hela tet on cells  (TaKaRa)


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    TaKaRa hela tet on cells
    <t>HeLa</t> <t>Tet-on</t> cells expressing H-2K b were nucleofected with 4×100 nM siRNA. Twenty-four hours later, the cells were electroporated with inducible plasmids encoding amyloid beta preceded by a signal peptide (+SP) or not (−SP), or encoding proinsulin, all tagged with the S8L peptide at the C-terminus. Protein expression was induced immediately by addition of 1 µg/ml doxycylin. Forty-eight hours later, K b /S8L complexes on the cell surface were detected using mAb 25D1.16 (Ab1) followed by FITC-labeled goat anti-mouse Abs (Ab2) and Alexa488-labeled goat anti-FITC Ab (Ab3). Control samples were HeLa-K b cells pulsed for 2 h with 10 −8 M S8L and stained with Abs 1, 2 and 3 or with Abs 2 and 3 only, as well as peptide-pulsed HeLa cells expressing H-2K d stained with Abs 1, 2 and 3. One of two experiments is shown.
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    Images

    1) Product Images from "No Major Role for Insulin-Degrading Enzyme in Antigen Presentation by MHC Molecules"

    Article Title: No Major Role for Insulin-Degrading Enzyme in Antigen Presentation by MHC Molecules

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088365

    HeLa Tet-on cells expressing H-2K b were nucleofected with 4×100 nM siRNA. Twenty-four hours later, the cells were electroporated with inducible plasmids encoding amyloid beta preceded by a signal peptide (+SP) or not (−SP), or encoding proinsulin, all tagged with the S8L peptide at the C-terminus. Protein expression was induced immediately by addition of 1 µg/ml doxycylin. Forty-eight hours later, K b /S8L complexes on the cell surface were detected using mAb 25D1.16 (Ab1) followed by FITC-labeled goat anti-mouse Abs (Ab2) and Alexa488-labeled goat anti-FITC Ab (Ab3). Control samples were HeLa-K b cells pulsed for 2 h with 10 −8 M S8L and stained with Abs 1, 2 and 3 or with Abs 2 and 3 only, as well as peptide-pulsed HeLa cells expressing H-2K d stained with Abs 1, 2 and 3. One of two experiments is shown.
    Figure Legend Snippet: HeLa Tet-on cells expressing H-2K b were nucleofected with 4×100 nM siRNA. Twenty-four hours later, the cells were electroporated with inducible plasmids encoding amyloid beta preceded by a signal peptide (+SP) or not (−SP), or encoding proinsulin, all tagged with the S8L peptide at the C-terminus. Protein expression was induced immediately by addition of 1 µg/ml doxycylin. Forty-eight hours later, K b /S8L complexes on the cell surface were detected using mAb 25D1.16 (Ab1) followed by FITC-labeled goat anti-mouse Abs (Ab2) and Alexa488-labeled goat anti-FITC Ab (Ab3). Control samples were HeLa-K b cells pulsed for 2 h with 10 −8 M S8L and stained with Abs 1, 2 and 3 or with Abs 2 and 3 only, as well as peptide-pulsed HeLa cells expressing H-2K d stained with Abs 1, 2 and 3. One of two experiments is shown.

    Techniques Used: Expressing, Labeling, Staining

    2) Product Images from "Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium"

    Article Title: Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI39740

    Forward trafficking of Cx43 to the plasma membrane is reduced during oxidative stress. ( A ) Timeline of Tet-inducible Cx43-EYFP trafficking assay. A tetracycline-inducible clonal HeLa cell line expressing Cx43-EYFP was induced with 2 μg/ml doxycycline 2.5 hours prior to imaging with TIRFm in the presence or absence of 200 μM H 2 O 2 , which was added 30 minutes prior to imaging. ( B ) TIRFm visualization of Cx43-EYFP delivery. TIRFm detection of Cx43-EYFP at 150, 195, and 240 minutes after the addition of doxycycline in the presence or absence of H 2 O 2 . White lines outline cell cortex. ( C ) Comparison of widefield epifluorescence and TIRFm detection of Cx43-EYFP at 240 minutes after the addition of doxycycline, showing that cells contain comparable levels of Cx43-EYFP. ( D ) Quantification of TIRFm-detectable Cx43-EYFP surface intensity. Error bars represent SEM. ( E ) Total and surface N-cadherin and Cx43-EYFP levels following total surface protein biotinylation and pulldown through neutravidin. Input lysates and pulldowns of biotinylated surface proteins were subjected to SDS-PAGE on the same gel. Original magnification, ×100. Scale bars: 10 μm. Values represent mean ± SEM.
    Figure Legend Snippet: Forward trafficking of Cx43 to the plasma membrane is reduced during oxidative stress. ( A ) Timeline of Tet-inducible Cx43-EYFP trafficking assay. A tetracycline-inducible clonal HeLa cell line expressing Cx43-EYFP was induced with 2 μg/ml doxycycline 2.5 hours prior to imaging with TIRFm in the presence or absence of 200 μM H 2 O 2 , which was added 30 minutes prior to imaging. ( B ) TIRFm visualization of Cx43-EYFP delivery. TIRFm detection of Cx43-EYFP at 150, 195, and 240 minutes after the addition of doxycycline in the presence or absence of H 2 O 2 . White lines outline cell cortex. ( C ) Comparison of widefield epifluorescence and TIRFm detection of Cx43-EYFP at 240 minutes after the addition of doxycycline, showing that cells contain comparable levels of Cx43-EYFP. ( D ) Quantification of TIRFm-detectable Cx43-EYFP surface intensity. Error bars represent SEM. ( E ) Total and surface N-cadherin and Cx43-EYFP levels following total surface protein biotinylation and pulldown through neutravidin. Input lysates and pulldowns of biotinylated surface proteins were subjected to SDS-PAGE on the same gel. Original magnification, ×100. Scale bars: 10 μm. Values represent mean ± SEM.

    Techniques Used: Expressing, Imaging, SDS Page

    3) Product Images from "Rapid purification of protein complexes from mammalian cells"

    Article Title: Rapid purification of protein complexes from mammalian cells

    Journal: Nucleic Acids Research

    doi:

    Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.

    Techniques Used:

    Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.
    Figure Legend Snippet: Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page, Western Blot, Staining

    Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.
    Figure Legend Snippet: Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.

    Techniques Used: SDS Page, Staining, Western Blot

    Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.
    Figure Legend Snippet: Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.

    Techniques Used: Purification, Incubation, Staining, Western Blot

    Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.

    Techniques Used: Transfection, Fluorescence, Expressing, Translocation Assay

    Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).
    Figure Legend Snippet: Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page

    4) Product Images from "Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2Shugoshin Prevents Dissociation of Cohesin from Centromeres During Mitosis in Vertebrate CellsChromosome Cohesion: A Cycle of Holding Together and Falling ApartSeparating Sisters: Shugoshin Protects SA2 at Centromeres but Not at Chromosome Arms"

    Article Title: Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2Shugoshin Prevents Dissociation of Cohesin from Centromeres During Mitosis in Vertebrate CellsChromosome Cohesion: A Cycle of Holding Together and Falling ApartSeparating Sisters: Shugoshin Protects SA2 at Centromeres but Not at Chromosome Arms

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0030069

    Characterization of HeLa Cell Lines Stably Expressing Wild-Type or Mutant Forms of Human Scc1 and SA2 (A) Wild-type Scc1 or SA2, or the indicated mutant proteins (see Figure 1 C), all tagged with 9xmyc at the C terminus, were stably and inducibly expressed in HeLa tet-on cells. After induction by treatment with 2 μg/ml doxycycline for 1–3 d, cell extracts were prepared from either logarithmically proliferating cells (i, interphase) or from cells arrested in mitosis by nocodazole (m, mitosis), then immunoblotted. In the case of Scc1 cell lines (upper blots), only data from interphase extracts are shown. Exogenous protein was detected by immunoblotting with myc antibodies (lower blots). Since the 9xmyc-tag caused a reduced mobility in SDS-PAGE compared to the endogenous protein, Scc1- and SA2-immunoblots (upper blots) revealed the relative amounts of exogenous and endogenous protein in the different cell lines. The position of molecular weight markers is indicated on the right side. (B) Extracts were prepared from the different cell lines as indicated. Immunoprecipitation was performed using myc antibodies, followed by SDS-PAGE and silver staining. As a control, the cohesin complex was immunoprecipitated from untransfected HeLa tet-on cells using antibodies to SA2. (C) Extracts were prepared from SA2-WT-myc or SA2–12xA-myc expressing cells, and fractionated by sucrose density gradient centrifugation (5%–30% sucrose), followed by immunoblotting with antibodies recognizing the proteins indicated on the right (inp. = input/unfractionated sample of the extract).
    Figure Legend Snippet: Characterization of HeLa Cell Lines Stably Expressing Wild-Type or Mutant Forms of Human Scc1 and SA2 (A) Wild-type Scc1 or SA2, or the indicated mutant proteins (see Figure 1 C), all tagged with 9xmyc at the C terminus, were stably and inducibly expressed in HeLa tet-on cells. After induction by treatment with 2 μg/ml doxycycline for 1–3 d, cell extracts were prepared from either logarithmically proliferating cells (i, interphase) or from cells arrested in mitosis by nocodazole (m, mitosis), then immunoblotted. In the case of Scc1 cell lines (upper blots), only data from interphase extracts are shown. Exogenous protein was detected by immunoblotting with myc antibodies (lower blots). Since the 9xmyc-tag caused a reduced mobility in SDS-PAGE compared to the endogenous protein, Scc1- and SA2-immunoblots (upper blots) revealed the relative amounts of exogenous and endogenous protein in the different cell lines. The position of molecular weight markers is indicated on the right side. (B) Extracts were prepared from the different cell lines as indicated. Immunoprecipitation was performed using myc antibodies, followed by SDS-PAGE and silver staining. As a control, the cohesin complex was immunoprecipitated from untransfected HeLa tet-on cells using antibodies to SA2. (C) Extracts were prepared from SA2-WT-myc or SA2–12xA-myc expressing cells, and fractionated by sucrose density gradient centrifugation (5%–30% sucrose), followed by immunoblotting with antibodies recognizing the proteins indicated on the right (inp. = input/unfractionated sample of the extract).

    Techniques Used: Stable Transfection, Expressing, Mutagenesis, SDS Page, Western Blot, Molecular Weight, Immunoprecipitation, Silver Staining, Gradient Centrifugation

    5) Product Images from "Lysine-Independent Turnover of Cyclin G1 Can Be Stabilized by B?? Subunits of Protein Phosphatase 2A ▿"

    Article Title: Lysine-Independent Turnover of Cyclin G1 Can Be Stabilized by B?? Subunits of Protein Phosphatase 2A ▿

    Journal:

    doi: 10.1128/MCB.00907-08

    B′α family subunits of PP2A stabilize human cyclin G1 protein. (A) HeLa Tet-on cells (clone 2) that were untreated or treated with doxycycline (Dox; 2 ng/ml) were transfected with empty pcDNA3 vector (Pc) or a vector expressing the Flag-tagged
    Figure Legend Snippet: B′α family subunits of PP2A stabilize human cyclin G1 protein. (A) HeLa Tet-on cells (clone 2) that were untreated or treated with doxycycline (Dox; 2 ng/ml) were transfected with empty pcDNA3 vector (Pc) or a vector expressing the Flag-tagged

    Techniques Used: Transfection, Plasmid Preparation, Expressing

    The B′α1 subunit of PP2A stabilizes human cyclin G1 protein under DNA damaging conditions. (A) HeLa Tet-on cells (clone 2) expressing Flag-tagged human cyclin G1 were plated with or without 2 ng/ml of doxycycline (Dox) for 48 h. The cells
    Figure Legend Snippet: The B′α1 subunit of PP2A stabilizes human cyclin G1 protein under DNA damaging conditions. (A) HeLa Tet-on cells (clone 2) expressing Flag-tagged human cyclin G1 were plated with or without 2 ng/ml of doxycycline (Dox) for 48 h. The cells

    Techniques Used: Expressing

    6) Product Images from "A novel clathrin homolog that co-distributes with cytoskeletal components functions in the trans-Golgi network"

    Article Title: A novel clathrin homolog that co-distributes with cytoskeletal components functions in the trans-Golgi network

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.1.272

    Fig. 4. Localization of CHC22 by immunoelectron microscopy. HeLa-tet/on cells transfected with pJM601CHC22 were induced for high level CHC22 expression for 24 h, then permeabilized, lightly fixed and labeled with antibodies to CHC22 (5 nm gold, small arrows), components of AP1 (10 nm gold, arrowheads) and clathrin light chains (15 nm gold, large arrows). Prior to embedding and sectioning, each antibody was sequentially applied and detected with protein A–gold attached to gold particles of the size indicated, followed by blocking with excess protein A. The images were selected to be representative of the intracellular distribution of CHC22 (statistics are provided in the text), showing vesicles of 80–100 nm in the TGN labeled for CHC22 and AP1 (γ subunit) ( A ) or labeled for CHC22 and AP1 (σ1 subunit) with notable protein coats ( B ). ( C ) Similar vesicles, with one labeled exclusively for CHC22 and the other for CHC22, clathrin light chain and AP1. ( D ) Peripheral vesicles, primarily labeled for clathrin light chain, with one co-labeled for CHC22. The labeling for AP1 (σ1 subunit) at the left suggests that these coated vesicles are near endosomes.
    Figure Legend Snippet: Fig. 4. Localization of CHC22 by immunoelectron microscopy. HeLa-tet/on cells transfected with pJM601CHC22 were induced for high level CHC22 expression for 24 h, then permeabilized, lightly fixed and labeled with antibodies to CHC22 (5 nm gold, small arrows), components of AP1 (10 nm gold, arrowheads) and clathrin light chains (15 nm gold, large arrows). Prior to embedding and sectioning, each antibody was sequentially applied and detected with protein A–gold attached to gold particles of the size indicated, followed by blocking with excess protein A. The images were selected to be representative of the intracellular distribution of CHC22 (statistics are provided in the text), showing vesicles of 80–100 nm in the TGN labeled for CHC22 and AP1 (γ subunit) ( A ) or labeled for CHC22 and AP1 (σ1 subunit) with notable protein coats ( B ). ( C ) Similar vesicles, with one labeled exclusively for CHC22 and the other for CHC22, clathrin light chain and AP1. ( D ) Peripheral vesicles, primarily labeled for clathrin light chain, with one co-labeled for CHC22. The labeling for AP1 (σ1 subunit) at the left suggests that these coated vesicles are near endosomes.

    Techniques Used: Immuno-Electron Microscopy, Transfection, Expressing, Labeling, Blocking Assay

    Fig. 2. Differential association of CHC22 with clathrin coat components. ( A ) Bacterial lysate containing recombinant CHC22Hub and co-expressed bovine neuronal LCa was separated by Superose 6 size exclusion chromatography. The column fractions were collected and resolved in sequence (left to right) by SDS–PAGE. The presence of CHC22Hub polypeptides or LCa was established by immunoblotting using rabbit serum against CHC22 (CHC22Hub) or MAb CON.1, which recognizes a determinant shared by both light chains (LCa and LCb). Arrows on the top indicate the fractions flanking the elution positions of molecular weight standard catalase (232 kDa). ( B ) Bacterial lysate containing recombinant CHC22Hub and co-expressed bovine neuronal LCb was separated by Superose 6 size exclusion chromatography and analyzed for the elution position of CHC22Hub polypeptides and LCb, as in (A). ( C ). The immunoprecipitates were then subjected to SDS–PAGE and probed with the following antibodies: anti-CHC22 polyclonal antiserum [CHC22 (PAb)], CHC17-specific monoclonal antibody TD.1, anti-clathrin light chain antiserum (CLC), anti-AP1 γ subunit monoclonal antibody 100/3 [AP1(γ)], anti-AP2 α subunit AC1M11 [AP2(α)] and anti-AP3 β3 antiserum [AP3(β)]. The CHC22 signals in CHC17 immunoprecipitates are due to cross-reactivity of X22 with CHC22, causing co-precipitation of CHC22 with CHC17 and possibly explaining the apparent association of AP3 with CHC17. ( D ) HeLa-tet/on cells were permanently transfected with T7-epitope-tagged full-length CHC22, under the tet operator (pJM601CHC22), and CHC22 expression was induced for 24 h with doxycycline. CHC22 full-length protein was then immunoprecipated using anti-T7 MAb and the sample was analyzed by SDS–PAGE and immunoblotting for CHC22 using a specific polyclonal antiserum, or for clathrin light chains (CLC) using the α-cons polyclonal antibody. Conventional clathrin (CHC17) was immunoprecipitated from the same sample using MAb X22 and similarly analyzed. Note that X22 immunoprecipitates some CHC22 along with CHC17 due to cross-reactivity (see C).
    Figure Legend Snippet: Fig. 2. Differential association of CHC22 with clathrin coat components. ( A ) Bacterial lysate containing recombinant CHC22Hub and co-expressed bovine neuronal LCa was separated by Superose 6 size exclusion chromatography. The column fractions were collected and resolved in sequence (left to right) by SDS–PAGE. The presence of CHC22Hub polypeptides or LCa was established by immunoblotting using rabbit serum against CHC22 (CHC22Hub) or MAb CON.1, which recognizes a determinant shared by both light chains (LCa and LCb). Arrows on the top indicate the fractions flanking the elution positions of molecular weight standard catalase (232 kDa). ( B ) Bacterial lysate containing recombinant CHC22Hub and co-expressed bovine neuronal LCb was separated by Superose 6 size exclusion chromatography and analyzed for the elution position of CHC22Hub polypeptides and LCb, as in (A). ( C ). The immunoprecipitates were then subjected to SDS–PAGE and probed with the following antibodies: anti-CHC22 polyclonal antiserum [CHC22 (PAb)], CHC17-specific monoclonal antibody TD.1, anti-clathrin light chain antiserum (CLC), anti-AP1 γ subunit monoclonal antibody 100/3 [AP1(γ)], anti-AP2 α subunit AC1M11 [AP2(α)] and anti-AP3 β3 antiserum [AP3(β)]. The CHC22 signals in CHC17 immunoprecipitates are due to cross-reactivity of X22 with CHC22, causing co-precipitation of CHC22 with CHC17 and possibly explaining the apparent association of AP3 with CHC17. ( D ) HeLa-tet/on cells were permanently transfected with T7-epitope-tagged full-length CHC22, under the tet operator (pJM601CHC22), and CHC22 expression was induced for 24 h with doxycycline. CHC22 full-length protein was then immunoprecipated using anti-T7 MAb and the sample was analyzed by SDS–PAGE and immunoblotting for CHC22 using a specific polyclonal antiserum, or for clathrin light chains (CLC) using the α-cons polyclonal antibody. Conventional clathrin (CHC17) was immunoprecipitated from the same sample using MAb X22 and similarly analyzed. Note that X22 immunoprecipitates some CHC22 along with CHC17 due to cross-reactivity (see C).

    Techniques Used: Recombinant, Size-exclusion Chromatography, Sequencing, SDS Page, Molecular Weight, Transfection, Expressing, Immunoprecipitation

    Fig. 7. Expression of CHC22 hub domain affects intracellular distribution of M6PR. HeLa-tet/on cells transfected with pJM601CHC22Hub ( A – F ) or with pJM601CHC22 ( G – J ) were induced for high level CHC22Hub expression or high level expression of full-length (FL) CHC22 for 24 h. Cells were stained for the expression of CHC22Hub (shown in A, C and E) or CHC22FL (shown in G and I) with anti-CHC22 MAb followed by LRSC-conjugated goat anti-mouse IgG. The distribution of endogenous M6PR (shown in B, D and H) was detected by double staining (A), (C) and (G) with antiserum to the cation-independent M6PR, and the distribution of endogenous clathrin (shown in F and J) was detected by double staining (E) and (I) with anti-clathrin LC antiserum α-cons, both followed by FITC-conjugated goat anti-rabbit IgG. The horizontal rows represent the same images viewed with different filters to see red and green staining on the left and right, respectively. Note that not all cells in the transfected cultures express CHC22Hub or CHC22FL, which was confirmed by staining with anti-T7 MAb, which reacts with the epitope tag on the transfected proteins (not shown). The non-expressing cells serve as negative controls for endogenous staining of M6PR or clathrin LC, and their staining patterns are identical to those of control cells that were either not transfected or not induced for expression of the transfected molecules.
    Figure Legend Snippet: Fig. 7. Expression of CHC22 hub domain affects intracellular distribution of M6PR. HeLa-tet/on cells transfected with pJM601CHC22Hub ( A – F ) or with pJM601CHC22 ( G – J ) were induced for high level CHC22Hub expression or high level expression of full-length (FL) CHC22 for 24 h. Cells were stained for the expression of CHC22Hub (shown in A, C and E) or CHC22FL (shown in G and I) with anti-CHC22 MAb followed by LRSC-conjugated goat anti-mouse IgG. The distribution of endogenous M6PR (shown in B, D and H) was detected by double staining (A), (C) and (G) with antiserum to the cation-independent M6PR, and the distribution of endogenous clathrin (shown in F and J) was detected by double staining (E) and (I) with anti-clathrin LC antiserum α-cons, both followed by FITC-conjugated goat anti-rabbit IgG. The horizontal rows represent the same images viewed with different filters to see red and green staining on the left and right, respectively. Note that not all cells in the transfected cultures express CHC22Hub or CHC22FL, which was confirmed by staining with anti-T7 MAb, which reacts with the epitope tag on the transfected proteins (not shown). The non-expressing cells serve as negative controls for endogenous staining of M6PR or clathrin LC, and their staining patterns are identical to those of control cells that were either not transfected or not induced for expression of the transfected molecules.

    Techniques Used: Expressing, Transfection, Staining, Double Staining

    7) Product Images from "Functional Implications in Apoptosis by Interferon Inducible Gene Product 1-8D, the Binding Protein to Adenovirus Preterminal Protein"

    Article Title: Functional Implications in Apoptosis by Interferon Inducible Gene Product 1-8D, the Binding Protein to Adenovirus Preterminal Protein

    Journal: Journal of microbiology (Seoul, Korea)

    doi:

    Inid induces apoptosis in uninfected cells. HeLa Tet-on cells transfected with pTRE-Inid were either infected with Ad2 (Ad2+) or remained uninfected (Ad2-). In Doxycyclin untreated cells (Dox-) minimal DNA fragmentation was detected, whereas, cells treated with Doxycyclin (Dox+) clearly showed apoptosis as determined by the smearing or fast moving fragmented DNA (A). The expressions of Inid and pTP were confirmed in the Western Blot analysis. Each protein was indicated by arrows (B).
    Figure Legend Snippet: Inid induces apoptosis in uninfected cells. HeLa Tet-on cells transfected with pTRE-Inid were either infected with Ad2 (Ad2+) or remained uninfected (Ad2-). In Doxycyclin untreated cells (Dox-) minimal DNA fragmentation was detected, whereas, cells treated with Doxycyclin (Dox+) clearly showed apoptosis as determined by the smearing or fast moving fragmented DNA (A). The expressions of Inid and pTP were confirmed in the Western Blot analysis. Each protein was indicated by arrows (B).

    Techniques Used: Transfection, Infection, Western Blot

    Inid induces apoptosis in Ad infected cells. HeLa Tet-on cells transfected with pTRE-Inid were infected with Ad2 for 20 h and the expression of Inid was induced with doxycyclin. The cells were fixed and were either probed with anti-Flag monoclonal antibody followed by FITC-conjugated goat anti-mouse antibody to detect the presence of Inid or anti-pTP polyclonal antibody followed by Texas-Red conjugated goat anti-rabbit antibody. Ad infected cells expressing pTP (A) and Inid (B) had a “rounded” morphology which was evident by phase contrast microscopy (C). Alternatively, the cells were tested for apoptosis using the TdT assay which employed the addition of FITC-dUTP, and in which case, Inid was detected with a Texas-Red conjugated goat anti-mouse secondary antibody. Infected cells transfected with pTRE-Inid in the absence of doxycyclin were assayed for the presence of (pTP; D) and for apoptosis by the TdT assay (F). Doxycyclin untreated cells showed little evidence of apoptosis (F). Cells treated with doxycyclin showed that the “rounded” cells stained strongly for Inid (E). These cells were also strongly positive for apoptosis (TdT assay; G). Magnification was 40X for A-C and 10X for D-G.
    Figure Legend Snippet: Inid induces apoptosis in Ad infected cells. HeLa Tet-on cells transfected with pTRE-Inid were infected with Ad2 for 20 h and the expression of Inid was induced with doxycyclin. The cells were fixed and were either probed with anti-Flag monoclonal antibody followed by FITC-conjugated goat anti-mouse antibody to detect the presence of Inid or anti-pTP polyclonal antibody followed by Texas-Red conjugated goat anti-rabbit antibody. Ad infected cells expressing pTP (A) and Inid (B) had a “rounded” morphology which was evident by phase contrast microscopy (C). Alternatively, the cells were tested for apoptosis using the TdT assay which employed the addition of FITC-dUTP, and in which case, Inid was detected with a Texas-Red conjugated goat anti-mouse secondary antibody. Infected cells transfected with pTRE-Inid in the absence of doxycyclin were assayed for the presence of (pTP; D) and for apoptosis by the TdT assay (F). Doxycyclin untreated cells showed little evidence of apoptosis (F). Cells treated with doxycyclin showed that the “rounded” cells stained strongly for Inid (E). These cells were also strongly positive for apoptosis (TdT assay; G). Magnification was 40X for A-C and 10X for D-G.

    Techniques Used: Infection, Transfection, Expressing, Microscopy, Staining

    8) Product Images from "Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein"

    Article Title: Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein

    Journal: Nucleic Acids Research

    doi:

    Tetracycline inducible expression of TBP-DR2 in HeLa cells. ( A ) Western blot analysis. Cellular extracts from uninduced (lane 1) and doxycycline-induced cells (lane 2) of one of the selected clones were probed with anti-histidine antibodies. Recombinant TBP-DR2 with a histidine tag (10 ng; lane 3), 25 µg HeLa whole-cell extract (WCE; lane 4) and 10 ng recombinant TBPwt without histidine tag (lane 5) served as controls. ( B ) Functional investigation of TBP-DR2 expressed in HeLa cells. To analyse whether the expressed TBP-DR2 was functionally active, 50 µg S100 from HeLa cells expressing TBP-DR2 was used to transcribe the P DR2 promoter (lanes 4–6) in vitro before (lane 5) and after (lane 6) doxycycline induction of these HeLa cells. Additionally, 50 µg S100 from untransfected HeLa Tet-on cells was used as a negative control (lane 4). As a control, all three extracts were simultaneously analysed by in vitro transcription of pUhU6 0.35 (lanes 1–3) and pUVAI DNA (lanes 7–9).
    Figure Legend Snippet: Tetracycline inducible expression of TBP-DR2 in HeLa cells. ( A ) Western blot analysis. Cellular extracts from uninduced (lane 1) and doxycycline-induced cells (lane 2) of one of the selected clones were probed with anti-histidine antibodies. Recombinant TBP-DR2 with a histidine tag (10 ng; lane 3), 25 µg HeLa whole-cell extract (WCE; lane 4) and 10 ng recombinant TBPwt without histidine tag (lane 5) served as controls. ( B ) Functional investigation of TBP-DR2 expressed in HeLa cells. To analyse whether the expressed TBP-DR2 was functionally active, 50 µg S100 from HeLa cells expressing TBP-DR2 was used to transcribe the P DR2 promoter (lanes 4–6) in vitro before (lane 5) and after (lane 6) doxycycline induction of these HeLa cells. Additionally, 50 µg S100 from untransfected HeLa Tet-on cells was used as a negative control (lane 4). As a control, all three extracts were simultaneously analysed by in vitro transcription of pUhU6 0.35 (lanes 1–3) and pUVAI DNA (lanes 7–9).

    Techniques Used: Expressing, Western Blot, Clone Assay, Recombinant, Functional Assay, In Vitro, Negative Control

    9) Product Images from "Lysine-Independent Turnover of Cyclin G1 Can Be Stabilized by B?? Subunits of Protein Phosphatase 2A ▿"

    Article Title: Lysine-Independent Turnover of Cyclin G1 Can Be Stabilized by B?? Subunits of Protein Phosphatase 2A ▿

    Journal:

    doi: 10.1128/MCB.00907-08

    B′α family subunits of PP2A stabilize human cyclin G1 protein. (A) HeLa Tet-on cells (clone 2) that were untreated or treated with doxycycline (Dox; 2 ng/ml) were transfected with empty pcDNA3 vector (Pc) or a vector expressing the Flag-tagged
    Figure Legend Snippet: B′α family subunits of PP2A stabilize human cyclin G1 protein. (A) HeLa Tet-on cells (clone 2) that were untreated or treated with doxycycline (Dox; 2 ng/ml) were transfected with empty pcDNA3 vector (Pc) or a vector expressing the Flag-tagged

    Techniques Used: Transfection, Plasmid Preparation, Expressing

    The B′α1 subunit of PP2A stabilizes human cyclin G1 protein under DNA damaging conditions. (A) HeLa Tet-on cells (clone 2) expressing Flag-tagged human cyclin G1 were plated with or without 2 ng/ml of doxycycline (Dox) for 48 h. The cells
    Figure Legend Snippet: The B′α1 subunit of PP2A stabilizes human cyclin G1 protein under DNA damaging conditions. (A) HeLa Tet-on cells (clone 2) expressing Flag-tagged human cyclin G1 were plated with or without 2 ng/ml of doxycycline (Dox) for 48 h. The cells

    Techniques Used: Expressing

    10) Product Images from "Conformation-specific binding of p31comet antagonizes the function of Mad2 in the spindle checkpoint"

    Article Title: Conformation-specific binding of p31comet antagonizes the function of Mad2 in the spindle checkpoint

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600322

    P31 comet does not bind to ΔC-Mad2 in living cells. ( A ) HeLa Tet-on cells were transfected with the indicated plasmids and lysed. The resulting lysates were immunoprecipicated with anti-Myc or anti-HA beads and the immunoprecipitates were then blotted with anti-Myc or anti-HA. ( B ) The cell lysates in (A) were blotted with anti-Myc or anti-HA.
    Figure Legend Snippet: P31 comet does not bind to ΔC-Mad2 in living cells. ( A ) HeLa Tet-on cells were transfected with the indicated plasmids and lysed. The resulting lysates were immunoprecipicated with anti-Myc or anti-HA beads and the immunoprecipitates were then blotted with anti-Myc or anti-HA. ( B ) The cell lysates in (A) were blotted with anti-Myc or anti-HA.

    Techniques Used: Transfection

    P31 comet is required for the inactivation of the spindle checkpoint. ( A ) HeLa cells transfected with the control or p31 comet siRNA duplexes were dissolved in SDS sample buffer, separated on SDS–PAGE, and blotted with anti-APC2 and anti-p31 comet antibodies. ( B ) HeLa cells transfected with control or p31 comet siRNA were treated with 100 ng/ml nocodazole for 18 h and released into fresh medium. The total cell lysates of log-phase cells and cell samples taken at the indicated time points were separated on SDS–PAGE and blotted with the indicated antibodies. ( C ) FACS analysis of some of the cell samples described in (B). The peaks corresponding to 2 N and 4 N DNA contents are labeled. ( D ) FACS analysis of HeLa cells that were transfected with control or p31 comet siRNA and treated with the indicated concentrations of nocodazole. The peaks corresponding to 2 N and 4 N DNA contents are labeled. ( E ) HeLa Tet-on cells transfected with the control or p31 comet siRNA were treated with varying concentrations of nocodazole for 16 h and stained with Hoechst 33342. The mitotic indices were determined by directly observing the cells with an inverted fluorescence microscope. The mitotic cells were round and contained condensed DNA, while the interphase cells were flat with decondensed DNA. This experiment was repeated three times and standard deviations are included as error bars.
    Figure Legend Snippet: P31 comet is required for the inactivation of the spindle checkpoint. ( A ) HeLa cells transfected with the control or p31 comet siRNA duplexes were dissolved in SDS sample buffer, separated on SDS–PAGE, and blotted with anti-APC2 and anti-p31 comet antibodies. ( B ) HeLa cells transfected with control or p31 comet siRNA were treated with 100 ng/ml nocodazole for 18 h and released into fresh medium. The total cell lysates of log-phase cells and cell samples taken at the indicated time points were separated on SDS–PAGE and blotted with the indicated antibodies. ( C ) FACS analysis of some of the cell samples described in (B). The peaks corresponding to 2 N and 4 N DNA contents are labeled. ( D ) FACS analysis of HeLa cells that were transfected with control or p31 comet siRNA and treated with the indicated concentrations of nocodazole. The peaks corresponding to 2 N and 4 N DNA contents are labeled. ( E ) HeLa Tet-on cells transfected with the control or p31 comet siRNA were treated with varying concentrations of nocodazole for 16 h and stained with Hoechst 33342. The mitotic indices were determined by directly observing the cells with an inverted fluorescence microscope. The mitotic cells were round and contained condensed DNA, while the interphase cells were flat with decondensed DNA. This experiment was repeated three times and standard deviations are included as error bars.

    Techniques Used: Transfection, SDS Page, FACS, Labeling, Staining, Fluorescence, Microscopy

    11) Product Images from "Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores"

    Article Title: Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201407074

    CENP-T promotes KMN kinetochore targeting through CENP-H-I-K. (A) HeLa Tet-On cells were mock transfected or transfected with siCENP-H, treated with thymidine for 14 h, released into nocodazole-containing medium for 12 h, and treated with or without ZM for 2 h. Their mitotic index was determined by flow cytometry. Means and SDs (error bars) are shown ( n = 3 independent experiments). (B) HeLa cells were transfected with the indicated plasmids and siRNAs (siCC, siCENP-C; siCC+CH, siCENP-C+siCENP-H), and treated with nocodazole. Their mitotic index (mean ± SD [error bars], n = 3) was quantified by flow cytometry. (C and D) Nocodazole-treated mitotic HeLa cells transfected with siCENP-H were further incubated with MG132 (NM) or with both MG132 and ZM (NM+Z), and stained with the indicated antibodies and DAPI. Boxed regions of merged images were magnified and shown in the rightmost column. The relative kinetochore intensities (mean ± SD, n = 400) in certain channels were quantified and shown. Bars, 5 µm (1 µm for magnified images). (E) Recombinant Ndc80C was preincubated with or without recombinant Mis12C, immobilized on beads, and incubated with 35 S-labeled CENP-H-I-K. Bound proteins and input were separated by SDS-PAGE, stained with CBB (left), and analyzed with a phosphorimager (right). CENP-H and -K co-migrate. The asterisk indicates a CENP-K fragment. Broken lines indicate that intervening lanes have been spliced out.
    Figure Legend Snippet: CENP-T promotes KMN kinetochore targeting through CENP-H-I-K. (A) HeLa Tet-On cells were mock transfected or transfected with siCENP-H, treated with thymidine for 14 h, released into nocodazole-containing medium for 12 h, and treated with or without ZM for 2 h. Their mitotic index was determined by flow cytometry. Means and SDs (error bars) are shown ( n = 3 independent experiments). (B) HeLa cells were transfected with the indicated plasmids and siRNAs (siCC, siCENP-C; siCC+CH, siCENP-C+siCENP-H), and treated with nocodazole. Their mitotic index (mean ± SD [error bars], n = 3) was quantified by flow cytometry. (C and D) Nocodazole-treated mitotic HeLa cells transfected with siCENP-H were further incubated with MG132 (NM) or with both MG132 and ZM (NM+Z), and stained with the indicated antibodies and DAPI. Boxed regions of merged images were magnified and shown in the rightmost column. The relative kinetochore intensities (mean ± SD, n = 400) in certain channels were quantified and shown. Bars, 5 µm (1 µm for magnified images). (E) Recombinant Ndc80C was preincubated with or without recombinant Mis12C, immobilized on beads, and incubated with 35 S-labeled CENP-H-I-K. Bound proteins and input were separated by SDS-PAGE, stained with CBB (left), and analyzed with a phosphorimager (right). CENP-H and -K co-migrate. The asterisk indicates a CENP-K fragment. Broken lines indicate that intervening lanes have been spliced out.

    Techniques Used: Transfection, Flow Cytometry, Cytometry, Incubation, Staining, Recombinant, Labeling, SDS Page

    12) Product Images from "The human SKA complex drives the metaphase-anaphase cell cycle transition by recruiting protein phosphatase 1 to kinetochores"

    Article Title: The human SKA complex drives the metaphase-anaphase cell cycle transition by recruiting protein phosphatase 1 to kinetochores

    Journal: eLife

    doi: 10.7554/eLife.12902

    Characterization of HeLa Tet-On cells expressing GFP-Ska1 or GFP-Ska1ΔCTD HeLa cells stably transfected with constructs for inducible expression of GFP-Ska1 or GFP Ska1ΔCTD cells were treated with Dox to induce transgene expression. Cells were imaged live to determine the localization of GFP-Ska1 and GFP-Ska1ΔCTD. As previously reported, GFP-Ska1 concentrates on spindle microtubules and at kinetochores while GFP-Ska1ΔCTD predominantly concentrates at kinetochores ( Abad et al., 2014 ; Schmidt et al., 2012 ). DOI: http://dx.doi.org/10.7554/eLife.12902.008
    Figure Legend Snippet: Characterization of HeLa Tet-On cells expressing GFP-Ska1 or GFP-Ska1ΔCTD HeLa cells stably transfected with constructs for inducible expression of GFP-Ska1 or GFP Ska1ΔCTD cells were treated with Dox to induce transgene expression. Cells were imaged live to determine the localization of GFP-Ska1 and GFP-Ska1ΔCTD. As previously reported, GFP-Ska1 concentrates on spindle microtubules and at kinetochores while GFP-Ska1ΔCTD predominantly concentrates at kinetochores ( Abad et al., 2014 ; Schmidt et al., 2012 ). DOI: http://dx.doi.org/10.7554/eLife.12902.008

    Techniques Used: Expressing, Stable Transfection, Transfection, Construct

    13) Product Images from "The ?-secretase-generated intracellular domain of ?-amyloid precursor protein binds Numb and inhibits Notch signaling"

    Article Title: The ?-secretase-generated intracellular domain of ?-amyloid precursor protein binds Numb and inhibits Notch signaling

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

    doi: 10.1073/pnas.102192599

    Processing of APP and release of AID inhibits Notch signaling. ( A ) HeLa cells were transfected with 4xCBF1-luciferase (CBF-luc) along with the indicated Notch1 and Nbl constructs. Nbl inhibits the transactivation of CBF-luc by both NICD and NΔE. ( B ) Western blotting (WB) with the α-Nbl antiserum shows expression of Nbl and/or Numb proteins in HeLa cells ( Top ). WB with α-APP (22C11) shows induction of APP by Dox in APP-HeLa Tet-on cells ( Middle ). An α-PARP antibody (2C10) was used to normalize for protein loading ( Bottom ). ( C and D ) APP-HeLa Tet-on cells were transfected with CBF-luc along with ( C ) NICD or ( D ) NΔE. Some samples were treated with Dox to induce APP expression and/or with the γ-secretase inhibitor DAPT (100 nM). The decrease in NICD activity by APP induction is inhibited by DAPT ( C ). DAPT treatment significantly reduces NΔE activity in HeLa Tet-on cells ( D ). ( E – H ) HeLa cells were transfected with CBF-luc along NICD, NΔE, AID, or mutant forms of AID as indicated. AID inhibits the activation of CBF-luc by both NICD and NΔE ( E ). The inhibition is dose-dependent ( F ) and correlates with the ability of AID to interact with Nbl ( G ). ( H ) HeLa cells were transfected with either CBF-luc or GAL4-luciferase (GAL4-luc) reporter genes along with APPCT-Gal4 in the presence or absence of cotransfected Fe65. APPCT-Gal4 reduces NICD activity but enhances Fe65 transactivation. Significance was determined by using a two-tailed Student's t test (*, P
    Figure Legend Snippet: Processing of APP and release of AID inhibits Notch signaling. ( A ) HeLa cells were transfected with 4xCBF1-luciferase (CBF-luc) along with the indicated Notch1 and Nbl constructs. Nbl inhibits the transactivation of CBF-luc by both NICD and NΔE. ( B ) Western blotting (WB) with the α-Nbl antiserum shows expression of Nbl and/or Numb proteins in HeLa cells ( Top ). WB with α-APP (22C11) shows induction of APP by Dox in APP-HeLa Tet-on cells ( Middle ). An α-PARP antibody (2C10) was used to normalize for protein loading ( Bottom ). ( C and D ) APP-HeLa Tet-on cells were transfected with CBF-luc along with ( C ) NICD or ( D ) NΔE. Some samples were treated with Dox to induce APP expression and/or with the γ-secretase inhibitor DAPT (100 nM). The decrease in NICD activity by APP induction is inhibited by DAPT ( C ). DAPT treatment significantly reduces NΔE activity in HeLa Tet-on cells ( D ). ( E – H ) HeLa cells were transfected with CBF-luc along NICD, NΔE, AID, or mutant forms of AID as indicated. AID inhibits the activation of CBF-luc by both NICD and NΔE ( E ). The inhibition is dose-dependent ( F ) and correlates with the ability of AID to interact with Nbl ( G ). ( H ) HeLa cells were transfected with either CBF-luc or GAL4-luciferase (GAL4-luc) reporter genes along with APPCT-Gal4 in the presence or absence of cotransfected Fe65. APPCT-Gal4 reduces NICD activity but enhances Fe65 transactivation. Significance was determined by using a two-tailed Student's t test (*, P

    Techniques Used: Transfection, Luciferase, Construct, Western Blot, Expressing, Activity Assay, Mutagenesis, Activation Assay, Inhibition, Two Tailed Test

    14) Product Images from "Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins *"

    Article Title: Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.338467

    Phosphorylation of ORC2 dissociates the ORC from chromatin and the replication origin. A , HeLa Tet-On cells expressing FLAG-tagged ORC2 wild type ( wt ), T116A/T226A ( AA ) mutant, or T116D/T226D ( DD ) mutant were incubated in media containing 2 μg/ml
    Figure Legend Snippet: Phosphorylation of ORC2 dissociates the ORC from chromatin and the replication origin. A , HeLa Tet-On cells expressing FLAG-tagged ORC2 wild type ( wt ), T116A/T226A ( AA ) mutant, or T116D/T226D ( DD ) mutant were incubated in media containing 2 μg/ml

    Techniques Used: Expressing, Mutagenesis, Incubation

    15) Product Images from "Human CST abundance determines recovery from diverse forms of DNA damage and replication stress"

    Article Title: Human CST abundance determines recovery from diverse forms of DNA damage and replication stress

    Journal: Cell Cycle

    doi: 10.4161/15384101.2014.964100

    Alteration in CST abundance does not change cell cycle entry following HU treatment. ( A ) Schematic showing the experimental design. HeLa Tet-On or HeLa 1.2.11 cells were labeled with EdU, treated with HU for 2 h and then released for 0, 30 or
    Figure Legend Snippet: Alteration in CST abundance does not change cell cycle entry following HU treatment. ( A ) Schematic showing the experimental design. HeLa Tet-On or HeLa 1.2.11 cells were labeled with EdU, treated with HU for 2 h and then released for 0, 30 or

    Techniques Used: Labeling

    16) Product Images from "Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis"

    Article Title: Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis

    Journal: Cells

    doi: 10.3390/cells10102792

    SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p
    Figure Legend Snippet: SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p

    Techniques Used: Expressing, Cell Culture, Western Blot, Immunofluorescence, Quantitative RT-PCR, Transfection, Plasmid Preparation, Confocal Microscopy, Staining, Fluorescence

    Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p
    Figure Legend Snippet: Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Western Blot

    17) Product Images from "Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis"

    Article Title: Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis

    Journal: Cells

    doi: 10.3390/cells10102792

    SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p
    Figure Legend Snippet: SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p

    Techniques Used: Expressing, Cell Culture, Western Blot, Immunofluorescence, Quantitative RT-PCR, Transfection, Plasmid Preparation, Confocal Microscopy, Staining, Fluorescence

    Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p
    Figure Legend Snippet: Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Western Blot

    18) Product Images from "Rapid purification of protein complexes from mammalian cells"

    Article Title: Rapid purification of protein complexes from mammalian cells

    Journal: Nucleic Acids Research

    doi:

    Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.

    Techniques Used:

    Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.
    Figure Legend Snippet: Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page, Western Blot, Staining

    Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.
    Figure Legend Snippet: Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.

    Techniques Used: SDS Page, Staining, Western Blot

    Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.
    Figure Legend Snippet: Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.

    Techniques Used: Purification, Incubation, Staining, Western Blot

    Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.

    Techniques Used: Transfection, Fluorescence, Expressing, Translocation Assay

    Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).
    Figure Legend Snippet: Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page

    19) Product Images from "Conformation-specific binding of p31comet antagonizes the function of Mad2 in the spindle checkpoint"

    Article Title: Conformation-specific binding of p31comet antagonizes the function of Mad2 in the spindle checkpoint

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600322

    P31 comet does not bind to ΔC-Mad2 in living cells. ( A ) HeLa Tet-on cells were transfected with the indicated plasmids and lysed. The resulting lysates were immunoprecipicated with anti-Myc or anti-HA beads and the immunoprecipitates were then blotted with anti-Myc or anti-HA. ( B ) The cell lysates in (A) were blotted with anti-Myc or anti-HA.
    Figure Legend Snippet: P31 comet does not bind to ΔC-Mad2 in living cells. ( A ) HeLa Tet-on cells were transfected with the indicated plasmids and lysed. The resulting lysates were immunoprecipicated with anti-Myc or anti-HA beads and the immunoprecipitates were then blotted with anti-Myc or anti-HA. ( B ) The cell lysates in (A) were blotted with anti-Myc or anti-HA.

    Techniques Used: Transfection

    P31 comet is required for the inactivation of the spindle checkpoint. ( A ) HeLa cells transfected with the control or p31 comet siRNA duplexes were dissolved in SDS sample buffer, separated on SDS–PAGE, and blotted with anti-APC2 and anti-p31 comet antibodies. ( B ) HeLa cells transfected with control or p31 comet siRNA were treated with 100 ng/ml nocodazole for 18 h and released into fresh medium. The total cell lysates of log-phase cells and cell samples taken at the indicated time points were separated on SDS–PAGE and blotted with the indicated antibodies. ( C ) FACS analysis of some of the cell samples described in (B). The peaks corresponding to 2 N and 4 N DNA contents are labeled. ( D ) FACS analysis of HeLa cells that were transfected with control or p31 comet siRNA and treated with the indicated concentrations of nocodazole. The peaks corresponding to 2 N and 4 N DNA contents are labeled. ( E ) HeLa Tet-on cells transfected with the control or p31 comet siRNA were treated with varying concentrations of nocodazole for 16 h and stained with Hoechst 33342. The mitotic indices were determined by directly observing the cells with an inverted fluorescence microscope. The mitotic cells were round and contained condensed DNA, while the interphase cells were flat with decondensed DNA. This experiment was repeated three times and standard deviations are included as error bars.
    Figure Legend Snippet: P31 comet is required for the inactivation of the spindle checkpoint. ( A ) HeLa cells transfected with the control or p31 comet siRNA duplexes were dissolved in SDS sample buffer, separated on SDS–PAGE, and blotted with anti-APC2 and anti-p31 comet antibodies. ( B ) HeLa cells transfected with control or p31 comet siRNA were treated with 100 ng/ml nocodazole for 18 h and released into fresh medium. The total cell lysates of log-phase cells and cell samples taken at the indicated time points were separated on SDS–PAGE and blotted with the indicated antibodies. ( C ) FACS analysis of some of the cell samples described in (B). The peaks corresponding to 2 N and 4 N DNA contents are labeled. ( D ) FACS analysis of HeLa cells that were transfected with control or p31 comet siRNA and treated with the indicated concentrations of nocodazole. The peaks corresponding to 2 N and 4 N DNA contents are labeled. ( E ) HeLa Tet-on cells transfected with the control or p31 comet siRNA were treated with varying concentrations of nocodazole for 16 h and stained with Hoechst 33342. The mitotic indices were determined by directly observing the cells with an inverted fluorescence microscope. The mitotic cells were round and contained condensed DNA, while the interphase cells were flat with decondensed DNA. This experiment was repeated three times and standard deviations are included as error bars.

    Techniques Used: Transfection, SDS Page, FACS, Labeling, Staining, Fluorescence, Microscopy

    20) Product Images from "N-Alpha-Acetyltransferases and Regulation of CFTR Expression"

    Article Title: N-Alpha-Acetyltransferases and Regulation of CFTR Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0155430

    Schematic of Steps in CFTR Conformational Maturation and Points of Regulation by NatA and NatB. Schematic diagram for the regulation of CFTR expression by NatA and NatB (A) where M = amount of mRNA, B = ER retained CFTR sensitive to proteasome, B* = ER retained CFTR insensitive to proteasome, C = trafficked CFTR, k prod = rate of production of B, k deg = rate of degradation of B, k conv = rate of conversion from B to B*, k traf = rate of trafficking of B* to C, B M = ER retained CFTR sensitive to proteasome, k mis = rate of misfolding of B. Representative Western blots after SDS PAGE of steady state protein levels of WT and deltaF508 CFTR in cultured HeLa-Tet-On cells upon depletion of C) NAA10 (NatA) or B) NAA20 (NatB). Actin or tubulin are loading controls. D) Quantitation by LiCor and statistics (n = 3, +/- SEM).
    Figure Legend Snippet: Schematic of Steps in CFTR Conformational Maturation and Points of Regulation by NatA and NatB. Schematic diagram for the regulation of CFTR expression by NatA and NatB (A) where M = amount of mRNA, B = ER retained CFTR sensitive to proteasome, B* = ER retained CFTR insensitive to proteasome, C = trafficked CFTR, k prod = rate of production of B, k deg = rate of degradation of B, k conv = rate of conversion from B to B*, k traf = rate of trafficking of B* to C, B M = ER retained CFTR sensitive to proteasome, k mis = rate of misfolding of B. Representative Western blots after SDS PAGE of steady state protein levels of WT and deltaF508 CFTR in cultured HeLa-Tet-On cells upon depletion of C) NAA10 (NatA) or B) NAA20 (NatB). Actin or tubulin are loading controls. D) Quantitation by LiCor and statistics (n = 3, +/- SEM).

    Techniques Used: Expressing, Western Blot, SDS Page, Cell Culture, Quantitation Assay

    Requirement for CFTR’s N-terminus in NatA/B Regulation. Steady state protein levels of CFTR in cultured HeLa-Tet-On cells expressing the Q2P mutation (A) as well as after depletion of NAA10 (B), or NAA20 (C) were monitored by Western blotting after separation of cell lysate by SDS-PAGE. Representative Western blots presented (A-C). Tubulin was a loading control. D-F) Quantitation and statistical analysis (n = 3, +/- SEM).
    Figure Legend Snippet: Requirement for CFTR’s N-terminus in NatA/B Regulation. Steady state protein levels of CFTR in cultured HeLa-Tet-On cells expressing the Q2P mutation (A) as well as after depletion of NAA10 (B), or NAA20 (C) were monitored by Western blotting after separation of cell lysate by SDS-PAGE. Representative Western blots presented (A-C). Tubulin was a loading control. D-F) Quantitation and statistical analysis (n = 3, +/- SEM).

    Techniques Used: Cell Culture, Expressing, Mutagenesis, Western Blot, SDS Page, Quantitation Assay

    Effects of NatA, NatE, and NatB Complex on G85E CFTR Protein Levels. Representative Western blots after SDS PAGE of steady state protein levels of G85E CFTR in cultured HeLa-Tet-On cells upon depletion of A) NAA16 (NatA), NAA15 (NatA/E), B) NAA10 (NatA), NAA50 (NatE), C) NAA20 (NatB), or NAA25 (NatB)). Actin or tubulin are loading controls. D) Quantitation by LiCor and statistics (n = 3, +/- SEM).
    Figure Legend Snippet: Effects of NatA, NatE, and NatB Complex on G85E CFTR Protein Levels. Representative Western blots after SDS PAGE of steady state protein levels of G85E CFTR in cultured HeLa-Tet-On cells upon depletion of A) NAA16 (NatA), NAA15 (NatA/E), B) NAA10 (NatA), NAA50 (NatE), C) NAA20 (NatB), or NAA25 (NatB)). Actin or tubulin are loading controls. D) Quantitation by LiCor and statistics (n = 3, +/- SEM).

    Techniques Used: Western Blot, SDS Page, Cell Culture, Quantitation Assay

    21) Product Images from "Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis"

    Article Title: Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis

    Journal: Cells

    doi: 10.3390/cells10102792

    SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p
    Figure Legend Snippet: SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p

    Techniques Used: Expressing, Cell Culture, Western Blot, Immunofluorescence, Quantitative RT-PCR, Transfection, Plasmid Preparation, Confocal Microscopy, Staining, Fluorescence

    Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p
    Figure Legend Snippet: Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Western Blot

    22) Product Images from "A Human Mitochondrial Transcription Factor Is Related to RNA Adenine Methyltransferases and Binds S-Adenosylmethionine"

    Article Title: A Human Mitochondrial Transcription Factor Is Related to RNA Adenine Methyltransferases and Binds S-Adenosylmethionine

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.4.1116-1125.2002

    Human CGI-75 is a mitochondrial protein. The mitochondrial localization of a CGI-75::EGFP fusion protein is shown. HeLa Tet-On cells were transiently transfected with a plasmid (pTRE2-HBGFP) that expresses a CGI-75::EGFP fusion protein and stained with the mitochondrion-specific dye Mitotracker Red. Shown are two representative transfected cells analyzed by fluorescence microscopy for EGFP fluorescence in green (labeled GFP), Mitotracker fluorescence in red (labeled MT), and a merge of the two signals (labeled merge), in which a yellow color indicates colocalization. In the panels on the right, the asterisks indicate the signals derived from a cell that was not transfected with the plasmid. As expected, this cell exhibited Mitotracker fluorescence but not EGFP fluorescence.
    Figure Legend Snippet: Human CGI-75 is a mitochondrial protein. The mitochondrial localization of a CGI-75::EGFP fusion protein is shown. HeLa Tet-On cells were transiently transfected with a plasmid (pTRE2-HBGFP) that expresses a CGI-75::EGFP fusion protein and stained with the mitochondrion-specific dye Mitotracker Red. Shown are two representative transfected cells analyzed by fluorescence microscopy for EGFP fluorescence in green (labeled GFP), Mitotracker fluorescence in red (labeled MT), and a merge of the two signals (labeled merge), in which a yellow color indicates colocalization. In the panels on the right, the asterisks indicate the signals derived from a cell that was not transfected with the plasmid. As expected, this cell exhibited Mitotracker fluorescence but not EGFP fluorescence.

    Techniques Used: Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy, Labeling, Derivative Assay

    23) Product Images from "A novel clathrin homolog that co-distributes with cytoskeletal components functions in the trans-Golgi network"

    Article Title: A novel clathrin homolog that co-distributes with cytoskeletal components functions in the trans-Golgi network

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.1.272

    Fig. 4. Localization of CHC22 by immunoelectron microscopy. HeLa-tet/on cells transfected with pJM601CHC22 were induced for high level CHC22 expression for 24 h, then permeabilized, lightly fixed and labeled with antibodies to CHC22 (5 nm gold, small arrows), components of AP1 (10 nm gold, arrowheads) and clathrin light chains (15 nm gold, large arrows). Prior to embedding and sectioning, each antibody was sequentially applied and detected with protein A–gold attached to gold particles of the size indicated, followed by blocking with excess protein A. The images were selected to be representative of the intracellular distribution of CHC22 (statistics are provided in the text), showing vesicles of 80–100 nm in the TGN labeled for CHC22 and AP1 (γ subunit) ( A ) or labeled for CHC22 and AP1 (σ1 subunit) with notable protein coats ( B ). ( C ) Similar vesicles, with one labeled exclusively for CHC22 and the other for CHC22, clathrin light chain and AP1. ( D ) Peripheral vesicles, primarily labeled for clathrin light chain, with one co-labeled for CHC22. The labeling for AP1 (σ1 subunit) at the left suggests that these coated vesicles are near endosomes.
    Figure Legend Snippet: Fig. 4. Localization of CHC22 by immunoelectron microscopy. HeLa-tet/on cells transfected with pJM601CHC22 were induced for high level CHC22 expression for 24 h, then permeabilized, lightly fixed and labeled with antibodies to CHC22 (5 nm gold, small arrows), components of AP1 (10 nm gold, arrowheads) and clathrin light chains (15 nm gold, large arrows). Prior to embedding and sectioning, each antibody was sequentially applied and detected with protein A–gold attached to gold particles of the size indicated, followed by blocking with excess protein A. The images were selected to be representative of the intracellular distribution of CHC22 (statistics are provided in the text), showing vesicles of 80–100 nm in the TGN labeled for CHC22 and AP1 (γ subunit) ( A ) or labeled for CHC22 and AP1 (σ1 subunit) with notable protein coats ( B ). ( C ) Similar vesicles, with one labeled exclusively for CHC22 and the other for CHC22, clathrin light chain and AP1. ( D ) Peripheral vesicles, primarily labeled for clathrin light chain, with one co-labeled for CHC22. The labeling for AP1 (σ1 subunit) at the left suggests that these coated vesicles are near endosomes.

    Techniques Used: Immuno-Electron Microscopy, Transfection, Expressing, Labeling, Blocking Assay

    Fig. 2. Differential association of CHC22 with clathrin coat components. ( A ) Bacterial lysate containing recombinant CHC22Hub and co-expressed bovine neuronal LCa was separated by Superose 6 size exclusion chromatography. The column fractions were collected and resolved in sequence (left to right) by SDS–PAGE. The presence of CHC22Hub polypeptides or LCa was established by immunoblotting using rabbit serum against CHC22 (CHC22Hub) or MAb CON.1, which recognizes a determinant shared by both light chains (LCa and LCb). Arrows on the top indicate the fractions flanking the elution positions of molecular weight standard catalase (232 kDa). ( B ) Bacterial lysate containing recombinant CHC22Hub and co-expressed bovine neuronal LCb was separated by Superose 6 size exclusion chromatography and analyzed for the elution position of CHC22Hub polypeptides and LCb, as in (A). ( C ). The immunoprecipitates were then subjected to SDS–PAGE and probed with the following antibodies: anti-CHC22 polyclonal antiserum [CHC22 (PAb)], CHC17-specific monoclonal antibody TD.1, anti-clathrin light chain antiserum (CLC), anti-AP1 γ subunit monoclonal antibody 100/3 [AP1(γ)], anti-AP2 α subunit AC1M11 [AP2(α)] and anti-AP3 β3 antiserum [AP3(β)]. The CHC22 signals in CHC17 immunoprecipitates are due to cross-reactivity of X22 with CHC22, causing co-precipitation of CHC22 with CHC17 and possibly explaining the apparent association of AP3 with CHC17. ( D ) HeLa-tet/on cells were permanently transfected with T7-epitope-tagged full-length CHC22, under the tet operator (pJM601CHC22), and CHC22 expression was induced for 24 h with doxycycline. CHC22 full-length protein was then immunoprecipated using anti-T7 MAb and the sample was analyzed by SDS–PAGE and immunoblotting for CHC22 using a specific polyclonal antiserum, or for clathrin light chains (CLC) using the α-cons polyclonal antibody. Conventional clathrin (CHC17) was immunoprecipitated from the same sample using MAb X22 and similarly analyzed. Note that X22 immunoprecipitates some CHC22 along with CHC17 due to cross-reactivity (see C).
    Figure Legend Snippet: Fig. 2. Differential association of CHC22 with clathrin coat components. ( A ) Bacterial lysate containing recombinant CHC22Hub and co-expressed bovine neuronal LCa was separated by Superose 6 size exclusion chromatography. The column fractions were collected and resolved in sequence (left to right) by SDS–PAGE. The presence of CHC22Hub polypeptides or LCa was established by immunoblotting using rabbit serum against CHC22 (CHC22Hub) or MAb CON.1, which recognizes a determinant shared by both light chains (LCa and LCb). Arrows on the top indicate the fractions flanking the elution positions of molecular weight standard catalase (232 kDa). ( B ) Bacterial lysate containing recombinant CHC22Hub and co-expressed bovine neuronal LCb was separated by Superose 6 size exclusion chromatography and analyzed for the elution position of CHC22Hub polypeptides and LCb, as in (A). ( C ). The immunoprecipitates were then subjected to SDS–PAGE and probed with the following antibodies: anti-CHC22 polyclonal antiserum [CHC22 (PAb)], CHC17-specific monoclonal antibody TD.1, anti-clathrin light chain antiserum (CLC), anti-AP1 γ subunit monoclonal antibody 100/3 [AP1(γ)], anti-AP2 α subunit AC1M11 [AP2(α)] and anti-AP3 β3 antiserum [AP3(β)]. The CHC22 signals in CHC17 immunoprecipitates are due to cross-reactivity of X22 with CHC22, causing co-precipitation of CHC22 with CHC17 and possibly explaining the apparent association of AP3 with CHC17. ( D ) HeLa-tet/on cells were permanently transfected with T7-epitope-tagged full-length CHC22, under the tet operator (pJM601CHC22), and CHC22 expression was induced for 24 h with doxycycline. CHC22 full-length protein was then immunoprecipated using anti-T7 MAb and the sample was analyzed by SDS–PAGE and immunoblotting for CHC22 using a specific polyclonal antiserum, or for clathrin light chains (CLC) using the α-cons polyclonal antibody. Conventional clathrin (CHC17) was immunoprecipitated from the same sample using MAb X22 and similarly analyzed. Note that X22 immunoprecipitates some CHC22 along with CHC17 due to cross-reactivity (see C).

    Techniques Used: Recombinant, Size-exclusion Chromatography, Sequencing, SDS Page, Molecular Weight, Transfection, Expressing, Immunoprecipitation

    Fig. 7. Expression of CHC22 hub domain affects intracellular distribution of M6PR. HeLa-tet/on cells transfected with pJM601CHC22Hub ( A – F ) or with pJM601CHC22 ( G – J ) were induced for high level CHC22Hub expression or high level expression of full-length (FL) CHC22 for 24 h. Cells were stained for the expression of CHC22Hub (shown in A, C and E) or CHC22FL (shown in G and I) with anti-CHC22 MAb followed by LRSC-conjugated goat anti-mouse IgG. The distribution of endogenous M6PR (shown in B, D and H) was detected by double staining (A), (C) and (G) with antiserum to the cation-independent M6PR, and the distribution of endogenous clathrin (shown in F and J) was detected by double staining (E) and (I) with anti-clathrin LC antiserum α-cons, both followed by FITC-conjugated goat anti-rabbit IgG. The horizontal rows represent the same images viewed with different filters to see red and green staining on the left and right, respectively. Note that not all cells in the transfected cultures express CHC22Hub or CHC22FL, which was confirmed by staining with anti-T7 MAb, which reacts with the epitope tag on the transfected proteins (not shown). The non-expressing cells serve as negative controls for endogenous staining of M6PR or clathrin LC, and their staining patterns are identical to those of control cells that were either not transfected or not induced for expression of the transfected molecules.
    Figure Legend Snippet: Fig. 7. Expression of CHC22 hub domain affects intracellular distribution of M6PR. HeLa-tet/on cells transfected with pJM601CHC22Hub ( A – F ) or with pJM601CHC22 ( G – J ) were induced for high level CHC22Hub expression or high level expression of full-length (FL) CHC22 for 24 h. Cells were stained for the expression of CHC22Hub (shown in A, C and E) or CHC22FL (shown in G and I) with anti-CHC22 MAb followed by LRSC-conjugated goat anti-mouse IgG. The distribution of endogenous M6PR (shown in B, D and H) was detected by double staining (A), (C) and (G) with antiserum to the cation-independent M6PR, and the distribution of endogenous clathrin (shown in F and J) was detected by double staining (E) and (I) with anti-clathrin LC antiserum α-cons, both followed by FITC-conjugated goat anti-rabbit IgG. The horizontal rows represent the same images viewed with different filters to see red and green staining on the left and right, respectively. Note that not all cells in the transfected cultures express CHC22Hub or CHC22FL, which was confirmed by staining with anti-T7 MAb, which reacts with the epitope tag on the transfected proteins (not shown). The non-expressing cells serve as negative controls for endogenous staining of M6PR or clathrin LC, and their staining patterns are identical to those of control cells that were either not transfected or not induced for expression of the transfected molecules.

    Techniques Used: Expressing, Transfection, Staining, Double Staining

    24) Product Images from "Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress"

    Article Title: Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh648

    Incorporation of MTS–EndoIII and MTS–EndoVIII inserts into HeLa Tet-On Cells. Southern blots of vector and ( A ) seven HeLa Tet-On clones transfected with MTS–EndoIII, hybridized with an MTS–EndoIII probe; ( B ) vector and four HeLa Tet-On/MTS–EndoVIII clones, hybridized with a MTS–EndoVIII-specific probe.
    Figure Legend Snippet: Incorporation of MTS–EndoIII and MTS–EndoVIII inserts into HeLa Tet-On Cells. Southern blots of vector and ( A ) seven HeLa Tet-On clones transfected with MTS–EndoIII, hybridized with an MTS–EndoIII probe; ( B ) vector and four HeLa Tet-On/MTS–EndoVIII clones, hybridized with a MTS–EndoVIII-specific probe.

    Techniques Used: Plasmid Preparation, Clone Assay, Transfection

    Conditional and targeted expression of EndoIII and EndoVIII into mitochondria. Western blots containing 20 μg of mitochondrial extracts isolated from ( A ) Dox ± vector and clone 3 of HeLa Tet-On/MTS–EndoIII cells; and ( B ) Dox ± vector and three separate clones (2, 3 and 4) of HeLa Tet-On/MTS–EndoVIII transfected cells using anti-HA antibodies. Immunodetection of cytochrome c was performed to show that the recombinant proteins are in mitochondria.
    Figure Legend Snippet: Conditional and targeted expression of EndoIII and EndoVIII into mitochondria. Western blots containing 20 μg of mitochondrial extracts isolated from ( A ) Dox ± vector and clone 3 of HeLa Tet-On/MTS–EndoIII cells; and ( B ) Dox ± vector and three separate clones (2, 3 and 4) of HeLa Tet-On/MTS–EndoVIII transfected cells using anti-HA antibodies. Immunodetection of cytochrome c was performed to show that the recombinant proteins are in mitochondria.

    Techniques Used: Expressing, Western Blot, Isolation, Plasmid Preparation, Clone Assay, Transfection, Immunodetection, Recombinant

    25) Product Images from "Rapid purification of protein complexes from mammalian cells"

    Article Title: Rapid purification of protein complexes from mammalian cells

    Journal: Nucleic Acids Research

    doi:

    Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.

    Techniques Used:

    Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.
    Figure Legend Snippet: Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page, Western Blot, Staining

    Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.
    Figure Legend Snippet: Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.

    Techniques Used: SDS Page, Staining, Western Blot

    Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.
    Figure Legend Snippet: Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.

    Techniques Used: Purification, Incubation, Staining, Western Blot

    Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.

    Techniques Used: Transfection, Fluorescence, Expressing, Translocation Assay

    Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).
    Figure Legend Snippet: Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page

    26) Product Images from "Casein Kinase 1 Functions as both Penultimate and Ultimate Kinase in Regulating Cdc25A Destruction"

    Article Title: Casein Kinase 1 Functions as both Penultimate and Ultimate Kinase in Regulating Cdc25A Destruction

    Journal: Oncogene

    doi: 10.1038/onc.2010.96

    CK1α negatively regulates Cdc25A stability in vivo (A) HeLa Tet-on Cdc25A-FLuc cells were mock transfected or transfected with control siRNA (Scr) or siRNAs specific for Chk1, GSK-3β or CK1α for 48 h and then cultured in the presence of 2 μg/ml Dox for an additional 16 h. Cells were incubated with D-Luciferin and imaged 10 min later. Quantification of bioluminescence signal plotted as mean ± SEM (n = 3). P-values from Student's t-test are shown when significantly different from control. Asterisks indicate significant p-values (**
    Figure Legend Snippet: CK1α negatively regulates Cdc25A stability in vivo (A) HeLa Tet-on Cdc25A-FLuc cells were mock transfected or transfected with control siRNA (Scr) or siRNAs specific for Chk1, GSK-3β or CK1α for 48 h and then cultured in the presence of 2 μg/ml Dox for an additional 16 h. Cells were incubated with D-Luciferin and imaged 10 min later. Quantification of bioluminescence signal plotted as mean ± SEM (n = 3). P-values from Student's t-test are shown when significantly different from control. Asterisks indicate significant p-values (**

    Techniques Used: In Vivo, Transfection, Cell Culture, Incubation

    CK1α regulates S82 phosphorylation in vivo (A) WT and mutant forms of Cdc25A were purified as GST fusion proteins from bacteria and kinase assays were performed in vitro in the presence of CK1. Reaction products were resolved by SDS-PAGE and analyzed by Western blotting for the indicated proteins. (B) HeLa cells transfected with plasmids encoding either CK1α or V5-LacZ for 20 h were treated with 50 μM MG132 for 4 h, lysed and analyzed by Western blotting. (C) MDA-MB-231 cells transfected with plasmids encoding tagged forms of Cdc25A and β-TrCP and untagged CK1α for 20 h were lysed and resolved directly by SDS-PAGE (WCE) or were incubated first with Myc agarose to precipitate β-TrCP. Precipitates were then resolved by SDS-PAGE followed by Western blotting. (D) HeLa cells transfected with plasmids encoding V5-LacZ (lane 1) or CK1α (lane 2) for 20 h were treated with 10 μg/ml cycloheximide (CHX) for 15 min in the presence or absence of MG132 (50 μM). Lysates were prepared and analyzed for the indicated proteins by Western blotting. (E) HeLa cells transfected with plasmids encoding Flag-Cdc25A for 20 h were mock-irradiated or exposed to 10 Gy IR followed by D4476 (75 μM) for 30 min. Lysates were prepared and analyzed by Western blotting. (F) HeLa Tet-on Cdc25A-FLuc cells were cultured in media containing 2 μg/ml Dox for 16 h. Cells were then placed in Dox-free media and exposed to 10 Gy IR in the presence of 10 μg/ml cycloheximide together with either DMSO or D4476 (75 μM). Lysates were prepared at the indicated times after IR and were analyzed by Western blotting for the indicated proteins.
    Figure Legend Snippet: CK1α regulates S82 phosphorylation in vivo (A) WT and mutant forms of Cdc25A were purified as GST fusion proteins from bacteria and kinase assays were performed in vitro in the presence of CK1. Reaction products were resolved by SDS-PAGE and analyzed by Western blotting for the indicated proteins. (B) HeLa cells transfected with plasmids encoding either CK1α or V5-LacZ for 20 h were treated with 50 μM MG132 for 4 h, lysed and analyzed by Western blotting. (C) MDA-MB-231 cells transfected with plasmids encoding tagged forms of Cdc25A and β-TrCP and untagged CK1α for 20 h were lysed and resolved directly by SDS-PAGE (WCE) or were incubated first with Myc agarose to precipitate β-TrCP. Precipitates were then resolved by SDS-PAGE followed by Western blotting. (D) HeLa cells transfected with plasmids encoding V5-LacZ (lane 1) or CK1α (lane 2) for 20 h were treated with 10 μg/ml cycloheximide (CHX) for 15 min in the presence or absence of MG132 (50 μM). Lysates were prepared and analyzed for the indicated proteins by Western blotting. (E) HeLa cells transfected with plasmids encoding Flag-Cdc25A for 20 h were mock-irradiated or exposed to 10 Gy IR followed by D4476 (75 μM) for 30 min. Lysates were prepared and analyzed by Western blotting. (F) HeLa Tet-on Cdc25A-FLuc cells were cultured in media containing 2 μg/ml Dox for 16 h. Cells were then placed in Dox-free media and exposed to 10 Gy IR in the presence of 10 μg/ml cycloheximide together with either DMSO or D4476 (75 μM). Lysates were prepared at the indicated times after IR and were analyzed by Western blotting for the indicated proteins.

    Techniques Used: In Vivo, Mutagenesis, Purification, In Vitro, SDS Page, Western Blot, Transfection, Multiple Displacement Amplification, Incubation, Irradiation, Cell Culture

    Validation of Cdc25A-FLuc fusion reporter (A) HeLa Tet-on Cdc25A-FLuc cells were cultured in the presence of 2 μg/ml doxycycline (Dox) for 16 h followed by D-Luciferin for 10 min. Cells were imaged using a charge-coupled device (CCD) camera-based bioluminescence imaging system (IVIS 100; Xenogen Corp). The color overlay on the images represents the photons/sec/cm 2 /steradian (p/s/cm 2 /sr) as indicated by the color scale next to the images. Cells were harvested immediately after imaging and analyzed for the Cdc25A-FLuc reporter protein by Western blotting using a Cdc25A-specific antibody. Lysates were probed for β-Catenin as a loading control. (B) HeLa Tet-on Cdc25A-FLuc cells were incubated in the culture media containing 2 μg/ml Dox for 13 h or 15 h. Cells were then cultured in the absence of Dox for the indicated times and analyzed for Cdc25A-FLuc by Western blotting or for luciferase activity by bioluminescence imaging. Lysates were probed for actin as a loading control. (C) HeLa Tet-on Cdc25A-FLuc cells were cultured in media containing 2 μg/ml Dox. After 16 h the culture media was removed and cells were cultured in Dox-free media containing 10 μg/ml cycloheximide and either MG132 (50 μM) or UCN-01 (500 nM) for 90 min, D-Luciferin was then added and cells were imaged 10 min later. Immediately after imaging, cells were lysed for Western blotting. Quantification of bioluminescence signal plotted as mean ± SEM (n = 3). P-values from Student's t-test are shown when significantly different from control. Asterisks indicate significant p-values (**
    Figure Legend Snippet: Validation of Cdc25A-FLuc fusion reporter (A) HeLa Tet-on Cdc25A-FLuc cells were cultured in the presence of 2 μg/ml doxycycline (Dox) for 16 h followed by D-Luciferin for 10 min. Cells were imaged using a charge-coupled device (CCD) camera-based bioluminescence imaging system (IVIS 100; Xenogen Corp). The color overlay on the images represents the photons/sec/cm 2 /steradian (p/s/cm 2 /sr) as indicated by the color scale next to the images. Cells were harvested immediately after imaging and analyzed for the Cdc25A-FLuc reporter protein by Western blotting using a Cdc25A-specific antibody. Lysates were probed for β-Catenin as a loading control. (B) HeLa Tet-on Cdc25A-FLuc cells were incubated in the culture media containing 2 μg/ml Dox for 13 h or 15 h. Cells were then cultured in the absence of Dox for the indicated times and analyzed for Cdc25A-FLuc by Western blotting or for luciferase activity by bioluminescence imaging. Lysates were probed for actin as a loading control. (C) HeLa Tet-on Cdc25A-FLuc cells were cultured in media containing 2 μg/ml Dox. After 16 h the culture media was removed and cells were cultured in Dox-free media containing 10 μg/ml cycloheximide and either MG132 (50 μM) or UCN-01 (500 nM) for 90 min, D-Luciferin was then added and cells were imaged 10 min later. Immediately after imaging, cells were lysed for Western blotting. Quantification of bioluminescence signal plotted as mean ± SEM (n = 3). P-values from Student's t-test are shown when significantly different from control. Asterisks indicate significant p-values (**

    Techniques Used: Cell Culture, Imaging, Western Blot, Incubation, Luciferase, Activity Assay

    27) Product Images from "Activation of the Ca2+/NFAT Pathway by Assembly of Hepatitis C Virus Core Protein into Nucleocapsid-like Particles"

    Article Title: Activation of the Ca2+/NFAT Pathway by Assembly of Hepatitis C Virus Core Protein into Nucleocapsid-like Particles

    Journal: Viruses

    doi: 10.3390/v14040761

    Analysis of nucleocapsid-like particles. HeLa Tet-On cells were transfected with plasmids expressing either full-length (wt) or C deletion mutants (∆61–68 or ∆70–79). ( A ) Sedimentation analysis of wt (1–194) and C mutants. Cellular extracts were loaded on 5–20% sucrose gradients and subjected to ultra-centrifugation. The gradients were fractionated and analyzed for the C protein accumulation as described above. The expected positions of nucleocapsid-like particles (NLPs) are indicated by arrows. ( B ) Map of internal part of D1. Amino acid deletions in ∆61–68 and ∆70–79 are indicated. ( C ) The densities of C containing fractions were determined by refractometry. ( D ) Buoyant density analysis of wt (1–194) and C mutants. Cellular extracts were mixed with CsCl and subjected to ultracentrifugation. The gradients were fractionated and the C protein was then detected by Western blotting using an anti HCV C antibody. The order of the samples is indicated with numbers from the bottom to the top of the gradient. ( E ) Electron microscopy of the NLPs. Fractions positive for NLPs in sedimentation centrifugation were dialyzed, negatively stained and examined by transmission electron microscopy.
    Figure Legend Snippet: Analysis of nucleocapsid-like particles. HeLa Tet-On cells were transfected with plasmids expressing either full-length (wt) or C deletion mutants (∆61–68 or ∆70–79). ( A ) Sedimentation analysis of wt (1–194) and C mutants. Cellular extracts were loaded on 5–20% sucrose gradients and subjected to ultra-centrifugation. The gradients were fractionated and analyzed for the C protein accumulation as described above. The expected positions of nucleocapsid-like particles (NLPs) are indicated by arrows. ( B ) Map of internal part of D1. Amino acid deletions in ∆61–68 and ∆70–79 are indicated. ( C ) The densities of C containing fractions were determined by refractometry. ( D ) Buoyant density analysis of wt (1–194) and C mutants. Cellular extracts were mixed with CsCl and subjected to ultracentrifugation. The gradients were fractionated and the C protein was then detected by Western blotting using an anti HCV C antibody. The order of the samples is indicated with numbers from the bottom to the top of the gradient. ( E ) Electron microscopy of the NLPs. Fractions positive for NLPs in sedimentation centrifugation were dialyzed, negatively stained and examined by transmission electron microscopy.

    Techniques Used: Transfection, Expressing, Sedimentation, Centrifugation, Western Blot, Electron Microscopy, Staining, Transmission Assay

    28) Product Images from "Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Protease Inhibits Host Cell Translation: a Novel Mechanism for Host Translation Shutoff"

    Article Title: Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Protease Inhibits Host Cell Translation: a Novel Mechanism for Host Translation Shutoff

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.4.1779-1790.2004

    Expression of 3Cpro inhibits translation in HeLa cells. (A) Schematic of DNAs cotransfected into HeLa Tet-On cells. (B) The graph shows levels of Luc expression in cells transiently transfected with pTRE-luc and cotransfected with control pTRE2 vector (C) or pTRE2-3C. Data are expressed as percentages of Luc expression in cells transfected with control pTRE2 vector. At 4 h posttransfection, cells were induced with doxycycline for 2 or 4 additional hours and then cell lysates were harvested and analyzed for Luc activity. Both 2- and 4-h control Luc RLU levels were set to 100% in the graph to calculate percentages of reduction in translation after 3Cpro expression. Black bars indicate cells induced with doxycycline for 4 h, and gray-shaded bars indicate cells left uninduced for 16 h before harvesting and analysis. (C) Graph showing [ 35 S]methionine-cysteine incorporated into newly synthesized proteins in doxycycline-induced or uninduced HeLa Tet-On cells transfected with pTRE2 control vector (C) or pTRE2-3C (3C). Black bars indicate cells treated for 4 h posttransfection or left untreated; gray-shaded bars indicate cells left untreated for 16 h. (D) Immunoblot analysis of PABP in HeLa Tet-On cells transfected with control vector or pTRE2-3C DNA that were either induced with doxycycline (+dox) or left uninduced (−dox). Cleavage products of PABP (3Calt cp and 3Calt cp c ) are indicated. Transfection rates determined with an enhanced green fluorescent protein expression vector and immunofluorescence analysis were 85 to 90% in the experiments represented here. Data represent the means ± SD of three individual experiments.
    Figure Legend Snippet: Expression of 3Cpro inhibits translation in HeLa cells. (A) Schematic of DNAs cotransfected into HeLa Tet-On cells. (B) The graph shows levels of Luc expression in cells transiently transfected with pTRE-luc and cotransfected with control pTRE2 vector (C) or pTRE2-3C. Data are expressed as percentages of Luc expression in cells transfected with control pTRE2 vector. At 4 h posttransfection, cells were induced with doxycycline for 2 or 4 additional hours and then cell lysates were harvested and analyzed for Luc activity. Both 2- and 4-h control Luc RLU levels were set to 100% in the graph to calculate percentages of reduction in translation after 3Cpro expression. Black bars indicate cells induced with doxycycline for 4 h, and gray-shaded bars indicate cells left uninduced for 16 h before harvesting and analysis. (C) Graph showing [ 35 S]methionine-cysteine incorporated into newly synthesized proteins in doxycycline-induced or uninduced HeLa Tet-On cells transfected with pTRE2 control vector (C) or pTRE2-3C (3C). Black bars indicate cells treated for 4 h posttransfection or left untreated; gray-shaded bars indicate cells left untreated for 16 h. (D) Immunoblot analysis of PABP in HeLa Tet-On cells transfected with control vector or pTRE2-3C DNA that were either induced with doxycycline (+dox) or left uninduced (−dox). Cleavage products of PABP (3Calt cp and 3Calt cp c ) are indicated. Transfection rates determined with an enhanced green fluorescent protein expression vector and immunofluorescence analysis were 85 to 90% in the experiments represented here. Data represent the means ± SD of three individual experiments.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Activity Assay, Synthesized, Immunofluorescence

    29) Product Images from "Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein"

    Article Title: Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein

    Journal: Nucleic Acids Research

    doi:

    Tetracycline inducible expression of TBP-DR2 in HeLa cells. ( A ) Western blot analysis. Cellular extracts from uninduced (lane 1) and doxycycline-induced cells (lane 2) of one of the selected clones were probed with anti-histidine antibodies. Recombinant TBP-DR2 with a histidine tag (10 ng; lane 3), 25 µg HeLa whole-cell extract (WCE; lane 4) and 10 ng recombinant TBPwt without histidine tag (lane 5) served as controls. ( B ) Functional investigation of TBP-DR2 expressed in HeLa cells. To analyse whether the expressed TBP-DR2 was functionally active, 50 µg S100 from HeLa cells expressing TBP-DR2 was used to transcribe the P DR2 promoter (lanes 4–6) in vitro before (lane 5) and after (lane 6) doxycycline induction of these HeLa cells. Additionally, 50 µg S100 from untransfected HeLa Tet-on cells was used as a negative control (lane 4). As a control, all three extracts were simultaneously analysed by in vitro transcription of pUhU6 0.35 (lanes 1–3) and pUVAI DNA (lanes 7–9).
    Figure Legend Snippet: Tetracycline inducible expression of TBP-DR2 in HeLa cells. ( A ) Western blot analysis. Cellular extracts from uninduced (lane 1) and doxycycline-induced cells (lane 2) of one of the selected clones were probed with anti-histidine antibodies. Recombinant TBP-DR2 with a histidine tag (10 ng; lane 3), 25 µg HeLa whole-cell extract (WCE; lane 4) and 10 ng recombinant TBPwt without histidine tag (lane 5) served as controls. ( B ) Functional investigation of TBP-DR2 expressed in HeLa cells. To analyse whether the expressed TBP-DR2 was functionally active, 50 µg S100 from HeLa cells expressing TBP-DR2 was used to transcribe the P DR2 promoter (lanes 4–6) in vitro before (lane 5) and after (lane 6) doxycycline induction of these HeLa cells. Additionally, 50 µg S100 from untransfected HeLa Tet-on cells was used as a negative control (lane 4). As a control, all three extracts were simultaneously analysed by in vitro transcription of pUhU6 0.35 (lanes 1–3) and pUVAI DNA (lanes 7–9).

    Techniques Used: Expressing, Western Blot, Clone Assay, Recombinant, Functional Assay, In Vitro, Negative Control

    30) Product Images from "Suppression of A? toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity"

    Article Title: Suppression of A? toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity

    Journal: Biochimica et Biophysica Acta

    doi: 10.1016/j.bbadis.2013.07.019

    PSA levels regulate autophagic flux. (A and D) HeLa Tet-On stable cell lines were treated with (+) or without (–) 1 μg/ml doxycycline (Dox) for 48 h to induce expression of GFP-PSA and GFP-ZBD. (B and E) Endogenous PSA was knocked down (PSA KD) in parental HeLa Tet-On cells (a mock transfection is shown as a control) by siRNA and lysed 48 h post-transfection. The samples were separated by SDS-PAGE and the immunoblot was probed with antibodies against PSA and LC3. β-actin is shown as a loading control. (D and E) The cells were treated with 400 nM bafilomycin A 1 (BFA) for 4 h before lysis. (C and F) Band intensities for total PSA comprised of endogenous PSA and GFP-PSA (black) or GFP-ZBD (gray), LC3 II, and β-actin were quantified by densitometry using ImageJ and normalized to the respective control: − Dox (A and D) or Mock (B and E). The results were displayed graphically by plotting PSA levels against the corresponding LC3 II levels both relative to β-actin. R 2 = 0.92 for the black PSA correlation in (C).
    Figure Legend Snippet: PSA levels regulate autophagic flux. (A and D) HeLa Tet-On stable cell lines were treated with (+) or without (–) 1 μg/ml doxycycline (Dox) for 48 h to induce expression of GFP-PSA and GFP-ZBD. (B and E) Endogenous PSA was knocked down (PSA KD) in parental HeLa Tet-On cells (a mock transfection is shown as a control) by siRNA and lysed 48 h post-transfection. The samples were separated by SDS-PAGE and the immunoblot was probed with antibodies against PSA and LC3. β-actin is shown as a loading control. (D and E) The cells were treated with 400 nM bafilomycin A 1 (BFA) for 4 h before lysis. (C and F) Band intensities for total PSA comprised of endogenous PSA and GFP-PSA (black) or GFP-ZBD (gray), LC3 II, and β-actin were quantified by densitometry using ImageJ and normalized to the respective control: − Dox (A and D) or Mock (B and E). The results were displayed graphically by plotting PSA levels against the corresponding LC3 II levels both relative to β-actin. R 2 = 0.92 for the black PSA correlation in (C).

    Techniques Used: Stable Transfection, Expressing, Transfection, SDS Page, Lysis

    31) Product Images from "Control of Centrin Stability by Aurora A"

    Article Title: Control of Centrin Stability by Aurora A

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021291

    Centrin interacts with APC/C during mitosis. (A) Levels of Aurora A, centrin, and cyclin B were compared in Western blots of lysates of HeLa cells harvested at the indicated time points after synchronization by double thymidine/nocodazole block and release. Cyclin B degradation is used to indicate the onset of anaphase, while the beta-actin blot serves as a loading control. Aurora A and centrin levels drop to basal levels by 150 minutes post-release, while cyclin B is at basal levels by 120 minutes post-release. (B) Equal volumes of immunoprecipitations of lysates from cycling (C) or nocodazole arrested (N) HeLa cells performed with Cdc20 and centrin and Western blotted with the indicated antibodies demonstrate that centrin is pulled down with cdc20 only in nocodazole arrested cells along with Cdc16 and Cdc27. Cdc20 is pulled down with centrin in nocodazole arrested cells and to a slightly lesser extent in cycling cells. The antibody heavy chain and cdc20 were indicated with ( ) and (◀), respectively. (C) In lysates from asynchronously growing HeLa cells only non-phosphorylated centrin immunoprecipitates with cdc20. Cdc20 does not pull down phosphorylated centrin even though p-S170 centrin is abundant, as seen in the lysate-only lane. (D) HeLa Tet-On cells expressing various centrin mutants treated overnight with DMSO (D), ALLnL (A), leupeptin (L), MG132 (M), and ammonium chloride (N) and Western blotted with antibodies directed against total centrin reveal that DMSO, leupeptin, and ammonium chloride do not prevent centrin degradation, whereas ALLnL and MG132, the two proteasome inhibitors, do prevent degradation of wildtype and mutant forms of centrin. Lamin B loading controls are shown for each mutant cell line. (E) Lysates from HeLa Tet-On cells expressing S170A centrin treated with DMSO (D) or ALLnL (A) for 16 hours were Western blotted for centrin and beta-actin show significant degradation products in the presence of ALLnL but not DMSO. Additionally, when the boxed area of the centrin blot is over-exposed, 7 kDa laddering indicative of ubiquitination is evident (Δ). Endogenous and HA-centrin are indicated with ( ) and (▶), respectively.
    Figure Legend Snippet: Centrin interacts with APC/C during mitosis. (A) Levels of Aurora A, centrin, and cyclin B were compared in Western blots of lysates of HeLa cells harvested at the indicated time points after synchronization by double thymidine/nocodazole block and release. Cyclin B degradation is used to indicate the onset of anaphase, while the beta-actin blot serves as a loading control. Aurora A and centrin levels drop to basal levels by 150 minutes post-release, while cyclin B is at basal levels by 120 minutes post-release. (B) Equal volumes of immunoprecipitations of lysates from cycling (C) or nocodazole arrested (N) HeLa cells performed with Cdc20 and centrin and Western blotted with the indicated antibodies demonstrate that centrin is pulled down with cdc20 only in nocodazole arrested cells along with Cdc16 and Cdc27. Cdc20 is pulled down with centrin in nocodazole arrested cells and to a slightly lesser extent in cycling cells. The antibody heavy chain and cdc20 were indicated with ( ) and (◀), respectively. (C) In lysates from asynchronously growing HeLa cells only non-phosphorylated centrin immunoprecipitates with cdc20. Cdc20 does not pull down phosphorylated centrin even though p-S170 centrin is abundant, as seen in the lysate-only lane. (D) HeLa Tet-On cells expressing various centrin mutants treated overnight with DMSO (D), ALLnL (A), leupeptin (L), MG132 (M), and ammonium chloride (N) and Western blotted with antibodies directed against total centrin reveal that DMSO, leupeptin, and ammonium chloride do not prevent centrin degradation, whereas ALLnL and MG132, the two proteasome inhibitors, do prevent degradation of wildtype and mutant forms of centrin. Lamin B loading controls are shown for each mutant cell line. (E) Lysates from HeLa Tet-On cells expressing S170A centrin treated with DMSO (D) or ALLnL (A) for 16 hours were Western blotted for centrin and beta-actin show significant degradation products in the presence of ALLnL but not DMSO. Additionally, when the boxed area of the centrin blot is over-exposed, 7 kDa laddering indicative of ubiquitination is evident (Δ). Endogenous and HA-centrin are indicated with ( ) and (▶), respectively.

    Techniques Used: Western Blot, Blocking Assay, Expressing, Mutagenesis

    S170D-centrin drives centrosome amplification. (A) Constant expression of S170D-centrin drives centrosome amplification in HeLa Tet-On S170D-centrin cells treated continuously with doxycycline. Cells were stained with an antibody against HA (green) and a polyclonal antibody against centrin (red). Scale bar = 10 microns. (B) Cells that were not induced with doxycycline have normal centrosomes. Cells were stained as in panel (A). Scale bar = 10 microns. (C–F) HeLa Tet-On HA-WT-centrin or HA-S170A-centrin cells transfected with GFP-WT Aurora A and allowed to recover for 24 hours. Cells were then transfected with control shRNA or centrin shRNA and treated with doxycycline to induce HA-centrin expression. Aurora A expression was noted by GFP positivity and cells were stained with centrin (red) and gamma-tubulin (turquoise). A vector control Western blot is shown in Figure S3 . Cells over-expressing HA-wildtype centrin in the presence of endogenous centrin have large, numerous centrosomes (C) in virtually all cells. Cells over-expressing HA-wildtype centrin in the absence of endogenous centrin have large, numerous centrosomes (D) in most cells. Conversely cells over-expressing HA-S170A centrin in the presence of endogenous centrin occasionally have slightly enlarged centrosomes (E) and cells over-expressing HA-S170A centrin in the absence of endogenous centrin rarely have slightly enlarged centrosomes (F). GFP positive cells were analyzed for centrosome amplification based on increased size and/or number of centrosomes. The number and percentage of cells with centrosome amplification is presented below each panel. Scale bar = 2.5 microns. (G–I) Frozen breast tissue including normal adjacent breast (G), ductal carcinoma in situ (H) and invasive breast cancers (I) were stained with antibodies directed against Aurora A (green), p-S170 centrin (turquoise), and gamma-tubulin (red). Cells were stained with DAPI to mark the DNA. Scale bar = 10 microns.
    Figure Legend Snippet: S170D-centrin drives centrosome amplification. (A) Constant expression of S170D-centrin drives centrosome amplification in HeLa Tet-On S170D-centrin cells treated continuously with doxycycline. Cells were stained with an antibody against HA (green) and a polyclonal antibody against centrin (red). Scale bar = 10 microns. (B) Cells that were not induced with doxycycline have normal centrosomes. Cells were stained as in panel (A). Scale bar = 10 microns. (C–F) HeLa Tet-On HA-WT-centrin or HA-S170A-centrin cells transfected with GFP-WT Aurora A and allowed to recover for 24 hours. Cells were then transfected with control shRNA or centrin shRNA and treated with doxycycline to induce HA-centrin expression. Aurora A expression was noted by GFP positivity and cells were stained with centrin (red) and gamma-tubulin (turquoise). A vector control Western blot is shown in Figure S3 . Cells over-expressing HA-wildtype centrin in the presence of endogenous centrin have large, numerous centrosomes (C) in virtually all cells. Cells over-expressing HA-wildtype centrin in the absence of endogenous centrin have large, numerous centrosomes (D) in most cells. Conversely cells over-expressing HA-S170A centrin in the presence of endogenous centrin occasionally have slightly enlarged centrosomes (E) and cells over-expressing HA-S170A centrin in the absence of endogenous centrin rarely have slightly enlarged centrosomes (F). GFP positive cells were analyzed for centrosome amplification based on increased size and/or number of centrosomes. The number and percentage of cells with centrosome amplification is presented below each panel. Scale bar = 2.5 microns. (G–I) Frozen breast tissue including normal adjacent breast (G), ductal carcinoma in situ (H) and invasive breast cancers (I) were stained with antibodies directed against Aurora A (green), p-S170 centrin (turquoise), and gamma-tubulin (red). Cells were stained with DAPI to mark the DNA. Scale bar = 10 microns.

    Techniques Used: Amplification, Expressing, Staining, Transfection, shRNA, Plasmid Preparation, Western Blot, In Situ

    32) Product Images from "Pitx2a Expression Alters Actin-Myosin Cytoskeleton and Migration of HeLa Cells through Rho GTPase Signaling V⃞"

    Article Title: Pitx2a Expression Alters Actin-Myosin Cytoskeleton and Migration of HeLa Cells through Rho GTPase Signaling V⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.01-07-0358

    Up-regulation of Trio by Pitx2a. (A) RT-PCR analysis of Trio expression in Pitx2a cells after addition of Dox for different periods of time. A 600-bp DNA fragment from Trio was amplified. Cont, no reverse transcriptase. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was amplified as a control. (B) Quantitation of the data from three independent RT-PCR experiments. (C) Immunoblot analysis of Trio expression after the expression of Pitx2a in HeLa cells by using an antibody specific for Trio. (D) Phase images of HeLa Tet-On cells transfected with empty vector (a), TrioGEF2 (b), or TrioGEF1 (c). Note that expression of TrioGEF1 induces cell spreading and cell morphological changes, but not TrioGEF2. (E) HeLa Tet-On cells were transiently transfected with empty vector (a and b) or plasmids encoding HA-TrioGEF2 (c and d) or Myc-TrioGEF1 (e and f). Twenty-four hours after transfection, cells were fixed and stained with phalloidin (red) and antibodies specific for Myc-tagged or HA-tagged (green) protein. Note that TrioGEF1 induced cell spreading and morphological changes (e and f), but not TrioGEF2. (F) TrioGEF1 induced β-catenin accumulation at the sites of cell-cell contacts. Bar, 20 μm.
    Figure Legend Snippet: Up-regulation of Trio by Pitx2a. (A) RT-PCR analysis of Trio expression in Pitx2a cells after addition of Dox for different periods of time. A 600-bp DNA fragment from Trio was amplified. Cont, no reverse transcriptase. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was amplified as a control. (B) Quantitation of the data from three independent RT-PCR experiments. (C) Immunoblot analysis of Trio expression after the expression of Pitx2a in HeLa cells by using an antibody specific for Trio. (D) Phase images of HeLa Tet-On cells transfected with empty vector (a), TrioGEF2 (b), or TrioGEF1 (c). Note that expression of TrioGEF1 induces cell spreading and cell morphological changes, but not TrioGEF2. (E) HeLa Tet-On cells were transiently transfected with empty vector (a and b) or plasmids encoding HA-TrioGEF2 (c and d) or Myc-TrioGEF1 (e and f). Twenty-four hours after transfection, cells were fixed and stained with phalloidin (red) and antibodies specific for Myc-tagged or HA-tagged (green) protein. Note that TrioGEF1 induced cell spreading and morphological changes (e and f), but not TrioGEF2. (F) TrioGEF1 induced β-catenin accumulation at the sites of cell-cell contacts. Bar, 20 μm.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Quantitation Assay, Transfection, Plasmid Preparation, Staining

    Cell cycle arrest at G0/G1 by serum starvation or dexamethasone inhibit cell migration. (A) After HeLa Tet-On cells were cultured with +serum, −serum, or the addition of 1 μM dexamethasone (+Dexa) for 24 h, a wound was introduced and the cells were continued in culture under the same conditions for another 15 min or 12 h. Cells were fixed and stained with phalloidin. (B) Enlarged images from panels above (12 h) showing no change in cell spreading and morphology.
    Figure Legend Snippet: Cell cycle arrest at G0/G1 by serum starvation or dexamethasone inhibit cell migration. (A) After HeLa Tet-On cells were cultured with +serum, −serum, or the addition of 1 μM dexamethasone (+Dexa) for 24 h, a wound was introduced and the cells were continued in culture under the same conditions for another 15 min or 12 h. Cells were fixed and stained with phalloidin. (B) Enlarged images from panels above (12 h) showing no change in cell spreading and morphology.

    Techniques Used: Migration, Cell Culture, Staining

    33) Product Images from "Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis"

    Article Title: Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis

    Journal: Cells

    doi: 10.3390/cells10102792

    SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p
    Figure Legend Snippet: SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p

    Techniques Used: Expressing, Cell Culture, Western Blot, Immunofluorescence, Quantitative RT-PCR, Transfection, Plasmid Preparation, Confocal Microscopy, Staining, Fluorescence

    Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p
    Figure Legend Snippet: Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Western Blot

    34) Product Images from "Activated RhoA Is a Positive Feedback Regulator of the Lbc Family of Rho Guanine Nucleotide Exchange Factor Proteins *"

    Article Title: Activated RhoA Is a Positive Feedback Regulator of the Lbc Family of Rho Guanine Nucleotide Exchange Factor Proteins *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.450056

    PH domains of Lbc-RhoGEFs block activated RhoA in cells. A , concentration dependence. Tet-On HeLa cells were transfected with increasing amounts of plasmid encoding the PH domain of PRG along with the SRE.L reporter plasmids. After 20 h, cells were treated
    Figure Legend Snippet: PH domains of Lbc-RhoGEFs block activated RhoA in cells. A , concentration dependence. Tet-On HeLa cells were transfected with increasing amounts of plasmid encoding the PH domain of PRG along with the SRE.L reporter plasmids. After 20 h, cells were treated

    Techniques Used: Blocking Assay, Concentration Assay, Transfection, Plasmid Preparation

    Binding of LbcRhoGEFs to activated RhoA in cells increases their tonic activity. A , HeLa Tet-On cells were co-transfected with SRE.L reporter plasmids and increasing amounts of pcDNA3.1-myc-DHPH (wild type or double mutant). Assays for luciferase activity
    Figure Legend Snippet: Binding of LbcRhoGEFs to activated RhoA in cells increases their tonic activity. A , HeLa Tet-On cells were co-transfected with SRE.L reporter plasmids and increasing amounts of pcDNA3.1-myc-DHPH (wild type or double mutant). Assays for luciferase activity

    Techniques Used: Binding Assay, Activity Assay, Transfection, Mutagenesis, Luciferase

    35) Product Images from "A Human Mitochondrial Transcription Factor Is Related to RNA Adenine Methyltransferases and Binds S-Adenosylmethionine"

    Article Title: A Human Mitochondrial Transcription Factor Is Related to RNA Adenine Methyltransferases and Binds S-Adenosylmethionine

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.4.1116-1125.2002

    Human CGI-75 is a mitochondrial protein. The mitochondrial localization of a CGI-75::EGFP fusion protein is shown. HeLa Tet-On cells were transiently transfected with a plasmid (pTRE2-HBGFP) that expresses a CGI-75::EGFP fusion protein and stained with the mitochondrion-specific dye Mitotracker Red. Shown are two representative transfected cells analyzed by fluorescence microscopy for EGFP fluorescence in green (labeled GFP), Mitotracker fluorescence in red (labeled MT), and a merge of the two signals (labeled merge), in which a yellow color indicates colocalization. In the panels on the right, the asterisks indicate the signals derived from a cell that was not transfected with the plasmid. As expected, this cell exhibited Mitotracker fluorescence but not EGFP fluorescence.
    Figure Legend Snippet: Human CGI-75 is a mitochondrial protein. The mitochondrial localization of a CGI-75::EGFP fusion protein is shown. HeLa Tet-On cells were transiently transfected with a plasmid (pTRE2-HBGFP) that expresses a CGI-75::EGFP fusion protein and stained with the mitochondrion-specific dye Mitotracker Red. Shown are two representative transfected cells analyzed by fluorescence microscopy for EGFP fluorescence in green (labeled GFP), Mitotracker fluorescence in red (labeled MT), and a merge of the two signals (labeled merge), in which a yellow color indicates colocalization. In the panels on the right, the asterisks indicate the signals derived from a cell that was not transfected with the plasmid. As expected, this cell exhibited Mitotracker fluorescence but not EGFP fluorescence.

    Techniques Used: Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy, Labeling, Derivative Assay

    36) Product Images from "Rapid purification of protein complexes from mammalian cells"

    Article Title: Rapid purification of protein complexes from mammalian cells

    Journal: Nucleic Acids Research

    doi:

    Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.

    Techniques Used:

    Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.
    Figure Legend Snippet: Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page, Western Blot, Staining

    Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.
    Figure Legend Snippet: Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.

    Techniques Used: SDS Page, Staining, Western Blot

    Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.
    Figure Legend Snippet: Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.

    Techniques Used: Purification, Incubation, Staining, Western Blot

    Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.

    Techniques Used: Transfection, Fluorescence, Expressing, Translocation Assay

    Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).
    Figure Legend Snippet: Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page

    37) Product Images from "Rapid purification of protein complexes from mammalian cells"

    Article Title: Rapid purification of protein complexes from mammalian cells

    Journal: Nucleic Acids Research

    doi:

    Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells harboring pTIP.HA1x3.EGFP. ( A ) Cells grown in the absence of doxycycline visualized under visible (left) and UV light with an EGFP filter (right). ( B ) HeLa Tet-On cells with pTIP.HA1x3.EGFP induced with 1 µg/ml doxycycline visualized under visible (left) and UV light with an EGFP filter (right). Magnification ×600.

    Techniques Used:

    Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.
    Figure Legend Snippet: Purification of p21-containing complexes from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP were induced and lysed as described in Materials and Methods. The p21 fusion protein was purified from the cleared cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with 10 mM glutathione and subjected to SDS–PAGE and western blotting. ( A ) Ponceau staining of the total cell lysate (T) and proteins eluted from the glutathione–Sepharose column (E). ( B ) Filter probed with anti-cyclin A antibody. ( C ) Filter probed with anti-Cdk2 antibody. ( D ) Filter probed with anti-actin antibody.

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page, Western Blot, Staining

    Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.
    Figure Legend Snippet: Cell lysates were prepared from HeLa Tet-On cells containing pTIP.HA1x3.EGFP and electrophoresed by SDS–PAGE. ( A ) Coomassie blue stained gel containing extracts from uninduced (UN) and induced (IN) cells. ( B ) Western blot of identical aliquots probed with a polyclonal anti-EGFP antibody. The appropriately sized HA1-tagged EGFP is expressed in the cells exposed to doxycycline (lane IN) but not in those grown without doxycycline.

    Techniques Used: SDS Page, Staining, Western Blot

    Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.
    Figure Legend Snippet: Purification of GST fusion proteins from crude extracts. An aliquot of the cleared lysate from HeLa Tet-On cells harboring pTIP.GEXP-EGFP and induced for 48 h with 1 µg/ml doxycycline was incubated with a slurry of glutathione–Sepharose, washed and then eluted with either 10 mM glutathione or 8 U of PreScission protease. ( A ) Coomassie blue staining; ( B ) Western blotting with anti-EGFP antibody. Lane 1, total cellular extract; lane 2, glutathione eluted GST–EGFP; lane 3, PreScission protease eluted EGFP.

    Techniques Used: Purification, Incubation, Staining, Western Blot

    Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.
    Figure Legend Snippet: Photomicrograph of HeLa Tet-On cells transfected with pTIP.GEX6P-1.p21.EGFP and induced with 1 µg/ml doxycycline. Approximately half of the cells exhibit green fluorescence of their nuclei, indicating expression and nuclear translocation of the tagged p21 fusion protein. Magnification ×600.

    Techniques Used: Transfection, Fluorescence, Expressing, Translocation Assay

    Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).
    Figure Legend Snippet: Purification of GST–EGFP, GST–cyclin A–EGFP and GST–HRS–EGFP from HeLa cells. HeLa Tet-On cells transfected with pTIP.GEX6P-1.EGFP, pTIP.GEX6P-1.cyclinA.EGFP and pTIP.GEX6P-1.HRS.EGFP were induced and lysed as described in Materials and Methods. The EGFP fusion proteins were purified from the cell lysates by glutathione–Sepharose affinity chromatography. After extensive washing, the bound proteins were eluted with PreScission protease digestion and subjected to SDS–PAGE. The lanes containing the control EGFP eluates were probed with anti-cyclin A antibody (lane 2) and anti-Cdk2 antibody (lane 4) while the lane containing the cyclin A–EGFP eluate was probed with anti-Cdk2 antibody (lane 6). The lane containing the HRS–EGFP eluate was probed with anti-STAM antibody (lane 8). The filters were then stripped and probed with anti-EGFP antibody to confirm the presence of the appropriate bait protein in the eluates (lanes 1, 3, 5 and 7).

    Techniques Used: Purification, Transfection, Affinity Chromatography, SDS Page

    38) Product Images from "Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2Shugoshin Prevents Dissociation of Cohesin from Centromeres During Mitosis in Vertebrate CellsChromosome Cohesion: A Cycle of Holding Together and Falling ApartSeparating Sisters: Shugoshin Protects SA2 at Centromeres but Not at Chromosome Arms"

    Article Title: Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2Shugoshin Prevents Dissociation of Cohesin from Centromeres During Mitosis in Vertebrate CellsChromosome Cohesion: A Cycle of Holding Together and Falling ApartSeparating Sisters: Shugoshin Protects SA2 at Centromeres but Not at Chromosome Arms

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0030069

    Characterization of HeLa Cell Lines Stably Expressing Wild-Type or Mutant Forms of Human Scc1 and SA2 (A) Wild-type Scc1 or SA2, or the indicated mutant proteins (see Figure 1 C), all tagged with 9xmyc at the C terminus, were stably and inducibly expressed in HeLa tet-on cells. After induction by treatment with 2 μg/ml doxycycline for 1–3 d, cell extracts were prepared from either logarithmically proliferating cells (i, interphase) or from cells arrested in mitosis by nocodazole (m, mitosis), then immunoblotted. In the case of Scc1 cell lines (upper blots), only data from interphase extracts are shown. Exogenous protein was detected by immunoblotting with myc antibodies (lower blots). Since the 9xmyc-tag caused a reduced mobility in SDS-PAGE compared to the endogenous protein, Scc1- and SA2-immunoblots (upper blots) revealed the relative amounts of exogenous and endogenous protein in the different cell lines. The position of molecular weight markers is indicated on the right side. (B) Extracts were prepared from the different cell lines as indicated. Immunoprecipitation was performed using myc antibodies, followed by SDS-PAGE and silver staining. As a control, the cohesin complex was immunoprecipitated from untransfected HeLa tet-on cells using antibodies to SA2. (C) Extracts were prepared from SA2-WT-myc or SA2–12xA-myc expressing cells, and fractionated by sucrose density gradient centrifugation (5%–30% sucrose), followed by immunoblotting with antibodies recognizing the proteins indicated on the right (inp. = input/unfractionated sample of the extract).
    Figure Legend Snippet: Characterization of HeLa Cell Lines Stably Expressing Wild-Type or Mutant Forms of Human Scc1 and SA2 (A) Wild-type Scc1 or SA2, or the indicated mutant proteins (see Figure 1 C), all tagged with 9xmyc at the C terminus, were stably and inducibly expressed in HeLa tet-on cells. After induction by treatment with 2 μg/ml doxycycline for 1–3 d, cell extracts were prepared from either logarithmically proliferating cells (i, interphase) or from cells arrested in mitosis by nocodazole (m, mitosis), then immunoblotted. In the case of Scc1 cell lines (upper blots), only data from interphase extracts are shown. Exogenous protein was detected by immunoblotting with myc antibodies (lower blots). Since the 9xmyc-tag caused a reduced mobility in SDS-PAGE compared to the endogenous protein, Scc1- and SA2-immunoblots (upper blots) revealed the relative amounts of exogenous and endogenous protein in the different cell lines. The position of molecular weight markers is indicated on the right side. (B) Extracts were prepared from the different cell lines as indicated. Immunoprecipitation was performed using myc antibodies, followed by SDS-PAGE and silver staining. As a control, the cohesin complex was immunoprecipitated from untransfected HeLa tet-on cells using antibodies to SA2. (C) Extracts were prepared from SA2-WT-myc or SA2–12xA-myc expressing cells, and fractionated by sucrose density gradient centrifugation (5%–30% sucrose), followed by immunoblotting with antibodies recognizing the proteins indicated on the right (inp. = input/unfractionated sample of the extract).

    Techniques Used: Stable Transfection, Expressing, Mutagenesis, SDS Page, Western Blot, Molecular Weight, Immunoprecipitation, Silver Staining, Gradient Centrifugation

    39) Product Images from "Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis"

    Article Title: Unexpected Implication of SRP and AGO2 in Parkinson’s Disease: Involvement in Alpha-Synuclein Biogenesis

    Journal: Cells

    doi: 10.3390/cells10102792

    SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p
    Figure Legend Snippet: SRP Regulates αSyn expression in cultured human cells. SRP54 knockdown leads to decrease in αSyn protein and αSyn mRNA expression in cultured human cells as detected by Western blot ( A , B ), immunofluorescence ( C – E ), and RT-qPCR ( F ). ( A ) Western blot analysis of total cell lysates using antibodies against αSyn, SRP54, and beta-Actin are shown. siSRP54 (siRNA specific for SRP54) was transfected into HeLa Tet-On cells, 24 h later, αSyn plasmid was transfected. Cells were analyzed 48 or 72 h post siRNA transfection. ( B ) Quantification of αSyn Western blots using Image J. αSyn levels were normalized to beta-Actin protein levels and then presented in a graph relative to αSyn protein levels in control cells taken as 1 in the respective time point. Black dashed line indicates αSyn protein levels in control cells. Graph shows mean values ± SE with n = 6 independent experiments at 48 h and n = 13 independent experiments at 72 h after siRNA transfection. ( C ) Immunofluorescence reveals decrease of αSyn expression in cultured human cells upon SRP54 depletion. Cells were transfected with siSRP54, and after 24 h with αSyn expressing plasmid (or mock transfected in controls). Confocal microscopy of αSyn in HeLa Tet-On cells was conducted 48 h after SRP54 siRNA was transfected. αSyn (shown in red) was detected with αSyn antibody and with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images are shown at 60× magnification. ( D ) Depletion of SRP54 expression following siRNA knockdown in HeLa Tet-On cells as observed by confocal microscopy. SRP54 (shown in red) was detected in the cells expressing αSyn in siSRP54 treated or control cells 48 h after siSRP54 was transfected. SRP54 antibody was used with Alexa 555 secondary antibody, nucleus stained with DAPI (shown in blue). Images shown at 60× magnification. ( E ) Corrected total cell fluorescence (CTCF) is expressed in relative fluorescence units and calculated as CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings). All measurements for CTCF calculations were performed in Image J. Graph shows mean values ± SE. n = 39 cells for αSyn mock samples, n = 19 cells for αSyn with siSRP54 samples, n = 18 cells for SRP54 mock and siSRP54 samples. ( F ) αSyn mRNA is downregulated in SRP54 knockdown cultured human cells. Quantification of mRNA expression levels at 48 and 72 h after SRP54 siRNA transfection is shown. mRNA levels measured by RT-qPCR, normalized to beta-Actin mRNA levels and presented relative to αSyn mRNA levels in control cells (black dashed line indicates αSyn mRNA levels in control cells). Graph shows mean values ± SE with a total of 9 independent experiments at 48 h and 12 independent experiments at 72 h after siRNA transfection. Significance determined by paired t test for protein and mRNA, * p

    Techniques Used: Expressing, Cell Culture, Western Blot, Immunofluorescence, Quantitative RT-PCR, Transfection, Plasmid Preparation, Confocal Microscopy, Staining, Fluorescence

    Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p
    Figure Legend Snippet: Depletion of AGO2 Leads to an Increase in αSyn Expression. ( A ) AGO2 expression is significantly decreased in the HeLa Tet-On cells treated with siAGO2. AGO2 mRNA levels were measured by RT-qPCR 48 h after siAGO2 transfection, normalized to HPRT mRNA levels and presented relative to AGO2 mRNA levels in control cells. ( B ) Quantification of αSyn mRNA expression levels at 48 h after AGO2 siRNA transfection. mRNA levels measured by RT-qPCR. αSyn mRNA levels were first normalized to HPRT mRNA levels and then to αSyn mRNA levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. ( C ) Western blot of total cell lysate using αSyn, AGO2, and beta-Actin antibodies (left panel). Quantification of αSyn Western blots using ImageJ (right panel). Normalized to beta-Actin protein levels and then to αSyn protein levels in control cells. Graph shows mean values ± SE with a total of n = 3 independent experiments. Significance determined by paired t test for protein and mRNA, ** p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Western Blot

    40) Product Images from "SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis"

    Article Title: SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

    Journal: Cell Cycle

    doi: 10.1080/15384101.2016.1191713

    SIMs in PICH regulates centromere localization of PICH. (A) PICH WT localizes at the centromere/KT region during mitosis as previously shown. Ectopically expressed EGFP-PICH WT induced by doxycycline visualized in different mitotic stages of HeLa Tet-ON cells. Cells were subjected to immunofluorescence staining for CENPA antibody as a centromere marker. Right panel is the merged images with PICH in green and CENPA in red. White bar on the top right panel indicates 10 μm. (B) Localization of PICH d3SIM is drastically reduced at the centromere during mitosis. Similar to PICH WT, EGFP-tagged PICH d3SIM expression was induced and detected along with CENPA by immunofluorescence. White bar on the top right panel indicates 10 μm.
    Figure Legend Snippet: SIMs in PICH regulates centromere localization of PICH. (A) PICH WT localizes at the centromere/KT region during mitosis as previously shown. Ectopically expressed EGFP-PICH WT induced by doxycycline visualized in different mitotic stages of HeLa Tet-ON cells. Cells were subjected to immunofluorescence staining for CENPA antibody as a centromere marker. Right panel is the merged images with PICH in green and CENPA in red. White bar on the top right panel indicates 10 μm. (B) Localization of PICH d3SIM is drastically reduced at the centromere during mitosis. Similar to PICH WT, EGFP-tagged PICH d3SIM expression was induced and detected along with CENPA by immunofluorescence. White bar on the top right panel indicates 10 μm.

    Techniques Used: Immunofluorescence, Staining, Marker, Expressing

    Polo-like kinase 1 interacting checkpoint helicase (PICH) has multiple SUMO-interacting motifs (SIMs). (A) C-terminus portion of human PICH (616–1250 a.a.) interacts robustly with SUMOylated-PARP1 (SPARP1) Xenoups laevis . Human PICH truncations with GFP tags were expressed in Xenopus egg extract by addition of mRNAs to the extract and pull-down assay was performed. The pulled-down samples were analyzed by immunoblotting using anti-GFP antibody. S-tag HRP depicted the amount of bait in the pull-down samples. (B) Schematic diagram of PICH with SIM sequences at a.a. 912–917 (VSIIEI), 1013–1016 (VVVK) and 1236–1239 (VMLL). Point mutations in each SIM are indicated in blue. (C) Mutations in all SIMs in PICH (PICH d3SIM) drastically reduced PICH binding to SUMOylated-PARP1 (SPARP1). XEE expressing GFP-tagged PICH WT and d3SIM were subjected to pull-down assays. Pulled-down samples were analyzed by immunoblotting using GFP antibody. S-protein HRP was used to detect the amount of bait in the samples. (D) PICH d3SIM is enzymatically active in vitro . Concentration- and time-dependent DNA translocase activity of PICH WT and d3SIM was tested in vitro by triplex assay. Alexa 488 tagged oligo was detected by Typhoon Imager. Positions of DNA triplex and released oligo are indicated at right side. (E and F) SIMs in PICH does not alter PICH's association with UFBs. Ectopically expressed EGFP-tagged PICH WT and PICH d3SIM induced in HeLa Tet-ON cells were detected by fluorescence microscopy. White bar on the merged images indicates 10 μm.
    Figure Legend Snippet: Polo-like kinase 1 interacting checkpoint helicase (PICH) has multiple SUMO-interacting motifs (SIMs). (A) C-terminus portion of human PICH (616–1250 a.a.) interacts robustly with SUMOylated-PARP1 (SPARP1) Xenoups laevis . Human PICH truncations with GFP tags were expressed in Xenopus egg extract by addition of mRNAs to the extract and pull-down assay was performed. The pulled-down samples were analyzed by immunoblotting using anti-GFP antibody. S-tag HRP depicted the amount of bait in the pull-down samples. (B) Schematic diagram of PICH with SIM sequences at a.a. 912–917 (VSIIEI), 1013–1016 (VVVK) and 1236–1239 (VMLL). Point mutations in each SIM are indicated in blue. (C) Mutations in all SIMs in PICH (PICH d3SIM) drastically reduced PICH binding to SUMOylated-PARP1 (SPARP1). XEE expressing GFP-tagged PICH WT and d3SIM were subjected to pull-down assays. Pulled-down samples were analyzed by immunoblotting using GFP antibody. S-protein HRP was used to detect the amount of bait in the samples. (D) PICH d3SIM is enzymatically active in vitro . Concentration- and time-dependent DNA translocase activity of PICH WT and d3SIM was tested in vitro by triplex assay. Alexa 488 tagged oligo was detected by Typhoon Imager. Positions of DNA triplex and released oligo are indicated at right side. (E and F) SIMs in PICH does not alter PICH's association with UFBs. Ectopically expressed EGFP-tagged PICH WT and PICH d3SIM induced in HeLa Tet-ON cells were detected by fluorescence microscopy. White bar on the merged images indicates 10 μm.

    Techniques Used: Pull Down Assay, Binding Assay, Expressing, In Vitro, Concentration Assay, Activity Assay, Fluorescence, Microscopy

    PICH's SIMs role in localization is different from its role in chromatin bridge resolution. (A) Schematic representation of the synchronization method used in the chromatin bridge analysis with thymidine (Thy), doxycycline (Dox), and nocodazole (Noc) addition. (B) Robust defective centromere localization of PICH dSIM3 does not result in chromatin bridge phenotype whereas mutations in SIM1 2 (PICH dSIM1 2) causes chromatin bridge phenotype. Stable HeLa Tet-ON cell lines expressing different PICH constructs were made. Endogenous PICH was depleted by siRNA and nocodazole-arrested cells were released to analyze for anaphase cells. Representative images of different PICH constructs cell lines are shown. White bar on the top right panel indicates 10 μm. (C) Percentage of anaphase cells with chromatin bridges per total anaphase cells were counted and graphed. At least 30 anaphase cells to a maximum of 360 anaphase cells were counted in each experiment (n = 3). Error bars indicate standard deviation and statistical analysis by paired student t-test is indicated as *p
    Figure Legend Snippet: PICH's SIMs role in localization is different from its role in chromatin bridge resolution. (A) Schematic representation of the synchronization method used in the chromatin bridge analysis with thymidine (Thy), doxycycline (Dox), and nocodazole (Noc) addition. (B) Robust defective centromere localization of PICH dSIM3 does not result in chromatin bridge phenotype whereas mutations in SIM1 2 (PICH dSIM1 2) causes chromatin bridge phenotype. Stable HeLa Tet-ON cell lines expressing different PICH constructs were made. Endogenous PICH was depleted by siRNA and nocodazole-arrested cells were released to analyze for anaphase cells. Representative images of different PICH constructs cell lines are shown. White bar on the top right panel indicates 10 μm. (C) Percentage of anaphase cells with chromatin bridges per total anaphase cells were counted and graphed. At least 30 anaphase cells to a maximum of 360 anaphase cells were counted in each experiment (n = 3). Error bars indicate standard deviation and statistical analysis by paired student t-test is indicated as *p

    Techniques Used: Expressing, Construct, Standard Deviation

    SIM3 is most critical for centromeric localization of PICH. (A) Mutation in SIM3 of PICH drastically reduces centromeric association of PICH. EGFP-tagged PICH dSIM3 transiently expressed and induced in HeLa Tet-ON cells. Different stages of mitotic cells isolated and EGFP-PICH dSIM3 visualized along with centromere marker, CENPA by immunofluorescence staining. White bar on the top right panel indicates 10 μm. (B) SIM1 and SIM2 of PICH does not affect centromere localization of PICH in HeLa Tet-ON cells. Similar to PICH dSIM3, PICH dSIM1 2 was ectopically induced and expressed in HeLa Tet-ON cells. PICH mutant was visualized by EGFP along with CENPA by immunostaining analysis. White bar on the top right panel indicates 10 μm. (C) PICH signals of the mutants (d3SIM, dSIM3, and dSIM1 2) at the centromeres were normalized to CENPA levels and quantified relative to PICH WT. Values represent the average of at least 30 different centromeres analyzed in at least 3 different cells in each set (n, n = 3). Error bars indicate standard deviation and statistical analysis by paired student t-test is indicated as **p
    Figure Legend Snippet: SIM3 is most critical for centromeric localization of PICH. (A) Mutation in SIM3 of PICH drastically reduces centromeric association of PICH. EGFP-tagged PICH dSIM3 transiently expressed and induced in HeLa Tet-ON cells. Different stages of mitotic cells isolated and EGFP-PICH dSIM3 visualized along with centromere marker, CENPA by immunofluorescence staining. White bar on the top right panel indicates 10 μm. (B) SIM1 and SIM2 of PICH does not affect centromere localization of PICH in HeLa Tet-ON cells. Similar to PICH dSIM3, PICH dSIM1 2 was ectopically induced and expressed in HeLa Tet-ON cells. PICH mutant was visualized by EGFP along with CENPA by immunostaining analysis. White bar on the top right panel indicates 10 μm. (C) PICH signals of the mutants (d3SIM, dSIM3, and dSIM1 2) at the centromeres were normalized to CENPA levels and quantified relative to PICH WT. Values represent the average of at least 30 different centromeres analyzed in at least 3 different cells in each set (n, n = 3). Error bars indicate standard deviation and statistical analysis by paired student t-test is indicated as **p

    Techniques Used: Mutagenesis, Isolation, Marker, Immunofluorescence, Staining, Immunostaining, Standard Deviation

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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
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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

    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

    A . Expression and production of Hcp in transfected HeLa Tet-Off cells. Panel I . Western blot analysis showing production of Hcp in whole cell lysates of HeLa Tet-Off cells after 24 hr of transfection with pBI-EGFP- hcp (lane 2) or pBI-EGFP (empty vector)

    Journal: Microbial pathogenesis

    Article Title: Molecular Characterization of a Functional Type VI Secretion System from a Clinical Isolate of Aeromonas hydrophila

    doi: 10.1016/j.micpath.2007.10.005

    Figure Lengend Snippet: A . Expression and production of Hcp in transfected HeLa Tet-Off cells. Panel I . Western blot analysis showing production of Hcp in whole cell lysates of HeLa Tet-Off cells after 24 hr of transfection with pBI-EGFP- hcp (lane 2) or pBI-EGFP (empty vector)

    Article Snippet: HeLa Tet-Off™ , a human cervical epithelial cell line, was obtained from Clontech (Mountain View, CA).

    Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation