panel) or L8P mutant Npro (L8P-1, right panel) were
    Figure Legend Snippet: Ectopic expression Npro reduces IRF-3 protein abundance and inhibits the induction of IRF-3 target genes. A. HeLa cell lines with tet-regulated expression of WT Npro (Npro-25 and Npro-29 cells, left panel) or L8P mutant Npro (L8P-1, right panel) were

    Techniques Used: Expressing, Mutagenesis

    11) Product Images from "p16INK4a Translation Suppressed by miR-24"

    Article Title: p16INK4a Translation Suppressed by miR-24

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001864

    Analysis of p16 predicted sites of miR-24 association using a heterologous reporter. Schematic ( left ) and names ( center ) of the EGFP and chimeric EGFP-p16 reporter plasmids tested ( Materials and Methods ). By 48 hr after co-transfection of HeLa Tet-off cells with Ctrl. siRNA or AS-miR-24, along with the plasmids indicated, the levels of EGFP expressed from each reporter vector was assessed; shown are representative Western blotting signals and quantification ( Fold , AS-miR-24 vs. Ctrl. siRNA) of EGFP reporter levels in each transfection group after normalization to α-tubulin signals.
    Figure Legend Snippet: Analysis of p16 predicted sites of miR-24 association using a heterologous reporter. Schematic ( left ) and names ( center ) of the EGFP and chimeric EGFP-p16 reporter plasmids tested ( Materials and Methods ). By 48 hr after co-transfection of HeLa Tet-off cells with Ctrl. siRNA or AS-miR-24, along with the plasmids indicated, the levels of EGFP expressed from each reporter vector was assessed; shown are representative Western blotting signals and quantification ( Fold , AS-miR-24 vs. Ctrl. siRNA) of EGFP reporter levels in each transfection group after normalization to α-tubulin signals.

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

    12) Product Images from "SGT1-HSP90 complex is required for CENP-A deposition at centromeres"

    Article Title: SGT1-HSP90 complex is required for CENP-A deposition at centromeres

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1325039

    The SGT1-HSP90 complex contributes to CENP-A monoubiquitylation and diubiquitylation in vivo. (A) Representative images of the in vivo ubiquitylation assay with the combination of SGT1 siRNA (#1 + #2), CUL4A siRNA (#1), or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods). HeLa Tet-Off cells were cotransfected with the indicated constructs and siRNAs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (**) Putative di-Ub-CENP-A-Flag, (*) Putative mono-Ub-CENP-A-Flag. (B) Representative images of the in vivo ubiquitylation assay performed as (A) with the combination of HSP90 siRNA (#1) or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods).
    Figure Legend Snippet: The SGT1-HSP90 complex contributes to CENP-A monoubiquitylation and diubiquitylation in vivo. (A) Representative images of the in vivo ubiquitylation assay with the combination of SGT1 siRNA (#1 + #2), CUL4A siRNA (#1), or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods). HeLa Tet-Off cells were cotransfected with the indicated constructs and siRNAs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (**) Putative di-Ub-CENP-A-Flag, (*) Putative mono-Ub-CENP-A-Flag. (B) Representative images of the in vivo ubiquitylation assay performed as (A) with the combination of HSP90 siRNA (#1) or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods).

    Techniques Used: In Vivo, Ubiquitin Assay, Construct, Western Blot

    SGT1-HSP90 complex is required for composition of the CUL4A complex and recognition of CENP-A by COPS8. (A) SGT1A interacts with CUL4A in vivo. Immunoblot analysis of CUL4A immunoprecipitates. Plasmids pTRM4-SGT1A-Flag and pcDNA3-myc3-CUL4A (Table S3) were cotransfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 1% of the total cell lysate (Input) and precipitate (IP) obtained by using anti-c-myc antibody (Table S1) were detected by Western blot analysis using anti-Flag antibody. (B) Interaction between CENP-A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of endogenous CENP-A immunoprecipitates. Indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using rabbit polyclonal anti-CENP-A antibody (Table S1) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (C) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of CUL4A immunoprecipitates. Plasmid pTRM4-CUL4A-Flag (Table S3) and indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using ANTI-FLAG M2 Affinity Gel (SIGMAALDRICH) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (D) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of COPS8 immunoprecipitates. The indicated plasmids (Table S3) and siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using Anti-HA.11 Epitope Tag Affinity Matrix (Covance) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input).
    Figure Legend Snippet: SGT1-HSP90 complex is required for composition of the CUL4A complex and recognition of CENP-A by COPS8. (A) SGT1A interacts with CUL4A in vivo. Immunoblot analysis of CUL4A immunoprecipitates. Plasmids pTRM4-SGT1A-Flag and pcDNA3-myc3-CUL4A (Table S3) were cotransfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 1% of the total cell lysate (Input) and precipitate (IP) obtained by using anti-c-myc antibody (Table S1) were detected by Western blot analysis using anti-Flag antibody. (B) Interaction between CENP-A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of endogenous CENP-A immunoprecipitates. Indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using rabbit polyclonal anti-CENP-A antibody (Table S1) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (C) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of CUL4A immunoprecipitates. Plasmid pTRM4-CUL4A-Flag (Table S3) and indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using ANTI-FLAG M2 Affinity Gel (SIGMAALDRICH) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (D) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of COPS8 immunoprecipitates. The indicated plasmids (Table S3) and siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using Anti-HA.11 Epitope Tag Affinity Matrix (Covance) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input).

    Techniques Used: In Vivo, Transfection, Western Blot, Plasmid Preparation

    13) Product Images from "Conditional Expression of a Truncated Fragment of Nonmuscle Myosin II-A Alters Cell Shape but Not Cytokinesis in HeLa Cells"

    Article Title: Conditional Expression of a Truncated Fragment of Nonmuscle Myosin II-A Alters Cell Shape but Not Cytokinesis in HeLa Cells

    Journal: Molecular Biology of the Cell

    doi:

    Disruption of myosin filaments induced by the expression of ΔN592 in HeLa Tet-Off Cells. (A) The cells were cultured in the presence of Dox (a–c) or in the absence of Dox for 1 d (d–f), 2 d (g–i), and 4 d (j–l) or Dox was added after 4 d in culture in the absence of Dox (m–o). An antibody to the NH 2 terminus of NMHC II-A was used to distinguish between full-length NMHC II-A (red) and the GFP-ΔN592 fragment (green) in f, i, and l. Yellow indicates colocalization of NMHC II-A and ΔN592. Note cell rounding by 4 d in the absence of Dox (j–l) and the reversal of the phenotype 16 h following addition of Dox (m–o). (B) Colocalization of GFP-ΔN592 and RFP-NMHC II-A. GFP-ΔN592 and RFP-NMHC II-A were transiently cotransfected with L63RhoA into HeLa Tet-Off cells. Transfected cells were fixed with 3.7% paraformaldehyde. GFP-ΔN592 and RFP-NMHC II-A were visualized by green (a and d) and red (b and e) fluorescence, respectively, by using confocal microscopy. The merged images (yellow, c and f) indicate colocalization of GFP-ΔN592 and RFP-NMHC II-A. Images of two different cells are shown. Bar, 20 μm.
    Figure Legend Snippet: Disruption of myosin filaments induced by the expression of ΔN592 in HeLa Tet-Off Cells. (A) The cells were cultured in the presence of Dox (a–c) or in the absence of Dox for 1 d (d–f), 2 d (g–i), and 4 d (j–l) or Dox was added after 4 d in culture in the absence of Dox (m–o). An antibody to the NH 2 terminus of NMHC II-A was used to distinguish between full-length NMHC II-A (red) and the GFP-ΔN592 fragment (green) in f, i, and l. Yellow indicates colocalization of NMHC II-A and ΔN592. Note cell rounding by 4 d in the absence of Dox (j–l) and the reversal of the phenotype 16 h following addition of Dox (m–o). (B) Colocalization of GFP-ΔN592 and RFP-NMHC II-A. GFP-ΔN592 and RFP-NMHC II-A were transiently cotransfected with L63RhoA into HeLa Tet-Off cells. Transfected cells were fixed with 3.7% paraformaldehyde. GFP-ΔN592 and RFP-NMHC II-A were visualized by green (a and d) and red (b and e) fluorescence, respectively, by using confocal microscopy. The merged images (yellow, c and f) indicate colocalization of GFP-ΔN592 and RFP-NMHC II-A. Images of two different cells are shown. Bar, 20 μm.

    Techniques Used: Expressing, Cell Culture, Transfection, Fluorescence, Confocal Microscopy

    Immunoblot analysis and actin-binding studies of NMHC-II and ΔN592. (A) Inducible expression of GFP-NMHC II-A, II-B, and GFP-ΔN592 in HeLa Tet-Off cells. The stable cell lines cultured with (+) or without (−) doxycycline (Dox) for 3 d were subjected to immunoblot analysis by using antibodies raised against amino acids at the carboxy terminus (anti-A, -COOH, lanes 1–4) or at the amino terminus (anti-A, NH 2 -, lanes 5 and 6) of the myosin II-A heavy chain as well as antibodies raised against the carboxy terminus (anti-B, -COOH, lanes 7 and 8) of the myosin II-B heavy chain. The HeLa Tet-Off cell line from Clontech does not contain endogenous NMHC II-B (lane 7), but introduction of a plasmid encoding NMHC-B into these cells by stable transfection resulted in NMHC II-B expression in the absence (lane 8), but not in the presence of Dox (lane 7). (B) Immunoblot demonstrating that ΔN592 does not bind to actin. Purified baculovirus-expressed ΔN592 does not cosediment with actin in the presence or in the absence of ATP (lanes 1 and 3). In contrast, endogenous NMHC II-A binds to actin in the absence of ATP (lane 5), but not in the presence of ATP (lane 8). P, pellet; S, supernatant. (C) GFP-ΔN592 does not dimerize with endogenous NMHC II-A. Cell extracts from HeLa cells expressing GFP-ΔN592 and endogenous NMHC II-A were incubated with F-actin in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of Mg 2+ -ATP, followed by sedimentation resulting in pellet (p) and supernatant (s) fractions. GFP-ΔN592 does not bind to actin in the absence or presence of ATP (lanes 2 and 4). Endogenous NMHC II-A bound to actin in the absence of ATP (lane 1). Note that only a very small amount of GFP-ΔN592 cosedimented with endogenous NMHC II-A (lane 1). This was most likely due to minor trapping of ΔN592 in the actin pellet.
    Figure Legend Snippet: Immunoblot analysis and actin-binding studies of NMHC-II and ΔN592. (A) Inducible expression of GFP-NMHC II-A, II-B, and GFP-ΔN592 in HeLa Tet-Off cells. The stable cell lines cultured with (+) or without (−) doxycycline (Dox) for 3 d were subjected to immunoblot analysis by using antibodies raised against amino acids at the carboxy terminus (anti-A, -COOH, lanes 1–4) or at the amino terminus (anti-A, NH 2 -, lanes 5 and 6) of the myosin II-A heavy chain as well as antibodies raised against the carboxy terminus (anti-B, -COOH, lanes 7 and 8) of the myosin II-B heavy chain. The HeLa Tet-Off cell line from Clontech does not contain endogenous NMHC II-B (lane 7), but introduction of a plasmid encoding NMHC-B into these cells by stable transfection resulted in NMHC II-B expression in the absence (lane 8), but not in the presence of Dox (lane 7). (B) Immunoblot demonstrating that ΔN592 does not bind to actin. Purified baculovirus-expressed ΔN592 does not cosediment with actin in the presence or in the absence of ATP (lanes 1 and 3). In contrast, endogenous NMHC II-A binds to actin in the absence of ATP (lane 5), but not in the presence of ATP (lane 8). P, pellet; S, supernatant. (C) GFP-ΔN592 does not dimerize with endogenous NMHC II-A. Cell extracts from HeLa cells expressing GFP-ΔN592 and endogenous NMHC II-A were incubated with F-actin in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of Mg 2+ -ATP, followed by sedimentation resulting in pellet (p) and supernatant (s) fractions. GFP-ΔN592 does not bind to actin in the absence or presence of ATP (lanes 2 and 4). Endogenous NMHC II-A bound to actin in the absence of ATP (lane 1). Note that only a very small amount of GFP-ΔN592 cosedimented with endogenous NMHC II-A (lane 1). This was most likely due to minor trapping of ΔN592 in the actin pellet.

    Techniques Used: Binding Assay, Expressing, Stable Transfection, Cell Culture, Plasmid Preparation, Purification, Incubation, Sedimentation

    Cell rounding induced by the expression of GFP-ΔN592, but not the full-length myosin heavy chain (NMHC II-A) in HeLa Tet-Off cells. Cells were cultured for 3 d without Dox (a, c, e, and g) or with Dox (b, d, f, and h). The images were taken from live cells by using an Axivert 35 microscope ( Carl Zeiss ). Note the presence of the GFP signal only in a and e (−Dox) and the rounded morphology of cells expressing GFP-ΔN592 (e and g) in contrast to cells expressing GFP-NMHC II-A (a and c). The original magnification was 320×.
    Figure Legend Snippet: Cell rounding induced by the expression of GFP-ΔN592, but not the full-length myosin heavy chain (NMHC II-A) in HeLa Tet-Off cells. Cells were cultured for 3 d without Dox (a, c, e, and g) or with Dox (b, d, f, and h). The images were taken from live cells by using an Axivert 35 microscope ( Carl Zeiss ). Note the presence of the GFP signal only in a and e (−Dox) and the rounded morphology of cells expressing GFP-ΔN592 (e and g) in contrast to cells expressing GFP-NMHC II-A (a and c). The original magnification was 320×.

    Techniques Used: Expressing, Cell Culture, Microscopy

    Colocalization of myosin II-B GFP-fusion protein and endogenous myosin II-A in HeLa Tet-Off cells and formation of myosin filaments following transfection of L63RhoA. (A) Full-length NMHC II-B was visualized by using GFP in the absence of doxycycline (a) and endogenous NMHC II-A was visualized by using antibodies to the carboxy-terminal sequence (b, e, and f). The distribution of both isoforms (II-B, green; II-A, red; colocalization, yellow) is shown in c; d, e, and f are from an experiment carried out in the presence of Dox. (B) Myosin filament formation induced by the transient expression of dominant active RhoA mutant L63RhoA. HeLa Tet-Off cells were transiently transfected with L63RhoA, along with NMHC II-A (a), ΔN592 (b), ΔC170 (c), or NMHC II-B (d), respectively. Transfected cells were visualized by GFP (green), phalloidin (red), and Myc antibody followed by Alexa 350 anti-mouse conjugate IgG (blue, to detect myc-tagged L63RhoA expression; our unpublished data). Arrows indicate the cells expressing both GFP-tagged myosin and L63RhoA. Note that NMHC II-A (a), ΔN592 (b), and NMHC II-B (d), but not ΔC170 (c) form filaments upon expression of L63RhoA. Bar, 20 μm.
    Figure Legend Snippet: Colocalization of myosin II-B GFP-fusion protein and endogenous myosin II-A in HeLa Tet-Off cells and formation of myosin filaments following transfection of L63RhoA. (A) Full-length NMHC II-B was visualized by using GFP in the absence of doxycycline (a) and endogenous NMHC II-A was visualized by using antibodies to the carboxy-terminal sequence (b, e, and f). The distribution of both isoforms (II-B, green; II-A, red; colocalization, yellow) is shown in c; d, e, and f are from an experiment carried out in the presence of Dox. (B) Myosin filament formation induced by the transient expression of dominant active RhoA mutant L63RhoA. HeLa Tet-Off cells were transiently transfected with L63RhoA, along with NMHC II-A (a), ΔN592 (b), ΔC170 (c), or NMHC II-B (d), respectively. Transfected cells were visualized by GFP (green), phalloidin (red), and Myc antibody followed by Alexa 350 anti-mouse conjugate IgG (blue, to detect myc-tagged L63RhoA expression; our unpublished data). Arrows indicate the cells expressing both GFP-tagged myosin and L63RhoA. Note that NMHC II-A (a), ΔN592 (b), and NMHC II-B (d), but not ΔC170 (c) form filaments upon expression of L63RhoA. Bar, 20 μm.

    Techniques Used: Transfection, Sequencing, Expressing, Mutagenesis

    14) Product Images from "Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay"

    Article Title: Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay

    Journal: Genes & Development

    doi: 10.1101/gad.913001

    SMG-1 is involved in the PTC-dependent degradation of β-globin mRNA. ( A ) Schematic representation of human β-globin (BGG) gene reporter constructs, BGG-WT and BGG-PTC. The ORF is represented by boxes, and introns and UTRs are represented by lines. ( B ) The BGG PTC transcript is less abundant than transcripts from the WT reporter. The WT or mutant reporter plasmid was transfected with the CAT plasmid into the HeLa Tet-Off or MEF Tet-Off cell lines. The same amount of total RNA from each cell was analyzed by Northern blotting using a BGG or a CAT probe. ( C ) Overexpression of hSMG-1 suppresses the accumulation of the BGG 39PTC transcript; whereas that of hSMG-1-DA enhances it. HeLa Tet-Off was transfected with the BGG WT reporter or BGG 39PTC (1.5 μg) with the hSMG-1-WT or hSMG-1-DA (3 μg) and a CAT construct (1.5 μg). Then the accumulated BGG transcripts were evaluated by Northern blotting using CAT as an internal control. The relative amount of transcripts is shown in the bar graph. ( D ) Overexpression of hSMG-1-WT enhances the decay of the BGG 39PTC transcript; whereas that of hSMG-1-DA suppresses it. The HeLa Tet-Off cell was transfected with the WT ( left ) or mutant ( right ) reporter gene, and cells were harvested at an indicated time point after the addition of 50 ng/mL doxycycline to the medium. Total RNA was isolated and analyzed by Northern blotting using the corresponding probe. The relative amount of transcripts is shown in the graph. The BGG RNA levels are normalized to those of GAPDH.
    Figure Legend Snippet: SMG-1 is involved in the PTC-dependent degradation of β-globin mRNA. ( A ) Schematic representation of human β-globin (BGG) gene reporter constructs, BGG-WT and BGG-PTC. The ORF is represented by boxes, and introns and UTRs are represented by lines. ( B ) The BGG PTC transcript is less abundant than transcripts from the WT reporter. The WT or mutant reporter plasmid was transfected with the CAT plasmid into the HeLa Tet-Off or MEF Tet-Off cell lines. The same amount of total RNA from each cell was analyzed by Northern blotting using a BGG or a CAT probe. ( C ) Overexpression of hSMG-1 suppresses the accumulation of the BGG 39PTC transcript; whereas that of hSMG-1-DA enhances it. HeLa Tet-Off was transfected with the BGG WT reporter or BGG 39PTC (1.5 μg) with the hSMG-1-WT or hSMG-1-DA (3 μg) and a CAT construct (1.5 μg). Then the accumulated BGG transcripts were evaluated by Northern blotting using CAT as an internal control. The relative amount of transcripts is shown in the bar graph. ( D ) Overexpression of hSMG-1-WT enhances the decay of the BGG 39PTC transcript; whereas that of hSMG-1-DA suppresses it. The HeLa Tet-Off cell was transfected with the WT ( left ) or mutant ( right ) reporter gene, and cells were harvested at an indicated time point after the addition of 50 ng/mL doxycycline to the medium. Total RNA was isolated and analyzed by Northern blotting using the corresponding probe. The relative amount of transcripts is shown in the graph. The BGG RNA levels are normalized to those of GAPDH.

    Techniques Used: Construct, Mutagenesis, Plasmid Preparation, Transfection, Northern Blot, Over Expression, Isolation

    15) Product Images from "Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2"

    Article Title: Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.20.7177-7188.2003

    Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.
    Figure Legend Snippet: Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.

    Techniques Used: Over Expression, Inhibition, Transfection, Plasmid Preparation, Northern Blot, Isolation, Labeling

    Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.
    Figure Legend Snippet: Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.

    Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Transfection, Incubation, SDS Page, Autoradiography, Labeling, Staining, Plasmid Preparation, Northern Blot, Quantitation Assay

    The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.
    Figure Legend Snippet: The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.

    Techniques Used: Functional Assay, Construct, Mutagenesis, Northern Blot, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Random Primed

    Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.
    Figure Legend Snippet: Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Isolation, Agarose Gel Electrophoresis, Produced, Northern Blot, Labeling, Quantitation Assay

    16) Product Images from "The C-Terminal RGG Domain of Human Lsm4 Promotes Processing Body Formation Stimulated by Arginine Dimethylation"

    Article Title: The C-Terminal RGG Domain of Human Lsm4 Promotes Processing Body Formation Stimulated by Arginine Dimethylation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01102-15

    The RGG domain of Lsm4 is not required for mRNA decay or translational repression. (A) Northern blot assays showing the decay of an ARE-containing reporter mRNA (β-c-fos) in HeLa Tet-off cells treated with the siRNAs indicated and transiently expressing the Lsm4 proteins indicated. β-c-fos half-lives ( t 1/2 ) were calculated with the constitutively transcribed β-GAP internal control mRNA for normalization. Fold stabilization was calculated relative to the siLuc control condition in three experiments, and the standard errors of the means are indicated. (B) Northern blot assays showing the decay of endogenous H2A mRNA induced by treatment with 5 mM hydroxyurea in HEK 293 T-REx cells treated with the siRNAs indicated and stably expressing the Lsm4 proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA served as a normalization control. (C) Luciferase luminescence assays of cells transiently transfected with plasmids encoding the MS2 fusion proteins indicated, as well as a firefly luciferase reporter with six MS2 coat protein binding sites in its 3′ UTR (F-Luc-6xMS2) and a Renilla luciferase reporter (R-Luc) as an internal control. F-Luc-6xMS2 was normalized to R-Luc, and all samples were normalized to cells transfected with MS2 and the reporters alone. Error bars represent the standard errors of the means. **, P
    Figure Legend Snippet: The RGG domain of Lsm4 is not required for mRNA decay or translational repression. (A) Northern blot assays showing the decay of an ARE-containing reporter mRNA (β-c-fos) in HeLa Tet-off cells treated with the siRNAs indicated and transiently expressing the Lsm4 proteins indicated. β-c-fos half-lives ( t 1/2 ) were calculated with the constitutively transcribed β-GAP internal control mRNA for normalization. Fold stabilization was calculated relative to the siLuc control condition in three experiments, and the standard errors of the means are indicated. (B) Northern blot assays showing the decay of endogenous H2A mRNA induced by treatment with 5 mM hydroxyurea in HEK 293 T-REx cells treated with the siRNAs indicated and stably expressing the Lsm4 proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA served as a normalization control. (C) Luciferase luminescence assays of cells transiently transfected with plasmids encoding the MS2 fusion proteins indicated, as well as a firefly luciferase reporter with six MS2 coat protein binding sites in its 3′ UTR (F-Luc-6xMS2) and a Renilla luciferase reporter (R-Luc) as an internal control. F-Luc-6xMS2 was normalized to R-Luc, and all samples were normalized to cells transfected with MS2 and the reporters alone. Error bars represent the standard errors of the means. **, P

    Techniques Used: Northern Blot, Expressing, Stable Transfection, Luciferase, Transfection, Protein Binding

    17) Product Images from "Coordinated Expression of Tristetraprolin Post-Transcriptionally Attenuates Mitogenic Induction of the Oncogenic Ser/Thr Kinase Pim-1"

    Article Title: Coordinated Expression of Tristetraprolin Post-Transcriptionally Attenuates Mitogenic Induction of the Oncogenic Ser/Thr Kinase Pim-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033194

    Functional association of TTP with PIM1 mRNA in HeLa cells. (A) RNP-IP experiments were performed using control IgG or anti-FLAG antibodies and lysates from untransfected HeLa/Tet-Off cells (ut) or stable clonal lines expressing FLAG-TTPwt or FLAG-TTP C147R as described in “ Materials and Methods ”. Immunoprecipitated material was then screened for PIM1 mRNA by quantitative real-time RT-PCR and normalized to GAPDH mRNA (mean ± SD of three reactions). Independent replicate experiments yielded similar results. (B) Relative levels of PIM1 mRNA were measured in untransfected versus FLAG-TTPwt- or FLAG-TTP C147R-expressing HeLa cells. Bars represent the mean ± SD of quadruplicate qRT-PCR reactions normalized to GAPDH mRNA. (C) The decay kinetics of PIM1 mRNA was measured in HeLa cell models using actD time course assays as described in Figure 2 . mRNA half-lives calculated from independent replicate experiments are provided in the text. (D) Western blot analyses using antibodies targeting specified proteins in HeLa/Tet-Off cell models, with positions of molecular weight markers (in kDa) shown at left.
    Figure Legend Snippet: Functional association of TTP with PIM1 mRNA in HeLa cells. (A) RNP-IP experiments were performed using control IgG or anti-FLAG antibodies and lysates from untransfected HeLa/Tet-Off cells (ut) or stable clonal lines expressing FLAG-TTPwt or FLAG-TTP C147R as described in “ Materials and Methods ”. Immunoprecipitated material was then screened for PIM1 mRNA by quantitative real-time RT-PCR and normalized to GAPDH mRNA (mean ± SD of three reactions). Independent replicate experiments yielded similar results. (B) Relative levels of PIM1 mRNA were measured in untransfected versus FLAG-TTPwt- or FLAG-TTP C147R-expressing HeLa cells. Bars represent the mean ± SD of quadruplicate qRT-PCR reactions normalized to GAPDH mRNA. (C) The decay kinetics of PIM1 mRNA was measured in HeLa cell models using actD time course assays as described in Figure 2 . mRNA half-lives calculated from independent replicate experiments are provided in the text. (D) Western blot analyses using antibodies targeting specified proteins in HeLa/Tet-Off cell models, with positions of molecular weight markers (in kDa) shown at left.

    Techniques Used: Functional Assay, Expressing, Immunoprecipitation, Quantitative RT-PCR, Western Blot, Molecular Weight

    Localization of TTP-responsive elements to an ARE-like sequence in the PIM1 mRNA 3′UTR. (A) Schematic of the 3′-end of the PIM1 mRNA coding sequence ( black box ) and complete 3′UTR, including the ARE domain ( white box ). The scale bar (top) is relative to the translational initiation codon. The positions of biotin-labeled riboprobes corresponding to the PIM1 ARE and coding sequence fragment (CDS) are shown as black bars above the mRNA schematic. Bars below delineate PIM1 3′UTR domains that were subcloned downstream of the translational termination codon of the βG gene for reporter mRNA decay assays. At the bottom is the sequence at the extreme 3′-end of the ARE domain that contains known high affinity TTP-binding motifs. In the βG- PIM1 AREmut reporter mRNA and biotin-labeled AREmut RNA probe, these motifs were disrupted by mutating underlined uridylate residues to cytidines. (B) Decay rates of βG- PIM1 chimeric reporter mRNAs were resolved by Dox time course assays in HeLa/Tet-Off cells co-transfected with an empty vector (pcDNA; solid circles , solid lines ) or vectors expressing FLAG-TTPwt ( open circles , dashed lines ) or FLAG-TTP C147R ( triangles , dotted lines ) as described under “ Materials and Methods ”. mRNA half-lives resolved from multiple independent experiments are summarized in Table 2 . (C) Western blots probed with indicated antibodies (Ab) show levels of FLAG-TTP wt and C147R mutant proteins (top panel) and GAPDH (second panel) in crude cytoplasmic extracts prepared from untransfected HeLa/Tet-Off cells (ut) or stable clonal lines expressing each FLAG-TTP variant. Samples of each lysate were fractionated using biotin-RNA pull-down assays programmed with riboprobes encoding a PIM1 coding sequence fragment (CDS), the PIM1 ARE or the ARE mutant containing the U→C substitutions specified above (AREmut). FLAG-TTP proteins co-purifying with each riboprobe were detected by Western blot (bottom panels). The positions of molecular weight markers (in kDa) are shown to the left of each Western blot panel.
    Figure Legend Snippet: Localization of TTP-responsive elements to an ARE-like sequence in the PIM1 mRNA 3′UTR. (A) Schematic of the 3′-end of the PIM1 mRNA coding sequence ( black box ) and complete 3′UTR, including the ARE domain ( white box ). The scale bar (top) is relative to the translational initiation codon. The positions of biotin-labeled riboprobes corresponding to the PIM1 ARE and coding sequence fragment (CDS) are shown as black bars above the mRNA schematic. Bars below delineate PIM1 3′UTR domains that were subcloned downstream of the translational termination codon of the βG gene for reporter mRNA decay assays. At the bottom is the sequence at the extreme 3′-end of the ARE domain that contains known high affinity TTP-binding motifs. In the βG- PIM1 AREmut reporter mRNA and biotin-labeled AREmut RNA probe, these motifs were disrupted by mutating underlined uridylate residues to cytidines. (B) Decay rates of βG- PIM1 chimeric reporter mRNAs were resolved by Dox time course assays in HeLa/Tet-Off cells co-transfected with an empty vector (pcDNA; solid circles , solid lines ) or vectors expressing FLAG-TTPwt ( open circles , dashed lines ) or FLAG-TTP C147R ( triangles , dotted lines ) as described under “ Materials and Methods ”. mRNA half-lives resolved from multiple independent experiments are summarized in Table 2 . (C) Western blots probed with indicated antibodies (Ab) show levels of FLAG-TTP wt and C147R mutant proteins (top panel) and GAPDH (second panel) in crude cytoplasmic extracts prepared from untransfected HeLa/Tet-Off cells (ut) or stable clonal lines expressing each FLAG-TTP variant. Samples of each lysate were fractionated using biotin-RNA pull-down assays programmed with riboprobes encoding a PIM1 coding sequence fragment (CDS), the PIM1 ARE or the ARE mutant containing the U→C substitutions specified above (AREmut). FLAG-TTP proteins co-purifying with each riboprobe were detected by Western blot (bottom panels). The positions of molecular weight markers (in kDa) are shown to the left of each Western blot panel.

    Techniques Used: Sequencing, Labeling, Binding Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Mutagenesis, Variant Assay, Molecular Weight

    18) Product Images from "Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2"

    Article Title: Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.20.7177-7188.2003

    Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.
    Figure Legend Snippet: Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.

    Techniques Used: Over Expression, Inhibition, Transfection, Plasmid Preparation, Northern Blot, Isolation, Labeling

    Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.
    Figure Legend Snippet: Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.

    Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Transfection, Incubation, SDS Page, Autoradiography, Labeling, Staining, Plasmid Preparation, Northern Blot, Quantitation Assay

    The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.
    Figure Legend Snippet: The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.

    Techniques Used: Functional Assay, Construct, Mutagenesis, Northern Blot, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Random Primed

    Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.
    Figure Legend Snippet: Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Isolation, Agarose Gel Electrophoresis, Produced, Northern Blot, Labeling, Quantitation Assay

    19) Product Images from "Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10 1"

    Article Title: Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0803883

    IL-10 reduces TTP phosphorylation and increases in vitro binding of TTP to ARE. A and B , HeLa-Tet-off cells were transiently transfected with pTRE-TTPfl and equally split into four 6cm dishes. In three dishes, the expression of TTP was allowed overnight
    Figure Legend Snippet: IL-10 reduces TTP phosphorylation and increases in vitro binding of TTP to ARE. A and B , HeLa-Tet-off cells were transiently transfected with pTRE-TTPfl and equally split into four 6cm dishes. In three dishes, the expression of TTP was allowed overnight

    Techniques Used: In Vitro, Binding Assay, Transfection, Expressing

    20) Product Images from "A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †"

    Article Title: A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01260-09

    Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.
    Figure Legend Snippet: Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection

    (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P
    Figure Legend Snippet: (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P

    Techniques Used: Western Blot, Expressing, Mouse Assay, Recombinant, Staining, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P
    Figure Legend Snippet: Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation

    21) Product Images from "LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner"

    Article Title: LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00804-15

    The predicted LARP4 ARE confers instability on β-globin reporter mRNA. HeLa Tet-Off cells were cotransfected with a vector containing GFP under the control of a constitutive CMV promoter, along with β-globin constructs under the control
    Figure Legend Snippet: The predicted LARP4 ARE confers instability on β-globin reporter mRNA. HeLa Tet-Off cells were cotransfected with a vector containing GFP under the control of a constitutive CMV promoter, along with β-globin constructs under the control

    Techniques Used: Plasmid Preparation, Construct

    22) Product Images from "A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †"

    Article Title: A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01260-09

    Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.
    Figure Legend Snippet: Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection

    (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P
    Figure Legend Snippet: (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P

    Techniques Used: Western Blot, Expressing, Mouse Assay, Recombinant, Staining, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P
    Figure Legend Snippet: Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation

    23) Product Images from "A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †"

    Article Title: A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01260-09

    Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.
    Figure Legend Snippet: Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection

    (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P
    Figure Legend Snippet: (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P

    Techniques Used: Western Blot, Expressing, Mouse Assay, Recombinant, Staining, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P
    Figure Legend Snippet: Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation

    24) Product Images from "Y-box protein-associated acidic protein (YBAP1/C1QBP) affects the localization and cytoplasmic functions of YB-1"

    Article Title: Y-box protein-associated acidic protein (YBAP1/C1QBP) affects the localization and cytoplasmic functions of YB-1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24401-3

    YBAP1 abrogates cytoplasmic functions of YB-1. ( a ) An experimental scheme to examine mRNA stability using a d2EGFP reporter mRNA in HeLa Tet-Off cells. ( b ) Cells were transfected with pTRE-d2EGFP-AU, pEGFP2C1, pCMV-YB-1-3xFLAG and pCI-neo-YBAP1. Transcription from the Tet-regulated promoter was induced for 4 h, and shut off by the addition of Tet. The cells were harvested at the indicated time points. d2EGFP mRNA and 2xEGFP mRNA as an internal control were detected by Northern blotting. ( c ) d2EGFP mRNA remaining at various time points after the Tet addition was quantified. Relative half-life of d2EGFP mRNA is shown. * P
    Figure Legend Snippet: YBAP1 abrogates cytoplasmic functions of YB-1. ( a ) An experimental scheme to examine mRNA stability using a d2EGFP reporter mRNA in HeLa Tet-Off cells. ( b ) Cells were transfected with pTRE-d2EGFP-AU, pEGFP2C1, pCMV-YB-1-3xFLAG and pCI-neo-YBAP1. Transcription from the Tet-regulated promoter was induced for 4 h, and shut off by the addition of Tet. The cells were harvested at the indicated time points. d2EGFP mRNA and 2xEGFP mRNA as an internal control were detected by Northern blotting. ( c ) d2EGFP mRNA remaining at various time points after the Tet addition was quantified. Relative half-life of d2EGFP mRNA is shown. * P

    Techniques Used: Transfection, Northern Blot

    25) Product Images from "A Novel Human Ada2 Homologue Functions with Gcn5 or Brg1 To Coactivate Transcription"

    Article Title: A Novel Human Ada2 Homologue Functions with Gcn5 or Brg1 To Coactivate Transcription

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.19.6944-6957.2003

    Examination of Ada2β association with PCAF. (A) Ada2β peptide antibodies. HeLa nuclear extract was fractionated on phosphocellulose p11 column, and bound material was step-eluted with increasing KCl (lanes 1 and 3 are a 0.3 M KCl elution, and lanes 2 and 4 are a 0.5 M KCl elution). The fractions were probed with anti-Ada2β sera either mock treated (left panel) or preincubated with the corresponding peptide (right panel). The position of the Ada2β-specific signal is shown. (B) Test of Ada2β association with PCAF in vivo. Flag-PCAF expression was induced in HeLa Tet-off cells by withdrawal of tetracycline (+). Cells treated with tetracycline served as negative control (−). Flag-agarose was used to immunoprecipitate FLAG-PCAF. Bound material was eluted with excess Flag-peptide. Immunoblotting was done with indicated antibodies. (C) Test of Ada2β interaction with PCAF or Gcn5 in the yeast two-hybrid assay. Yeast were cotransformed with PCAF, Gcn5, or Rho fused to LexA DBD or LexA DBD alone, along with the PCAF interacting region of Ada2β fused to VP16 activation domain. The LacZ reporter contained LexA binding sites. β-Galactosidase activity was measured in units per milligram of protein. The numbers are averages of three independent experiments.
    Figure Legend Snippet: Examination of Ada2β association with PCAF. (A) Ada2β peptide antibodies. HeLa nuclear extract was fractionated on phosphocellulose p11 column, and bound material was step-eluted with increasing KCl (lanes 1 and 3 are a 0.3 M KCl elution, and lanes 2 and 4 are a 0.5 M KCl elution). The fractions were probed with anti-Ada2β sera either mock treated (left panel) or preincubated with the corresponding peptide (right panel). The position of the Ada2β-specific signal is shown. (B) Test of Ada2β association with PCAF in vivo. Flag-PCAF expression was induced in HeLa Tet-off cells by withdrawal of tetracycline (+). Cells treated with tetracycline served as negative control (−). Flag-agarose was used to immunoprecipitate FLAG-PCAF. Bound material was eluted with excess Flag-peptide. Immunoblotting was done with indicated antibodies. (C) Test of Ada2β interaction with PCAF or Gcn5 in the yeast two-hybrid assay. Yeast were cotransformed with PCAF, Gcn5, or Rho fused to LexA DBD or LexA DBD alone, along with the PCAF interacting region of Ada2β fused to VP16 activation domain. The LacZ reporter contained LexA binding sites. β-Galactosidase activity was measured in units per milligram of protein. The numbers are averages of three independent experiments.

    Techniques Used: In Vivo, Expressing, Negative Control, Y2H Assay, Activation Assay, Binding Assay, Activity Assay

    26) Product Images from "CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex"

    Article Title: CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

    Journal: bioRxiv

    doi: 10.1101/811018

    The CASC3 N-terminus promotes but is not necessary to elicit NMD. A : Schematic depiction of the TPI-MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). B : Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). C : Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1-480 has a C-terminal deletion, whereas in the construct 110-480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). D : Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C. Rescue protein expression is confirmed in Figure 6E and F. Individual data points and means are plotted from n=3 experiments. E and F: Western blot of samples shown in Figure 6D. The expression of rescue proteins was confirmed by an antibody against CASC3 (E) and an antibody recognizing the V5 tag (F).
    Figure Legend Snippet: The CASC3 N-terminus promotes but is not necessary to elicit NMD. A : Schematic depiction of the TPI-MS2V5-SMG5 tethering reporter. The reporter consists of the TPI ORF (blue boxes) followed by 4 MS2 stem loops (SL). Downstream the SMG5 3′ untranslated region (UTR) is inserted to increase the size of 3′ fragments that result from cleavage at the termination codon. Reporter and 3′ fragment mRNAs can be detected via the probe binding cassette (gray boxes). B : Northern blot of a tethering assay performed in HeLa Tet-Off cells. The cells stably express the tethering reporter shown in Figure 6A together with the indicated MS2V5-tagged proteins. When the cells are additionally treated with XRN1 siRNA, a 3′ degradation fragment can be detected below the full-length reporter. The reporter and 3′ fragment mRNA levels are normalized to the 7SL RNA. For the calculation of the relative mRNA levels in each condition (Luc vs. XRN1) the levels were normalized to the MS2V5-GST control (lanes 1 and 5). C : Schematic depiction of CASC3 rescue protein constructs. The full-length (FL) protein consists of an N-terminal (blue), C-terminal (orange) and central SELOR domain (purple). The construct 1-480 has a C-terminal deletion, whereas in the construct 110-480 both the N- and C-terminus are truncated. Both deletion constructs were also rendered EJC-binding deficient by mutating the amino acid residues 188 and 218 (F188D, W218D). D : Relative quantification of the CLN6 (top) and TOE1 (bottom) transcript isoforms by qPCR in the indicated cell lines. The V5-tagged rescue proteins expressed in the KO condition are shown schematically in Figure 6C. Rescue protein expression is confirmed in Figure 6E and F. Individual data points and means are plotted from n=3 experiments. E and F: Western blot of samples shown in Figure 6D. The expression of rescue proteins was confirmed by an antibody against CASC3 (E) and an antibody recognizing the V5 tag (F).

    Techniques Used: Binding Assay, Northern Blot, Stable Transfection, Construct, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    27) Product Images from "Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2"

    Article Title: Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.20.7177-7188.2003

    Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.
    Figure Legend Snippet: Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.

    Techniques Used: Over Expression, Inhibition, Transfection, Plasmid Preparation, Northern Blot, Isolation, Labeling

    Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.
    Figure Legend Snippet: Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.

    Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Transfection, Incubation, SDS Page, Autoradiography, Labeling, Staining, Plasmid Preparation, Northern Blot, Quantitation Assay

    The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.
    Figure Legend Snippet: The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.

    Techniques Used: Functional Assay, Construct, Mutagenesis, Northern Blot, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Random Primed

    Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.
    Figure Legend Snippet: Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Isolation, Agarose Gel Electrophoresis, Produced, Northern Blot, Labeling, Quantitation Assay

    28) Product Images from "SGT1-HSP90 complex is required for CENP-A deposition at centromeres"

    Article Title: SGT1-HSP90 complex is required for CENP-A deposition at centromeres

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1325039

    The SGT1-HSP90 complex contributes to CENP-A monoubiquitylation and diubiquitylation in vivo. (A) Representative images of the in vivo ubiquitylation assay with the combination of SGT1 siRNA (#1 + #2), CUL4A siRNA (#1), or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods). HeLa Tet-Off cells were cotransfected with the indicated constructs and siRNAs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (**) Putative di-Ub-CENP-A-Flag, (*) Putative mono-Ub-CENP-A-Flag. (B) Representative images of the in vivo ubiquitylation assay performed as (A) with the combination of HSP90 siRNA (#1) or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods).
    Figure Legend Snippet: The SGT1-HSP90 complex contributes to CENP-A monoubiquitylation and diubiquitylation in vivo. (A) Representative images of the in vivo ubiquitylation assay with the combination of SGT1 siRNA (#1 + #2), CUL4A siRNA (#1), or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods). HeLa Tet-Off cells were cotransfected with the indicated constructs and siRNAs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (**) Putative di-Ub-CENP-A-Flag, (*) Putative mono-Ub-CENP-A-Flag. (B) Representative images of the in vivo ubiquitylation assay performed as (A) with the combination of HSP90 siRNA (#1) or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods).

    Techniques Used: In Vivo, Ubiquitin Assay, Construct, Western Blot

    SGT1-HSP90 complex is required for composition of the CUL4A complex and recognition of CENP-A by COPS8. (A) SGT1A interacts with CUL4A in vivo. Immunoblot analysis of CUL4A immunoprecipitates. Plasmids pTRM4-SGT1A-Flag and pcDNA3-myc3-CUL4A (Table S3) were cotransfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 1% of the total cell lysate (Input) and precipitate (IP) obtained by using anti-c-myc antibody (Table S1) were detected by Western blot analysis using anti-Flag antibody. (B) Interaction between CENP-A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of endogenous CENP-A immunoprecipitates. Indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using rabbit polyclonal anti-CENP-A antibody (Table S1) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (C) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of CUL4A immunoprecipitates. Plasmid pTRM4-CUL4A-Flag (Table S3) and indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using ANTI-FLAG M2 Affinity Gel (SIGMAALDRICH) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (D) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of COPS8 immunoprecipitates. The indicated plasmids (Table S3) and siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using Anti-HA.11 Epitope Tag Affinity Matrix (Covance) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input).
    Figure Legend Snippet: SGT1-HSP90 complex is required for composition of the CUL4A complex and recognition of CENP-A by COPS8. (A) SGT1A interacts with CUL4A in vivo. Immunoblot analysis of CUL4A immunoprecipitates. Plasmids pTRM4-SGT1A-Flag and pcDNA3-myc3-CUL4A (Table S3) were cotransfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 1% of the total cell lysate (Input) and precipitate (IP) obtained by using anti-c-myc antibody (Table S1) were detected by Western blot analysis using anti-Flag antibody. (B) Interaction between CENP-A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of endogenous CENP-A immunoprecipitates. Indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using rabbit polyclonal anti-CENP-A antibody (Table S1) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (C) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of CUL4A immunoprecipitates. Plasmid pTRM4-CUL4A-Flag (Table S3) and indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using ANTI-FLAG M2 Affinity Gel (SIGMAALDRICH) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (D) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of COPS8 immunoprecipitates. The indicated plasmids (Table S3) and siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using Anti-HA.11 Epitope Tag Affinity Matrix (Covance) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input).

    Techniques Used: In Vivo, Transfection, Western Blot, Plasmid Preparation

    29) Product Images from "Y-box protein-associated acidic protein (YBAP1/C1QBP) affects the localization and cytoplasmic functions of YB-1"

    Article Title: Y-box protein-associated acidic protein (YBAP1/C1QBP) affects the localization and cytoplasmic functions of YB-1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24401-3

    YBAP1 abrogates cytoplasmic functions of YB-1. ( a ) An experimental scheme to examine mRNA stability using a d2EGFP reporter mRNA in HeLa Tet-Off cells. ( b ) Cells were transfected with pTRE-d2EGFP-AU, pEGFP2C1, pCMV-YB-1-3xFLAG and pCI-neo-YBAP1. Transcription from the Tet-regulated promoter was induced for 4 h, and shut off by the addition of Tet. The cells were harvested at the indicated time points. d2EGFP mRNA and 2xEGFP mRNA as an internal control were detected by Northern blotting. ( c ) d2EGFP mRNA remaining at various time points after the Tet addition was quantified. Relative half-life of d2EGFP mRNA is shown. * P
    Figure Legend Snippet: YBAP1 abrogates cytoplasmic functions of YB-1. ( a ) An experimental scheme to examine mRNA stability using a d2EGFP reporter mRNA in HeLa Tet-Off cells. ( b ) Cells were transfected with pTRE-d2EGFP-AU, pEGFP2C1, pCMV-YB-1-3xFLAG and pCI-neo-YBAP1. Transcription from the Tet-regulated promoter was induced for 4 h, and shut off by the addition of Tet. The cells were harvested at the indicated time points. d2EGFP mRNA and 2xEGFP mRNA as an internal control were detected by Northern blotting. ( c ) d2EGFP mRNA remaining at various time points after the Tet addition was quantified. Relative half-life of d2EGFP mRNA is shown. * P

    Techniques Used: Transfection, Northern Blot

    30) Product Images from "p16INK4a Translation Suppressed by miR-24"

    Article Title: p16INK4a Translation Suppressed by miR-24

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001864

    Analysis of p16 predicted sites of miR-24 association using a heterologous reporter. Schematic ( left ) and names ( center ) of the EGFP and chimeric EGFP-p16 reporter plasmids tested ( Materials and Methods ). By 48 hr after co-transfection of HeLa Tet-off cells with Ctrl. siRNA or AS-miR-24, along with the plasmids indicated, the levels of EGFP expressed from each reporter vector was assessed; shown are representative Western blotting signals and quantification ( Fold , AS-miR-24 vs. Ctrl. siRNA) of EGFP reporter levels in each transfection group after normalization to α-tubulin signals.
    Figure Legend Snippet: Analysis of p16 predicted sites of miR-24 association using a heterologous reporter. Schematic ( left ) and names ( center ) of the EGFP and chimeric EGFP-p16 reporter plasmids tested ( Materials and Methods ). By 48 hr after co-transfection of HeLa Tet-off cells with Ctrl. siRNA or AS-miR-24, along with the plasmids indicated, the levels of EGFP expressed from each reporter vector was assessed; shown are representative Western blotting signals and quantification ( Fold , AS-miR-24 vs. Ctrl. siRNA) of EGFP reporter levels in each transfection group after normalization to α-tubulin signals.

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

    31) Product Images from "Differential host cell gene expression regulated by the porcine reproductive and respiratory syndrome virus GP4 and GP5 glycoproteins"

    Article Title: Differential host cell gene expression regulated by the porcine reproductive and respiratory syndrome virus GP4 and GP5 glycoproteins

    Journal: Veterinary Immunology and Immunopathology

    doi: 10.1016/j.vetimm.2004.09.020

    Establishment of cells stably expressing the PRRSV GP4 or GP5 protein. Immunofluorescent cell staining was conducted in Marc-GP4 and HeLa-GP5 cells to confirm the expression of GP4 and GP5 proteins. Cells were grown on microscope coverslips, fixed with methanol, and incubated with anti-PRRSV-2 (North American) pig serum followed by FITC-conjugated goat anti-swine antibody. The fluorescence was visualized by a fluorescent microscope at 40× magnification. (A) Marc-145 cells infected with PRRSV-2 at 24 h post-infection; (B) Marc-GP4 cells at 48 h post-seeding; (C) HeLa-GP5 cells at 48 h post-induction in the absence of doxycline; (D) immunoprecipitation of GP4 and GP5 using anti-PRRSV-2 pig serum; lane 1: PRRSV-infected Marc-145 cells; lanes 2 and 3: un-infected Marc-145 cells; lane 4: Marc-GP4 cells; lane 5: HeLa Tet-off cells; lane 6: HeLa-GP5 cells.
    Figure Legend Snippet: Establishment of cells stably expressing the PRRSV GP4 or GP5 protein. Immunofluorescent cell staining was conducted in Marc-GP4 and HeLa-GP5 cells to confirm the expression of GP4 and GP5 proteins. Cells were grown on microscope coverslips, fixed with methanol, and incubated with anti-PRRSV-2 (North American) pig serum followed by FITC-conjugated goat anti-swine antibody. The fluorescence was visualized by a fluorescent microscope at 40× magnification. (A) Marc-145 cells infected with PRRSV-2 at 24 h post-infection; (B) Marc-GP4 cells at 48 h post-seeding; (C) HeLa-GP5 cells at 48 h post-induction in the absence of doxycline; (D) immunoprecipitation of GP4 and GP5 using anti-PRRSV-2 pig serum; lane 1: PRRSV-infected Marc-145 cells; lanes 2 and 3: un-infected Marc-145 cells; lane 4: Marc-GP4 cells; lane 5: HeLa Tet-off cells; lane 6: HeLa-GP5 cells.

    Techniques Used: Stable Transfection, Expressing, Staining, Microscopy, Incubation, Fluorescence, Infection, Immunoprecipitation

    32) Product Images from "Ubiquitination and Proteasomal Degradation of Interferon Regulatory Factor-3 induced by Npro from a Cytopathic Bovine Viral Diarrhea Virus"

    Article Title: Ubiquitination and Proteasomal Degradation of Interferon Regulatory Factor-3 induced by Npro from a Cytopathic Bovine Viral Diarrhea Virus

    Journal: Virology

    doi: 10.1016/j.virol.2007.04.023

    Ectopic expression Npro reduces IRF-3 protein abundance and inhibits the induction of IRF-3 target genes. A. HeLa cell lines with tet-regulated expression of WT Npro (Npro-25 and Npro-29 cells, left panel) or L8P mutant Npro (L8P-1, right panel) were
    Figure Legend Snippet: Ectopic expression Npro reduces IRF-3 protein abundance and inhibits the induction of IRF-3 target genes. A. HeLa cell lines with tet-regulated expression of WT Npro (Npro-25 and Npro-29 cells, left panel) or L8P mutant Npro (L8P-1, right panel) were

    Techniques Used: Expressing, Mutagenesis

    33) Product Images from "A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †"

    Article Title: A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01260-09

    Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.
    Figure Legend Snippet: Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection

    (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P
    Figure Legend Snippet: (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P

    Techniques Used: Western Blot, Expressing, Mouse Assay, Recombinant, Staining, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P
    Figure Legend Snippet: Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation

    34) Product Images from "Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿ †"

    Article Title: Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00624-10

    CUGBP1 is responsible for GU-repeat-mediated mRNA decay. (A) The decay of the GSN-GU beta-globin reporter was measured in cells that expressed a control siRNA (Ctrl-si) or two different siRNAs (siA and siB) directed against CUGBP1. HeLa Tet-Off cells were transfected with the GSN-GU-repeat-containing reporter and the indicated siRNAs. Knockdown efficiency was monitored for each experiment by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. (B) Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at the 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C) The experiment shown in panel B was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are given in minutes.
    Figure Legend Snippet: CUGBP1 is responsible for GU-repeat-mediated mRNA decay. (A) The decay of the GSN-GU beta-globin reporter was measured in cells that expressed a control siRNA (Ctrl-si) or two different siRNAs (siA and siB) directed against CUGBP1. HeLa Tet-Off cells were transfected with the GSN-GU-repeat-containing reporter and the indicated siRNAs. Knockdown efficiency was monitored for each experiment by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. (B) Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at the 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C) The experiment shown in panel B was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are given in minutes.

    Techniques Used: Transfection, Western Blot, Northern Blot

    The GU-repeat sequence mediates rapid decay of the beta-globin reporter transcript. (A) HeLa Tet-Off cells were transfected with the pTetBBB beta-globin reporter construct or with reporter constructs in which GRE-containing sequences from the 3′ UTR of the JUNB transcript (JUNB-GRE) or GU-repeat sequences from the 3′ UTRs of the NDUFS2 (NDUFS2-GU), GSN (GSN-GU), or PPIC (PPIC-GU) were inserted into the beta-globin 3′ UTR. Doxycycline was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (B) The experiment shown in panel A was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.
    Figure Legend Snippet: The GU-repeat sequence mediates rapid decay of the beta-globin reporter transcript. (A) HeLa Tet-Off cells were transfected with the pTetBBB beta-globin reporter construct or with reporter constructs in which GRE-containing sequences from the 3′ UTR of the JUNB transcript (JUNB-GRE) or GU-repeat sequences from the 3′ UTRs of the NDUFS2 (NDUFS2-GU), GSN (GSN-GU), or PPIC (PPIC-GU) were inserted into the beta-globin 3′ UTR. Doxycycline was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (B) The experiment shown in panel A was performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.

    Techniques Used: Sequencing, Transfection, Construct, Northern Blot

    Mutation of the GU-repeat sequence abrogates mRNA decay. (A and B) The decay of the NDUFS2-GU (A) or the GSN-GU (B) beta-globin reporter was compared to the decay of these reporters in which the GU-repeats were deleted (NDUFS2-ΔGU and GSN-ΔGU) or mutated (NDUFS2-mGU and GSN-mGU). HeLa Tet-Off cells were transfected with the indicated beta-globin reporter constructs. Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C and D) The experiments shown in panels A and B were performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.
    Figure Legend Snippet: Mutation of the GU-repeat sequence abrogates mRNA decay. (A and B) The decay of the NDUFS2-GU (A) or the GSN-GU (B) beta-globin reporter was compared to the decay of these reporters in which the GU-repeats were deleted (NDUFS2-ΔGU and GSN-ΔGU) or mutated (NDUFS2-mGU and GSN-mGU). HeLa Tet-Off cells were transfected with the indicated beta-globin reporter constructs. Doxycycline (Dox) was added to the medium to stop transcription from the Tet-responsive promoter, and total cellular RNA was collected at 0-, 2-, 4-, and 6-h time points. Northern blot analyses were performed to monitor the levels of the GAPDH transcripts and the beta-globin reporter transcripts. (C and D) The experiments shown in panels A and B were performed four times, and the Northern blot signals were quantified using a phosphorimager. For each time point, the intensity of the beta-globin band was normalized to the intensity of the GAPDH band, and the band intensity at the zero time point was set to 100%. The percentage of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean from three experiments. Half-lives are indicated in minutes.

    Techniques Used: Mutagenesis, Sequencing, Transfection, Construct, Northern Blot

    Posttranscriptional regulation of the CUGBP1 target network. (A) The network diagram depicts the coordinate regulation of CUGBP1 target transcripts involved in apoptosis. Transcripts depicted in orange represent GRE-containing transcripts that were enriched in the CUGBP1 targets. These pathways were identi fied by Ingenuity Pathway Assistant software (Ingenuity Systems, CA). (B) HeLa Tet-Off cells were transfected twice within 24 h with either a control siRNA or two different siRNAs specific for CUGBP1 (siA and siB). Knockdown efficiency was monitored by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. Apoptosis was assessed with a specific anti-PARP antibody. (C) Cells were stained for FACS analysis with a PE-labeled anti-active caspase 3 antibody. A representative experiment is shown. Four independent experiments were analyzed. Average percentages of apoptosis, standard error, and P values are indicated.
    Figure Legend Snippet: Posttranscriptional regulation of the CUGBP1 target network. (A) The network diagram depicts the coordinate regulation of CUGBP1 target transcripts involved in apoptosis. Transcripts depicted in orange represent GRE-containing transcripts that were enriched in the CUGBP1 targets. These pathways were identi fied by Ingenuity Pathway Assistant software (Ingenuity Systems, CA). (B) HeLa Tet-Off cells were transfected twice within 24 h with either a control siRNA or two different siRNAs specific for CUGBP1 (siA and siB). Knockdown efficiency was monitored by Western blotting with a specific anti-CUGBP1 antibody. A GAPDH antibody was used as the loading control. Apoptosis was assessed with a specific anti-PARP antibody. (C) Cells were stained for FACS analysis with a PE-labeled anti-active caspase 3 antibody. A representative experiment is shown. Four independent experiments were analyzed. Average percentages of apoptosis, standard error, and P values are indicated.

    Techniques Used: Software, Transfection, Western Blot, Staining, FACS, Labeling

    35) Product Images from "ALTERATION IN THE ACTIVATION STATE OF NEW INFLAMMATION-ASSOCIATED TARGETS BY PHOSPHOLIPASE A2-ACTIVATING PROTEIN (PLAA)"

    Article Title: ALTERATION IN THE ACTIVATION STATE OF NEW INFLAMMATION-ASSOCIATED TARGETS BY PHOSPHOLIPASE A2-ACTIVATING PROTEIN (PLAA)

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2008.01.004

    Overexpression of the plaa gene increases COX-2 expression by TNF-α in HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene increases COX-2 expression by TNF-α in HeLa Tet-off cells

    Techniques Used: Over Expression, Expressing

    Overexpression of the plaa gene increases PGE 2 production and PLA 2 activation by TNF-α in HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene increases PGE 2 production and PLA 2 activation by TNF-α in HeLa Tet-off cells

    Techniques Used: Over Expression, Proximity Ligation Assay, Activation Assay

    Overexpression of the plaa gene increases IL-6 production in TNF-α-stimulated HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene increases IL-6 production in TNF-α-stimulated HeLa Tet-off cells

    Techniques Used: Over Expression

    Overexpression of the plaa gene enhances TNF-α-induced NF-κB activation in HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene enhances TNF-α-induced NF-κB activation in HeLa Tet-off cells

    Techniques Used: Over Expression, Activation Assay

    Overexpression of the plaa gene increases cPLA 2 membrane translocation by TNF-α in HeLa Tet-off cells
    Figure Legend Snippet: Overexpression of the plaa gene increases cPLA 2 membrane translocation by TNF-α in HeLa Tet-off cells

    Techniques Used: Over Expression, Translocation Assay

    Overexpression of the plaa gene in HeLa Tet-off cells.
    Figure Legend Snippet: Overexpression of the plaa gene in HeLa Tet-off cells.

    Techniques Used: Over Expression

    36) Product Images from "Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10 1"

    Article Title: Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0803883

    IL-10 reduces TTP phosphorylation and increases in vitro binding of TTP to ARE. A and B , HeLa-Tet-off cells were transiently transfected with pTRE-TTPfl and equally split into four 6cm dishes. In three dishes, the expression of TTP was allowed overnight
    Figure Legend Snippet: IL-10 reduces TTP phosphorylation and increases in vitro binding of TTP to ARE. A and B , HeLa-Tet-off cells were transiently transfected with pTRE-TTPfl and equally split into four 6cm dishes. In three dishes, the expression of TTP was allowed overnight

    Techniques Used: In Vitro, Binding Assay, Transfection, Expressing

    37) Product Images from "A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †"

    Article Title: A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ That Induces Host Cell Toxicity by ADP Ribosylation of Actin ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01260-09

    Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.
    Figure Legend Snippet: Viability of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1 from A. hydrophila ATCC 7966. Percentages of dead and/or dying cells were quantified by incorporation of 7-AAD and flow cytometry of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or producing different fragments of VgrG1 (NH 2 - and COOH-terminal domains and full-length) after 24 h and 72 h of transfection. Means ± standard deviations of the results from three different assays are shown, and statistical significance is indicated. The designation ter refers to NH 2 - or COOH-terminal domains.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection

    (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P
    Figure Legend Snippet: (A) Western blot analysis of HeLa Tet-Off cell lysates expressing and producing different fragments of VgrG1. The various forms of VgrG1 were detected by using two types of sera. We used sera from mice immunized with rVgrG2 of A. hydrophila SSU which cross-reacted with the NH 2 -terminal portion of VgrG1 (left panel) and from mice immunized with the recombinant VIP-2 domain of VgrG1 of A. hydrophila ATCC 7966 which recognized only VgrG1 and its COOH-terminal domain. The samples loaded in the lanes are depicted on the top. α, anti. (B) Morphological changes of HeLa Tet-Off cells induced by the expression of different VgrG1 fragments of A. hydrophila ATCC 7966. Host cells were stained for actin cytoskeleton by using Alexa Fluor 568-phalloidin (red), and the expression of the enhanced fluorescent green protein (EGFP) gene was detected in cells successfully transfected with the pBI-EGFP vector alone (I) or with the vector containing genes encoding NH 2 -terminal (II), full-length (III), or COOH-terminal (IV) fragments of VgrG1. The blue strain (DAPI) shows nuclei. Original magnification, ×400. (C) Quantification of actin cytoskeleton (F-actin) as measured by fluorescent phalloidin staining of HeLa Tet-Off cells expressing and producing different VgrG1 fragments. Flow cytometry dot plots show results for HeLa Tet-Off cells stained with Alexa Fluor 568-phalloidin and expressing different fragments of VgrG1. The analysis was performed on EGFP-positive cells. The percentages of positive cells from representative experiments (72 h) are shown in the plotted quadrants (left), and mean fluorescent intensity values (MFI) from three different assays at 24 h and 72 h were determined (right). Statistical differences at 24 h ( P

    Techniques Used: Western Blot, Expressing, Mouse Assay, Recombinant, Staining, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P
    Figure Legend Snippet: Apoptosis of HeLa Tet-Off cells expressing genes encoding different fragments of VgrG1. Apoptosis rates were measured by quantification of cytoplasmic nucleosomes (A) and levels of caspase 3 and caspase 9 activity (B) in lysates of HeLa Tet-Off cells expressing vector alone (pBI-EGFP) or genes encoding NH 2 - and COOH-terminal fragments and full-length VgrG1 from A. hydrophila ATCC 7966. Means ± standard deviations of the results from three different assays are shown, and statistical significance ( P

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation

    38) Product Images from "Deviant Expression of Rab5 on Phagosomes Containing the Intracellular Pathogens Mycobacterium tuberculosis and Legionella pneumophila Is Associated with Altered Phagosomal Fate"

    Article Title: Deviant Expression of Rab5 on Phagosomes Containing the Intracellular Pathogens Mycobacterium tuberculosis and Legionella pneumophila Is Associated with Altered Phagosomal Fate

    Journal: Infection and Immunity

    doi:

    Phagosomes containing wild-type L. pneumophila but not avirulent L. pneumophila exclude Rab5c. Suspensions of wild-type (A) or avirulent (B and C) L. pneumophila were spun down onto monolayers of pTRE/rab5c-HeLa Tet-off cells at 4°C, incubated at 37°C for 15 min, and either fixed immediately (A and B) or washed, incubated in fresh culture medium for 6 h, and then fixed (C). Cells were processed for cryoimmunoelectron microscopy. Rab5c has been stained using 15-nm gold particles (arrowheads), and L. pneumophila LPS has been stained using 5-nm gold particles (arrows). Rab5c is absent from the wild-type L. pneumophila phagosome (A), despite the presence of Rab5c immunogold staining on vesicles adjacent to the phagosome. Rab5c is present on the avirulent L. pneumophila phagosome at 15 min (B) but is absent by 6 h (C). Magnifications, ×72,827 (A), ×38,235 (B), and ×50,664 (C).
    Figure Legend Snippet: Phagosomes containing wild-type L. pneumophila but not avirulent L. pneumophila exclude Rab5c. Suspensions of wild-type (A) or avirulent (B and C) L. pneumophila were spun down onto monolayers of pTRE/rab5c-HeLa Tet-off cells at 4°C, incubated at 37°C for 15 min, and either fixed immediately (A and B) or washed, incubated in fresh culture medium for 6 h, and then fixed (C). Cells were processed for cryoimmunoelectron microscopy. Rab5c has been stained using 15-nm gold particles (arrowheads), and L. pneumophila LPS has been stained using 5-nm gold particles (arrows). Rab5c is absent from the wild-type L. pneumophila phagosome (A), despite the presence of Rab5c immunogold staining on vesicles adjacent to the phagosome. Rab5c is present on the avirulent L. pneumophila phagosome at 15 min (B) but is absent by 6 h (C). Magnifications, ×72,827 (A), ×38,235 (B), and ×50,664 (C).

    Techniques Used: Incubation, Microscopy, Staining

    Growth of L. pneumophila and M. tuberculosis in THP-1, HeLa Tet-off, and HeLa-Rab5c cells. Monolayers of THP-1 macrophage-like cells, HeLa Tet-off cells, and HeLa-Rab5c cells expressing Rab5c were coincubated with L. pneumophila at a high MOI (2 × 10 8 /ml) or a low MOI (2 × 10 7 /ml) for 1 h (A), with L. pneumophila (2 × 10 8 /ml) for 2 h (C), with M. tuberculosis (10 6 /ml) for 2 h (B), or with M. tuberculosis (10 7 /ml) for 2 h (D) at 37°C, washed, and incubated in fresh medium at 37°C. At sequential times thereafter, the monolayers were lysed and combined with the culture supernatant, and the number of CFU was determined by plating serial dilutions on CYE (A and C) or 7H11 (B and D) agar plates. The capacity of the bacteria to grow extracellularly in the culture medium was assessed by inoculating L. pneumophila (C) or M. tuberculosis (D) into wells containing only the culture medium or into parabiotic chambers in which the bacteria were separated from the HeLa cells by a 0.2-μm-pore-size filter. Although the bacteria are taken up much less efficiently by HeLa cells, they multiply, once inside, with a similar doubling time in HeLa cells and THP-1 cells (A and B). Overexpression of Rab5c does not alter the intracellular growth rate of L. pneumophila or M. tuberculosis in HeLa cells (C and D). The bacteria do not grow in the absence of cell monolayers or when separated from the monolayer in a parabiotic chamber (C and D). Data shown are the means ± the standard deviations of triplicate determinations.
    Figure Legend Snippet: Growth of L. pneumophila and M. tuberculosis in THP-1, HeLa Tet-off, and HeLa-Rab5c cells. Monolayers of THP-1 macrophage-like cells, HeLa Tet-off cells, and HeLa-Rab5c cells expressing Rab5c were coincubated with L. pneumophila at a high MOI (2 × 10 8 /ml) or a low MOI (2 × 10 7 /ml) for 1 h (A), with L. pneumophila (2 × 10 8 /ml) for 2 h (C), with M. tuberculosis (10 6 /ml) for 2 h (B), or with M. tuberculosis (10 7 /ml) for 2 h (D) at 37°C, washed, and incubated in fresh medium at 37°C. At sequential times thereafter, the monolayers were lysed and combined with the culture supernatant, and the number of CFU was determined by plating serial dilutions on CYE (A and C) or 7H11 (B and D) agar plates. The capacity of the bacteria to grow extracellularly in the culture medium was assessed by inoculating L. pneumophila (C) or M. tuberculosis (D) into wells containing only the culture medium or into parabiotic chambers in which the bacteria were separated from the HeLa cells by a 0.2-μm-pore-size filter. Although the bacteria are taken up much less efficiently by HeLa cells, they multiply, once inside, with a similar doubling time in HeLa cells and THP-1 cells (A and B). Overexpression of Rab5c does not alter the intracellular growth rate of L. pneumophila or M. tuberculosis in HeLa cells (C and D). The bacteria do not grow in the absence of cell monolayers or when separated from the monolayer in a parabiotic chamber (C and D). Data shown are the means ± the standard deviations of triplicate determinations.

    Techniques Used: Expressing, Incubation, Over Expression

    39) Product Images from "Mycobacterium tuberculosis and Legionella pneumophila Phagosomes Exhibit Arrested Maturation despite Acquisition of Rab7"

    Article Title: Mycobacterium tuberculosis and Legionella pneumophila Phagosomes Exhibit Arrested Maturation despite Acquisition of Rab7

    Journal: Infection and Immunity

    doi:

    Growth of L. pneumophila and M. tuberculosis in HeLa–Tet-off cells or in Hela-Rab7 or HeLa-Rab7 Q67L cells overexpressing Rab7 or Rab7 Q67L. Monolayers of HeLa–Tet-off, HeLa-Rab7, and HeLa-Rab7 Q67L cells in 2-cm 2 wells were grown for 2 days (A) or 1 day (B) in the absence of tetracycline to induce expression of Rab7 or Rab7 Q67L. The cells were incubated with L. pneumophila (2 × 10 7 /ml [A]) or M. tuberculosis (10 6 /ml [B]) for 2 h at 37°C, were washed, and were incubated in fresh medium at 37°C. At sequential times thereafter, the monolayers were lysed and combined with the culture supernatant, and CFU were determined by plating serial dilutions on CYE (A) and 7H11 (B) agar plates. Data shown represent the means ± the standard deviations of triplicate determinations.
    Figure Legend Snippet: Growth of L. pneumophila and M. tuberculosis in HeLa–Tet-off cells or in Hela-Rab7 or HeLa-Rab7 Q67L cells overexpressing Rab7 or Rab7 Q67L. Monolayers of HeLa–Tet-off, HeLa-Rab7, and HeLa-Rab7 Q67L cells in 2-cm 2 wells were grown for 2 days (A) or 1 day (B) in the absence of tetracycline to induce expression of Rab7 or Rab7 Q67L. The cells were incubated with L. pneumophila (2 × 10 7 /ml [A]) or M. tuberculosis (10 6 /ml [B]) for 2 h at 37°C, were washed, and were incubated in fresh medium at 37°C. At sequential times thereafter, the monolayers were lysed and combined with the culture supernatant, and CFU were determined by plating serial dilutions on CYE (A) and 7H11 (B) agar plates. Data shown represent the means ± the standard deviations of triplicate determinations.

    Techniques Used: Expressing, Incubation

    40) Product Images from "Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2"

    Article Title: Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.20.7177-7188.2003

    Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.
    Figure Legend Snippet: Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.

    Techniques Used: Over Expression, Inhibition, Transfection, Plasmid Preparation, Northern Blot, Isolation, Labeling

    Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.
    Figure Legend Snippet: Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.

    Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Transfection, Incubation, SDS Page, Autoradiography, Labeling, Staining, Plasmid Preparation, Northern Blot, Quantitation Assay

    The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.
    Figure Legend Snippet: The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.

    Techniques Used: Functional Assay, Construct, Mutagenesis, Northern Blot, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Random Primed

    Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.
    Figure Legend Snippet: Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Isolation, Agarose Gel Electrophoresis, Produced, Northern Blot, Labeling, Quantitation Assay

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    TaKaRa tet off advanced gpl cell line
    Characterization of <t>GPL</t> <t>Tet-Off</t> cell lines via luciferase reporter gene transactivation. GPL cells (A to D) or candidate GP Tet-Off cells (E to H) were transfected with a luciferase reporter plasmid under TREtight promoter control (pTREtightLUC). Wells
    Tet Off Advanced Gpl Cell Line, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characterization of GPL Tet-Off cell lines via luciferase reporter gene transactivation. GPL cells (A to D) or candidate GP Tet-Off cells (E to H) were transfected with a luciferase reporter plasmid under TREtight promoter control (pTREtightLUC). Wells

    Journal: Journal of Virology

    Article Title: A Novel Non-Replication-Competent Cytomegalovirus Capsid Mutant Vaccine Strategy Is Effective in Reducing Congenital Infection

    doi: 10.1128/JVI.00283-16

    Figure Lengend Snippet: Characterization of GPL Tet-Off cell lines via luciferase reporter gene transactivation. GPL cells (A to D) or candidate GP Tet-Off cells (E to H) were transfected with a luciferase reporter plasmid under TREtight promoter control (pTREtightLUC). Wells

    Article Snippet: In order to generate a Tet-Off Advanced GPL cell line, GPL cells in 6-well plates were transfected with pTet-Off Advanced plasmid (4 μg/well; Clontech Laboratories), and cells were maintained under neomycin, G418 (Life Technologies), and antibiotic (400 to 600 μg/ml) selection in complete F-12 medium as described above.

    Techniques: Luciferase, Transfection, Plasmid Preparation

    Growth kinetics of DISC GP85 GPCMV versus wild-type GPCMV on GPL Tet-Off cells. GPL Tet-Off cells were infected at a multiplicity of infection of 1 PFU/cell with each respective virus in separate wells of six-well dishes. Samples were taken on different

    Journal: Journal of Virology

    Article Title: A Novel Non-Replication-Competent Cytomegalovirus Capsid Mutant Vaccine Strategy Is Effective in Reducing Congenital Infection

    doi: 10.1128/JVI.00283-16

    Figure Lengend Snippet: Growth kinetics of DISC GP85 GPCMV versus wild-type GPCMV on GPL Tet-Off cells. GPL Tet-Off cells were infected at a multiplicity of infection of 1 PFU/cell with each respective virus in separate wells of six-well dishes. Samples were taken on different

    Article Snippet: In order to generate a Tet-Off Advanced GPL cell line, GPL cells in 6-well plates were transfected with pTet-Off Advanced plasmid (4 μg/well; Clontech Laboratories), and cells were maintained under neomycin, G418 (Life Technologies), and antibiotic (400 to 600 μg/ml) selection in complete F-12 medium as described above.

    Techniques: Infection

    Regeneration of a DISC GP85 GPCMV from GP85 BAC mutants requires a TRE promoter and a cell line expressing a Tet-Off transactivator (tTA2). (A to C) GP85 mutant GPCMV BACs were individually transfected into GPL cells: GP85/GP86 KmR (A), GP85/86 KmR poly(A)

    Journal: Journal of Virology

    Article Title: A Novel Non-Replication-Competent Cytomegalovirus Capsid Mutant Vaccine Strategy Is Effective in Reducing Congenital Infection

    doi: 10.1128/JVI.00283-16

    Figure Lengend Snippet: Regeneration of a DISC GP85 GPCMV from GP85 BAC mutants requires a TRE promoter and a cell line expressing a Tet-Off transactivator (tTA2). (A to C) GP85 mutant GPCMV BACs were individually transfected into GPL cells: GP85/GP86 KmR (A), GP85/86 KmR poly(A)

    Article Snippet: In order to generate a Tet-Off Advanced GPL cell line, GPL cells in 6-well plates were transfected with pTet-Off Advanced plasmid (4 μg/well; Clontech Laboratories), and cells were maintained under neomycin, G418 (Life Technologies), and antibiotic (400 to 600 μg/ml) selection in complete F-12 medium as described above.

    Techniques: BAC Assay, Expressing, Mutagenesis, Transfection

    Generation of brain region-specific inducible mitoPstI mice. ( A ) Dox-regulated induction of mitoPstI in tet-off HeLa cells. Cells were transiently transfected with the mitoPstI construct in the absence (upper panels) or presence (lower panels) of Dox.

    Journal: Human Molecular Genetics

    Article Title: Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons

    doi: 10.1093/hmg/ddn437

    Figure Lengend Snippet: Generation of brain region-specific inducible mitoPstI mice. ( A ) Dox-regulated induction of mitoPstI in tet-off HeLa cells. Cells were transiently transfected with the mitoPstI construct in the absence (upper panels) or presence (lower panels) of Dox.

    Article Snippet: The tet-off HeLa cell line that constitutively expresses tTA was obtained from Clontech.

    Techniques: Mouse Assay, Transfection, Construct

    Comparative transcriptional chase analyses of a short-lived study mRNA. tTA-expressing HeLa cells were engineered to stably express a short-lived derivative human β-globin mRNA (β ARE ). Experiments were conducted as described in Fig. 3 , including the calculation of mRNA t 1/2 values. (A) Aggregate analysis using the conventional method. Cell aliquots were amended with tet at t 0 , and sacrificed at 30-min intervals. Points represent the mean ± S.D. from three replicate experiments. (B) Aggregate analysis using the reverse-chase method. Cell aliquots were amended with tet at 5-min intervals and sacrificed simultaneously after 120 min. Points represent the mean ± S.D. of three independent experiments. (C, D) Analyses of individual replicates. Curves illustrate results from individual replicates that were analyzed in aggregate in panels A and B. Data are presented as described in Fig. 3 . (C) Conventional method. (D) Reverse-chase method.

    Journal: PLoS ONE

    Article Title: A Reverse Time-Course Method for Transcriptional Chase Analyses of mRNA Half-Lives in Cultured Cells

    doi: 10.1371/journal.pone.0040827

    Figure Lengend Snippet: Comparative transcriptional chase analyses of a short-lived study mRNA. tTA-expressing HeLa cells were engineered to stably express a short-lived derivative human β-globin mRNA (β ARE ). Experiments were conducted as described in Fig. 3 , including the calculation of mRNA t 1/2 values. (A) Aggregate analysis using the conventional method. Cell aliquots were amended with tet at t 0 , and sacrificed at 30-min intervals. Points represent the mean ± S.D. from three replicate experiments. (B) Aggregate analysis using the reverse-chase method. Cell aliquots were amended with tet at 5-min intervals and sacrificed simultaneously after 120 min. Points represent the mean ± S.D. of three independent experiments. (C, D) Analyses of individual replicates. Curves illustrate results from individual replicates that were analyzed in aggregate in panels A and B. Data are presented as described in Fig. 3 . (C) Conventional method. (D) Reverse-chase method.

    Article Snippet: Cell Culture HeLa cells expressing the tetracycline trans -activator (tTA) fusion protein (HeLa Tet-Off cell line, Clontech) were maintained in DMEM/F12 media supplemented with 10% fetal bovine serum and antibiotics.

    Techniques: Expressing, Stable Transfection

    NMD is inhibited during doxorubicin treatment. ( a ) mRNA decay assays in MCF7 cells. MCF7 cells either were (red) or were not (black) pre-treated with 5 μM doxorubicin for 1 h before addition of 3 μg/ml actinomycin D to halt transcription. Cells were collected at the indicated times after actinomycin D addition. Levels of the indicated NMD-targeted mRNAs were assessed by RT-qPCR, normalized to 18s rRNA levels, and displayed as a percentage of the levels at t=0 h. Error bars=S.E.M., n=4 independent biological quadruplicates. ( b ) Human β-Gl mRNA half-life studies in HeLa Tet-off cells. HeLa Tet-off cells were transfected with plasmids encoding human β-Gl Norm mRNA and MUP mRNA or β-Gl Ter mRNA and MUP mRNA. β-Gl Norm and β-Gl 39 Ter mRNA transcription occurs under the agency of the non-stress-responsive Tet-off promoter. Cells were either pre-treated with nothing (top), 50 μM doxorubicin for 1 h (middle), or 50 μg/ml puromycin for 3 h (bottom) before transcriptional shut-off with 2 μg/ml doxycycline. Cell aliquots were removed at the indicated “chase” time points, and RT-qPCR was used to assess the remaining levels of β-Gl Norm and β-Gl Ter mRNAs, each after normalization to MUP mRNA. ( c ) Western blots of lysates of MCF7 cells from a (blots derive from and are representative of the three biological replicates in a ) that had been exposed to doxorubicin (5 μM) for the indicated times. CP, cleavage product. GAPDH levels serve as loading controls. Three-fold serial dilutions (wedge) reveal the dynamic range of analysis. ( d ) As in c , but cells were exposed to a 10-fold higher concentration of doxorubicin and analyzed at earlier time points. Representative of 2 biological replicates.

    Journal: Nature communications

    Article Title: Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics

    doi: 10.1038/ncomms7632

    Figure Lengend Snippet: NMD is inhibited during doxorubicin treatment. ( a ) mRNA decay assays in MCF7 cells. MCF7 cells either were (red) or were not (black) pre-treated with 5 μM doxorubicin for 1 h before addition of 3 μg/ml actinomycin D to halt transcription. Cells were collected at the indicated times after actinomycin D addition. Levels of the indicated NMD-targeted mRNAs were assessed by RT-qPCR, normalized to 18s rRNA levels, and displayed as a percentage of the levels at t=0 h. Error bars=S.E.M., n=4 independent biological quadruplicates. ( b ) Human β-Gl mRNA half-life studies in HeLa Tet-off cells. HeLa Tet-off cells were transfected with plasmids encoding human β-Gl Norm mRNA and MUP mRNA or β-Gl Ter mRNA and MUP mRNA. β-Gl Norm and β-Gl 39 Ter mRNA transcription occurs under the agency of the non-stress-responsive Tet-off promoter. Cells were either pre-treated with nothing (top), 50 μM doxorubicin for 1 h (middle), or 50 μg/ml puromycin for 3 h (bottom) before transcriptional shut-off with 2 μg/ml doxycycline. Cell aliquots were removed at the indicated “chase” time points, and RT-qPCR was used to assess the remaining levels of β-Gl Norm and β-Gl Ter mRNAs, each after normalization to MUP mRNA. ( c ) Western blots of lysates of MCF7 cells from a (blots derive from and are representative of the three biological replicates in a ) that had been exposed to doxorubicin (5 μM) for the indicated times. CP, cleavage product. GAPDH levels serve as loading controls. Three-fold serial dilutions (wedge) reveal the dynamic range of analysis. ( d ) As in c , but cells were exposed to a 10-fold higher concentration of doxorubicin and analyzed at earlier time points. Representative of 2 biological replicates.

    Article Snippet: For Tet-off assays, HeLa Tet-off cells (Clontech) were plated at 40,000 cells/well in 24-well dishes.

    Techniques: Quantitative RT-PCR, Transfection, Western Blot, Concentration Assay