monoclonal anti flag  (Millipore)


Bioz Verified Symbol Millipore is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Monoclonal ANTI FLAG antibody
    Description:
    This rabbit ANTI FLAG antibody is produced from the hybridoma SIG1 25 The hybridoma is derived by the fusion of myeloma cells and splenocytes from rabbits immunized with the FLAG R peptide sequence conjugated to KLH
    Catalog Number:
    f2555
    Price:
    None
    Applications:
    Antibody suitable for use in immunoblotting,immunocytochemistry, and indirect ELISA.Browse additional application references in our FLAG(R) Literature portal.
    Buy from Supplier


    Structured Review

    Millipore monoclonal anti flag
    Monoclonal ANTI FLAG antibody
    This rabbit ANTI FLAG antibody is produced from the hybridoma SIG1 25 The hybridoma is derived by the fusion of myeloma cells and splenocytes from rabbits immunized with the FLAG R peptide sequence conjugated to KLH
    https://www.bioz.com/result/monoclonal anti flag/product/Millipore
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Phosphorylation of the arginine/serine dipeptide‐rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization"

    Article Title: Phosphorylation of the arginine/serine dipeptide‐rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization

    Journal: The Febs Journal

    doi: 10.1111/j.1742-4658.2008.06564.x

    Translocation of the SARS‐CoV N protein to cytoplasmic granules can be induced by cell stress and modulated by phosphorylation. (A) Expression vector encoding FLAG‐tagged full‐length (N), RS motif‐deleted (NΔRS), two serine‐to‐alanine mutants (N‐6A and N‐14A), N‐terminal‐half (N NT ) or C‐terminal‐half (N CT ) N protein was transiently transfected into HeLa cells. Upper panel: representative fluorescence images. Lower panel: percentage of granule‐positive cells; approximately 100 transfected cells were counted for each protein. (B) HeLa cells transiently expressing HA‐tagged N or NΔRS or coexpressing HA‐N and GFP‐TIA‐1 were mock treated (−) or treated (+) with 0.5 m m arsenite for 1 h. Double immunofluorescence was performed using anti‐HA and anti‐PABP. A merged image is shown in the right‐hand panel.
    Figure Legend Snippet: Translocation of the SARS‐CoV N protein to cytoplasmic granules can be induced by cell stress and modulated by phosphorylation. (A) Expression vector encoding FLAG‐tagged full‐length (N), RS motif‐deleted (NΔRS), two serine‐to‐alanine mutants (N‐6A and N‐14A), N‐terminal‐half (N NT ) or C‐terminal‐half (N CT ) N protein was transiently transfected into HeLa cells. Upper panel: representative fluorescence images. Lower panel: percentage of granule‐positive cells; approximately 100 transfected cells were counted for each protein. (B) HeLa cells transiently expressing HA‐tagged N or NΔRS or coexpressing HA‐N and GFP‐TIA‐1 were mock treated (−) or treated (+) with 0.5 m m arsenite for 1 h. Double immunofluorescence was performed using anti‐HA and anti‐PABP. A merged image is shown in the right‐hand panel.

    Techniques Used: Translocation Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Immunofluorescence

    Overexpression of SRPK1 prevents N protein translocation to stress granules. HeLa cells were transiently cotransfected with vectors encoding FLAG‐tagged full‐length N, N‐14A or NΔRS and HA‐tagged SRPK1, and treated with arsenite as in Fig. 4B . Immunofluorescence using anti‐HA and anti‐FLAG was performed; two representative images are shown for the N protein. Arrowheads indicate cells that expressed FLAG‐N protein alone, and white arrows indicate cells expressing both FLAG‐N and HA‐SRPK1. Cell nuclei were stained with 4′,6′‐diamidino‐2‐phenylindole (DAPI). The lower panel shows double immunofluorescence of HA‐SRPK1‐overexpressing HeLa cells using anti‐HA and anti‐PABP. Yellow arrows indicate cells that overexpressed HA‐SRPK1.
    Figure Legend Snippet: Overexpression of SRPK1 prevents N protein translocation to stress granules. HeLa cells were transiently cotransfected with vectors encoding FLAG‐tagged full‐length N, N‐14A or NΔRS and HA‐tagged SRPK1, and treated with arsenite as in Fig. 4B . Immunofluorescence using anti‐HA and anti‐FLAG was performed; two representative images are shown for the N protein. Arrowheads indicate cells that expressed FLAG‐N protein alone, and white arrows indicate cells expressing both FLAG‐N and HA‐SRPK1. Cell nuclei were stained with 4′,6′‐diamidino‐2‐phenylindole (DAPI). The lower panel shows double immunofluorescence of HA‐SRPK1‐overexpressing HeLa cells using anti‐HA and anti‐PABP. Yellow arrows indicate cells that overexpressed HA‐SRPK1.

    Techniques Used: Over Expression, Translocation Assay, Immunofluorescence, Expressing, Staining

    2) Product Images from "Serine 111 Phosphorylation Regulates OCT4A Protein Subcellular Distribution and Degradation"

    Article Title: Serine 111 Phosphorylation Regulates OCT4A Protein Subcellular Distribution and Degradation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.386755

    OCT4A is phosphorylated at Ser-111. A , lysates from NTera2 cells in proliferation ( Prol ) or serum-starved overnight and treated with CIP or stimulated with FGF2 for 30 min were subjected to immunoblotting. B , HeLa cells transfected with FLAG-OCT4A and
    Figure Legend Snippet: OCT4A is phosphorylated at Ser-111. A , lysates from NTera2 cells in proliferation ( Prol ) or serum-starved overnight and treated with CIP or stimulated with FGF2 for 30 min were subjected to immunoblotting. B , HeLa cells transfected with FLAG-OCT4A and

    Techniques Used: Transfection

    3) Product Images from "Characterizing WW Domain Interactions of Tumor Suppressor WWOX Reveals Its Association with Multiprotein Networks *"

    Article Title: Characterizing WW Domain Interactions of Tumor Suppressor WWOX Reveals Its Association with Multiprotein Networks *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.506790

    Physical association between ITCH and WWOX. A , HEK293 cells were transiently cotransfected with MYC-ITCH and GST-WW1 or GST-WW1-WFPA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. B , HEK293 cells were transiently transfected with MYC-WWOX ( M ) or MYC-WWOX and FLAG-ITCH ( M + F ). After 24 h, cells were lysed, and immunoprecipitation ( IP ) using anti-MYC ( M ), anti-FLAG ( F ), or anti-IgG ( G ) was performed. Precipitates and lysates (input) were immunoblotted with antibodies against FLAG (ITCH) or MYC (WWOX). C , HEK293 cells were co-transfected with MYC-WWOX and GST-ITCH or GST-NEDD4. Cells were treated as in A . Densitometry shows band quantification. D , endogenous interaction. Lysates of HEK293 cells were immunoprecipitated with anti-WWOX or anti-IgG, and complexes were immunoblotted with anti-WWOX and anti-ITCH. E , HEK293 cells were transiently cotransfected with the indicated expression vector ( top ). At 24 h, cell lysates were probed with anti-FLAG (ITCH). GAPDH was used for normalization. DM , double mutant (Y623A,Y839A). Densitometry shows band quantification. F , HEK293 cells were transiently cotransfected with GST-WWOX and the indicated ITCH expression vector ( top ). At 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. Densitometry shows band quantification.
    Figure Legend Snippet: Physical association between ITCH and WWOX. A , HEK293 cells were transiently cotransfected with MYC-ITCH and GST-WW1 or GST-WW1-WFPA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. B , HEK293 cells were transiently transfected with MYC-WWOX ( M ) or MYC-WWOX and FLAG-ITCH ( M + F ). After 24 h, cells were lysed, and immunoprecipitation ( IP ) using anti-MYC ( M ), anti-FLAG ( F ), or anti-IgG ( G ) was performed. Precipitates and lysates (input) were immunoblotted with antibodies against FLAG (ITCH) or MYC (WWOX). C , HEK293 cells were co-transfected with MYC-WWOX and GST-ITCH or GST-NEDD4. Cells were treated as in A . Densitometry shows band quantification. D , endogenous interaction. Lysates of HEK293 cells were immunoprecipitated with anti-WWOX or anti-IgG, and complexes were immunoblotted with anti-WWOX and anti-ITCH. E , HEK293 cells were transiently cotransfected with the indicated expression vector ( top ). At 24 h, cell lysates were probed with anti-FLAG (ITCH). GAPDH was used for normalization. DM , double mutant (Y623A,Y839A). Densitometry shows band quantification. F , HEK293 cells were transiently cotransfected with GST-WWOX and the indicated ITCH expression vector ( top ). At 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. Densitometry shows band quantification.

    Techniques Used: Transfection, Immunoprecipitation, Expressing, Plasmid Preparation, Mutagenesis

    Validation of putative PP X Y-containing partners of WWOX identified by MS. A , HEK293 cells were transiently cotransfected with FLAG-DVL1 and GST-WW1 or GST-WW1-WFPA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. B , HEK293 cells were transiently cotransfected with FLAG-DVL1 or FLAG-DVL1Y553A and GST-WWOX or GST-WWOX-WFPA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. Numbers below the DVL1 blots represent quantification of bands relative to the GST blot. C , HEK293 cells were transiently cotransfected with FLAG-DVL1 and GST-WWOX or GST-WWOX-WFPA or GST-SDR. Cells were treated as in B. D , HEK293 cells were transiently transfected with FLAG-AMOTL1 and GST-WWOX or GST-WWOX-WFPA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. Densitometry shows band quantification. E , HEK293 cells were transiently transfected with GST-WWOX and FLAG-AMOTL1 or FLAG-AMOTL1-PPEA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. Densitometry shows band quantification. F , endogenous interaction. HEK293 cells were lysed and immunoprecipitated using rabbit ( r ) or mouse ( m ) anti-WWOX antibody. Precipitates were blotted using anti-WWOX, anti-angiomotin, anti-ITCH, or anti-DVL antibodies. Anti-IgG ( r or m ) was used as a control.
    Figure Legend Snippet: Validation of putative PP X Y-containing partners of WWOX identified by MS. A , HEK293 cells were transiently cotransfected with FLAG-DVL1 and GST-WW1 or GST-WW1-WFPA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. B , HEK293 cells were transiently cotransfected with FLAG-DVL1 or FLAG-DVL1Y553A and GST-WWOX or GST-WWOX-WFPA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. Numbers below the DVL1 blots represent quantification of bands relative to the GST blot. C , HEK293 cells were transiently cotransfected with FLAG-DVL1 and GST-WWOX or GST-WWOX-WFPA or GST-SDR. Cells were treated as in B. D , HEK293 cells were transiently transfected with FLAG-AMOTL1 and GST-WWOX or GST-WWOX-WFPA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. Densitometry shows band quantification. E , HEK293 cells were transiently transfected with GST-WWOX and FLAG-AMOTL1 or FLAG-AMOTL1-PPEA. After 24 h, cells were lysed, and GST pull-down was performed. Precipitates and lysates (input) were immunoblotted with the indicated antibodies. Densitometry shows band quantification. F , endogenous interaction. HEK293 cells were lysed and immunoprecipitated using rabbit ( r ) or mouse ( m ) anti-WWOX antibody. Precipitates were blotted using anti-WWOX, anti-angiomotin, anti-ITCH, or anti-DVL antibodies. Anti-IgG ( r or m ) was used as a control.

    Techniques Used: Mass Spectrometry, Transfection, Immunoprecipitation

    4) Product Images from "The C-Terminal Domain of CENP-C Displays Multiple and Critical Functions for Mammalian Centromere Formation"

    Article Title: The C-Terminal Domain of CENP-C Displays Multiple and Critical Functions for Mammalian Centromere Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005832

    The Mif2p homology domain III of CENP-C possesses a self-associating activity in vivo. Cells HEK-293T were transiently transfected with the entire C-terminal region of CENP-C (HA::638/943) and FLAG::CENP-C constructs as indicated in the diagrams (A, FLAG::23/410; B, FLAG::638/943; C, FLAG::638/819 and D, FLAG::760/943). After 24 hours of expression, nuclear extracts were performed and proteins were immunoprecipitated through an anti-HA affinity matrix. Interactions between CENP-C domains are revealed by the presence of FLAG-tagged proteins in the IP anti-HA sample. The diagram in each panel illustrates the HA and FLAG CENP-C derivatives as compared to the full length protein: Mif2p homology domain II (light grey), Mif2p homology domain III (dark grey), central DNA binding domain (dotted box), HA-tag (black box), FLAG-tag (white box).
    Figure Legend Snippet: The Mif2p homology domain III of CENP-C possesses a self-associating activity in vivo. Cells HEK-293T were transiently transfected with the entire C-terminal region of CENP-C (HA::638/943) and FLAG::CENP-C constructs as indicated in the diagrams (A, FLAG::23/410; B, FLAG::638/943; C, FLAG::638/819 and D, FLAG::760/943). After 24 hours of expression, nuclear extracts were performed and proteins were immunoprecipitated through an anti-HA affinity matrix. Interactions between CENP-C domains are revealed by the presence of FLAG-tagged proteins in the IP anti-HA sample. The diagram in each panel illustrates the HA and FLAG CENP-C derivatives as compared to the full length protein: Mif2p homology domain II (light grey), Mif2p homology domain III (dark grey), central DNA binding domain (dotted box), HA-tag (black box), FLAG-tag (white box).

    Techniques Used: Activity Assay, In Vivo, Transfection, Construct, Expressing, Immunoprecipitation, Binding Assay, FLAG-tag

    CENP-C can interact with CENP-A and histone H3 through its C-terminal domain. (A) HA::CENP-A coimmunoprecipitates the endogenous CENP-C. HEK-293 cells were transfected with HA::CENP-A or with the HA empty vector (pcDNA3.1HA) and HA proteins were purified from nuclear extracts with anti-HA affinity matrix. The presence of the endogenous CENP-C in the immunoprecipitated samples was determined by western blot with an anti-CENP-C antibody (B) Overexpressed HA::CENP-A coimmunoprecipitates FLAG C-terminal CENP-C domain. (C) Coimmunoprecipitation of overexpressed HA::CENP-C domains along with FLAG-tagged histone proteins and c-Myc protein. Only the construct that contains the C-terminal CENP-C region is able to interact with both CENP-A and histone H3.
    Figure Legend Snippet: CENP-C can interact with CENP-A and histone H3 through its C-terminal domain. (A) HA::CENP-A coimmunoprecipitates the endogenous CENP-C. HEK-293 cells were transfected with HA::CENP-A or with the HA empty vector (pcDNA3.1HA) and HA proteins were purified from nuclear extracts with anti-HA affinity matrix. The presence of the endogenous CENP-C in the immunoprecipitated samples was determined by western blot with an anti-CENP-C antibody (B) Overexpressed HA::CENP-A coimmunoprecipitates FLAG C-terminal CENP-C domain. (C) Coimmunoprecipitation of overexpressed HA::CENP-C domains along with FLAG-tagged histone proteins and c-Myc protein. Only the construct that contains the C-terminal CENP-C region is able to interact with both CENP-A and histone H3.

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

    The Mif2p homology domain III mediates the interaction of CENP-C with CENP-A and histone H3. Coimmunoprecipitation of overexpressed HA::CENP-C C-terminal domains along with FLAG-tagged CENP-A and histone H3. Constructs containing the Mif2 homology domain III shows the ability to interact with both CENP-A and histone H3.
    Figure Legend Snippet: The Mif2p homology domain III mediates the interaction of CENP-C with CENP-A and histone H3. Coimmunoprecipitation of overexpressed HA::CENP-C C-terminal domains along with FLAG-tagged CENP-A and histone H3. Constructs containing the Mif2 homology domain III shows the ability to interact with both CENP-A and histone H3.

    Techniques Used: Construct

    5) Product Images from "SCFβ-TRCP suppresses angiogenesis and thyroid cancer cell migration by promoting ubiquitination and destruction of VEGF receptor 2"

    Article Title: SCFβ-TRCP suppresses angiogenesis and thyroid cancer cell migration by promoting ubiquitination and destruction of VEGF receptor 2

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20112446

    CKI is involved in the regulation of VEGFR2 stability. (A) Immunoblot analysis of 293T cells transfected with HA-VEGFR2, Flag–β-TRCP1, and indicated kinases. Where indicated, cells were treated with the proteasome inhibitor MG132. Data shown is representative of two independent experiments. (B) Immunoblot analysis of 293T cells transfected with HA-VEGFR2 and/or Myc-CKIδ together with Flag-WT–β-TRCP1 or Flag-R474A–β-TRCP1. Data shown is representative of two independent experiments. (C and D) Immunoblot analysis of HMVECs treated with the CKI inhibitor D4476 at the indicated concentrations for 12 h. In D, where indicated, 100 ng/ml VEGF-A was added for 2 h before harvesting. Data shown is representative of two independent experiments. (E) Immunoblot analysis of WCLs and immunoprecipitates (IP) derived from 293T cells transfected with HA-VEGFR2 and Myc-tagged versions of indicated CKI isoforms. Data shown is representative of two independent experiments. (F) Immunoblot analysis of HMVECs infected with shRNA specific for GFP or the indicated CKI isoforms. Data shown is representative of two independent experiments. (G) HMVECs were transfected with the indicated shRNA constructs. 40 h after infection, cells were treated with 1 µg/ml puromycin for 72 h to eliminate the noninfected cells. Afterward, the resulting cells were split into 60-mm dishes and, after another 20 h, were treated with 20 µg/ml CHX. At the indicated time points, WCLs were prepared, and immunoblots were probed with the indicated antibodies. Data shown is representative of three independent experiments. (H) Quantification of the band intensities in G. VEGFR2 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD ( n = 3). (I) 293T cells were transfected with EV or a construct encoding CKIδ. 20 h after transfection, cells were split into 60-mm dishes, and after another 20 h cells were treated with 20 µg/ml CHX. At the indicated time points, WCLs were prepared, and immunoblots were probed with the indicated antibodies. Data shown is representative of two independent experiments.
    Figure Legend Snippet: CKI is involved in the regulation of VEGFR2 stability. (A) Immunoblot analysis of 293T cells transfected with HA-VEGFR2, Flag–β-TRCP1, and indicated kinases. Where indicated, cells were treated with the proteasome inhibitor MG132. Data shown is representative of two independent experiments. (B) Immunoblot analysis of 293T cells transfected with HA-VEGFR2 and/or Myc-CKIδ together with Flag-WT–β-TRCP1 or Flag-R474A–β-TRCP1. Data shown is representative of two independent experiments. (C and D) Immunoblot analysis of HMVECs treated with the CKI inhibitor D4476 at the indicated concentrations for 12 h. In D, where indicated, 100 ng/ml VEGF-A was added for 2 h before harvesting. Data shown is representative of two independent experiments. (E) Immunoblot analysis of WCLs and immunoprecipitates (IP) derived from 293T cells transfected with HA-VEGFR2 and Myc-tagged versions of indicated CKI isoforms. Data shown is representative of two independent experiments. (F) Immunoblot analysis of HMVECs infected with shRNA specific for GFP or the indicated CKI isoforms. Data shown is representative of two independent experiments. (G) HMVECs were transfected with the indicated shRNA constructs. 40 h after infection, cells were treated with 1 µg/ml puromycin for 72 h to eliminate the noninfected cells. Afterward, the resulting cells were split into 60-mm dishes and, after another 20 h, were treated with 20 µg/ml CHX. At the indicated time points, WCLs were prepared, and immunoblots were probed with the indicated antibodies. Data shown is representative of three independent experiments. (H) Quantification of the band intensities in G. VEGFR2 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD ( n = 3). (I) 293T cells were transfected with EV or a construct encoding CKIδ. 20 h after transfection, cells were split into 60-mm dishes, and after another 20 h cells were treated with 20 µg/ml CHX. At the indicated time points, WCLs were prepared, and immunoblots were probed with the indicated antibodies. Data shown is representative of two independent experiments.

    Techniques Used: Transfection, Derivative Assay, Infection, shRNA, Construct, Western Blot

    6) Product Images from "Autoinhibitory Regulation of p73 by ?Np73 To Modulate Cell Survival and Death through a p73-Specific Target Element within the ?Np73 Promoter"

    Article Title: Autoinhibitory Regulation of p73 by ?Np73 To Modulate Cell Survival and Death through a p73-Specific Target Element within the ?Np73 Promoter

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.8.2575-2585.2002

    Functional interactions between p73 and ΔNp73. (A) Immunoprecipitation and Western blot analysis. 293 cells were transiently transfected with the indicated expression plasmids. Whole-cell lysates (400 μg of protein) were subjected to immunoprecipitation (IP) with anti-HA antibody, and the precipitated proteins were analyzed by immunoblotting (IB) with anti-FLAG M2 antibody. ΔNp73α and ΔNp73β are indicated by closed and open arrowheads, respectively. The asterisk indicates the position of heavy-chain immunoglobulin G. (B) p53 interacts with ΔNp73α or ΔNp73β in the COS7 cells. The cells were transfected with 8 μg each of the indicated expression plasmids. At 48 h after transfection, whole-cell lysates (1.5 mg of protein) were prepared, followed by immunoprecipitation with anti-p53 (DO-1/PAb1801) antibodies and immunoblotting with the anti-ΔNp73 antibody (top). ΔNp73α and ΔNp73β are indicated by closed and open arrowheads, respectively. The expression of ΔNp73 and endogenous p53 was examined by immunoblotting with the anti-ΔNp73 and anti-p53 antibodies, respectively (middle and bottom, respectively). (C) p53 interacts with ΔNp73α or ΔNp73β in H1299 cells. The cells were transiently transfected with 4 μg each of the indicated expression plasmids. At 48 h after transfection, whole-cell lysates (1.5 mg of protein) were prepared, followed by immunoprecipitation with the anti-ΔNp73 antibody and immunoblotting with the anti-p53 antibody (top). The expression of ΔNp73 and p53 was examined by immunoblotting with the anti-ΔNp73 and anti-p53 antibody, respectively (middle and bottom, respectively). ΔNp73α and ΔNp73β are indicated by closed and open arrowheads, respectively. For luciferase assays, SAOS-2 cells were cotransfected with the indicated expression plasmids, together with a reporter plasmid containing the MDM2 (D), Bax (E), or ΔNp73 (F) promoter driving luciferase expression. At 48 h posttransfection, cells were lysed and subjected to the luciferase assays. The data shown are mean values ± SD.
    Figure Legend Snippet: Functional interactions between p73 and ΔNp73. (A) Immunoprecipitation and Western blot analysis. 293 cells were transiently transfected with the indicated expression plasmids. Whole-cell lysates (400 μg of protein) were subjected to immunoprecipitation (IP) with anti-HA antibody, and the precipitated proteins were analyzed by immunoblotting (IB) with anti-FLAG M2 antibody. ΔNp73α and ΔNp73β are indicated by closed and open arrowheads, respectively. The asterisk indicates the position of heavy-chain immunoglobulin G. (B) p53 interacts with ΔNp73α or ΔNp73β in the COS7 cells. The cells were transfected with 8 μg each of the indicated expression plasmids. At 48 h after transfection, whole-cell lysates (1.5 mg of protein) were prepared, followed by immunoprecipitation with anti-p53 (DO-1/PAb1801) antibodies and immunoblotting with the anti-ΔNp73 antibody (top). ΔNp73α and ΔNp73β are indicated by closed and open arrowheads, respectively. The expression of ΔNp73 and endogenous p53 was examined by immunoblotting with the anti-ΔNp73 and anti-p53 antibodies, respectively (middle and bottom, respectively). (C) p53 interacts with ΔNp73α or ΔNp73β in H1299 cells. The cells were transiently transfected with 4 μg each of the indicated expression plasmids. At 48 h after transfection, whole-cell lysates (1.5 mg of protein) were prepared, followed by immunoprecipitation with the anti-ΔNp73 antibody and immunoblotting with the anti-p53 antibody (top). The expression of ΔNp73 and p53 was examined by immunoblotting with the anti-ΔNp73 and anti-p53 antibody, respectively (middle and bottom, respectively). ΔNp73α and ΔNp73β are indicated by closed and open arrowheads, respectively. For luciferase assays, SAOS-2 cells were cotransfected with the indicated expression plasmids, together with a reporter plasmid containing the MDM2 (D), Bax (E), or ΔNp73 (F) promoter driving luciferase expression. At 48 h posttransfection, cells were lysed and subjected to the luciferase assays. The data shown are mean values ± SD.

    Techniques Used: Functional Assay, Immunoprecipitation, Western Blot, Transfection, Expressing, Luciferase, Plasmid Preparation

    Specificity of the anti-ΔNp73 antibody and identification of ΔNp73α in SH-SY5Y cells infected with recombinant adenovirus for HA-p73α. (A) FLAG-tagged p73α, p73β, ΔNp73α, and ΔNp73β were generated in vitro by using the rabbit reticulocyte lysate, subjected to SDS-PAGE (10% polyacrylamide), and transferred to a nitrocellulose membrane, and the membrane was probed with the monoclonal anti-FLAG antibody at a dilution of 1:3,000 (top). Arrowheads indicate the position of each product. Similarly, the in vitro-translated products were immunoblotted with the polyclonal anti-ΔNp73 antibody at a dilution of 1:10,000 (bottom). Arrowheads indicate the positions of ΔNp73α and ΔNp73β. The asterisk indicates a nonspecific protein. The positions of molecular mass markers are marked at the left of each panel in kilodaltons. (B) At the indicated times after infection with recombinant adenovirus for HA-p73α, SH-SY5Y cell lysates were prepared, subjected to SDS-8% PAGE, and immunoblotted with the monoclonal anti-p73α antibody (Ab-1; Oncogene Research Products) (top) or with the polyclonal anti-ΔNp73 antibody (middle). The p73α blot was stripped and reprobed with the anti-actin antibody to ensure equal protein loading (bottom). The positions of the molecular size standards are indicated on the left in kilodaltons.
    Figure Legend Snippet: Specificity of the anti-ΔNp73 antibody and identification of ΔNp73α in SH-SY5Y cells infected with recombinant adenovirus for HA-p73α. (A) FLAG-tagged p73α, p73β, ΔNp73α, and ΔNp73β were generated in vitro by using the rabbit reticulocyte lysate, subjected to SDS-PAGE (10% polyacrylamide), and transferred to a nitrocellulose membrane, and the membrane was probed with the monoclonal anti-FLAG antibody at a dilution of 1:3,000 (top). Arrowheads indicate the position of each product. Similarly, the in vitro-translated products were immunoblotted with the polyclonal anti-ΔNp73 antibody at a dilution of 1:10,000 (bottom). Arrowheads indicate the positions of ΔNp73α and ΔNp73β. The asterisk indicates a nonspecific protein. The positions of molecular mass markers are marked at the left of each panel in kilodaltons. (B) At the indicated times after infection with recombinant adenovirus for HA-p73α, SH-SY5Y cell lysates were prepared, subjected to SDS-8% PAGE, and immunoblotted with the monoclonal anti-p73α antibody (Ab-1; Oncogene Research Products) (top) or with the polyclonal anti-ΔNp73 antibody (middle). The p73α blot was stripped and reprobed with the anti-actin antibody to ensure equal protein loading (bottom). The positions of the molecular size standards are indicated on the left in kilodaltons.

    Techniques Used: Infection, Recombinant, Generated, In Vitro, SDS Page, Polyacrylamide Gel Electrophoresis

    7) Product Images from "The M-phase specific hyperphosphorylation of Staufen2 involved the cyclin-dependent kinase CDK1"

    Article Title: The M-phase specific hyperphosphorylation of Staufen2 involved the cyclin-dependent kinase CDK1

    Journal: BMC Cell Biology

    doi: 10.1186/s12860-017-0142-z

    STAU2 is hyperphosphorylated during the M-phase. a shRNA control (shCtrl) or against STAU2 (shSTAU2) were cloned in a retrovirus vector to infect hTert-RPE1 and HeLa cells. Cells were synchronized in prometaphase with nocodazole and mitotic cells were enriched by gentle shake off (Ndz + S.off). Protein extracts from asynchronous (−) and synchronized cells (+) were analyzed by western blotting to detect STAU2 migration and cell cycle markers (MPM2 and cyclins). β-actin was used as loading control. b Schematic representation of STAU2 expression vectors, STAU2 52 -FLAG 3 and STAU2 59 -FLAG 3 . Dark gray boxes, double-stranded RNA-binding domains (dsRBD); light gray boxes, tubulin-binding domain (TBD); white boxes, FLAG 3 . c hTert-RPE1 cells infected with viruses expressing the empty pMSCV vector (pMSCV), STAU2 52 -FLAG 3 , STAU2 59 -FLAG 3 or both were synchronized (+) by nocodazole and shake off (Ndz + S.off). Migration of STAU2 proteins was detected by SDS-PAGE and western blotting. Both endogenous and overexpressed STAU2 59 -FLAG 3 were analyzed with anti-STAU2 antibody, while anti-FLAG antibody was used to specifically recognize FLAG 3 -tagged STAU2 isoforms. Mitotic marker accumulation was assessed with anti-MPM2 and anti-cyclin B1 antibodies. Loading was normalized with β-actin antibody. d Asynchronous (−) and nocodazole-treated (Ndz)(+) hTert-RPE1 cells were lysed and protein extracts were subjected to separation on phospho-columns. Input from total extracts (I), flow through (F) and phospho-eluates (P) were analyzed by western blotting using anti-STAU2. Anti-RSK1, anti-nucleolin and anti-β-actin antibodies were used as controls for phosphorylated and unphosphorylated proteins, respectively. e Protein extracts from nocodazole-treated (+) hTert-RPE1 and HeLa were incubated in vitro with either water (H2O), Lambda Phosphatase (λPP), inactivated Lambda Phosphatase (λPPin), Calf Intestinal Alkaline Phosphatase (CIP) or inactivated Calf Intestinal Alkaline Phosphatase (CIPin). STAU2 phosphorylation status was analyzed by SDS-PAGE and western blotting. Untreated cells (Mock) were used as control for dephosphorylated STAU2. All western blots are representatives of three independently performed experiments that showed similar profiles
    Figure Legend Snippet: STAU2 is hyperphosphorylated during the M-phase. a shRNA control (shCtrl) or against STAU2 (shSTAU2) were cloned in a retrovirus vector to infect hTert-RPE1 and HeLa cells. Cells were synchronized in prometaphase with nocodazole and mitotic cells were enriched by gentle shake off (Ndz + S.off). Protein extracts from asynchronous (−) and synchronized cells (+) were analyzed by western blotting to detect STAU2 migration and cell cycle markers (MPM2 and cyclins). β-actin was used as loading control. b Schematic representation of STAU2 expression vectors, STAU2 52 -FLAG 3 and STAU2 59 -FLAG 3 . Dark gray boxes, double-stranded RNA-binding domains (dsRBD); light gray boxes, tubulin-binding domain (TBD); white boxes, FLAG 3 . c hTert-RPE1 cells infected with viruses expressing the empty pMSCV vector (pMSCV), STAU2 52 -FLAG 3 , STAU2 59 -FLAG 3 or both were synchronized (+) by nocodazole and shake off (Ndz + S.off). Migration of STAU2 proteins was detected by SDS-PAGE and western blotting. Both endogenous and overexpressed STAU2 59 -FLAG 3 were analyzed with anti-STAU2 antibody, while anti-FLAG antibody was used to specifically recognize FLAG 3 -tagged STAU2 isoforms. Mitotic marker accumulation was assessed with anti-MPM2 and anti-cyclin B1 antibodies. Loading was normalized with β-actin antibody. d Asynchronous (−) and nocodazole-treated (Ndz)(+) hTert-RPE1 cells were lysed and protein extracts were subjected to separation on phospho-columns. Input from total extracts (I), flow through (F) and phospho-eluates (P) were analyzed by western blotting using anti-STAU2. Anti-RSK1, anti-nucleolin and anti-β-actin antibodies were used as controls for phosphorylated and unphosphorylated proteins, respectively. e Protein extracts from nocodazole-treated (+) hTert-RPE1 and HeLa were incubated in vitro with either water (H2O), Lambda Phosphatase (λPP), inactivated Lambda Phosphatase (λPPin), Calf Intestinal Alkaline Phosphatase (CIP) or inactivated Calf Intestinal Alkaline Phosphatase (CIPin). STAU2 phosphorylation status was analyzed by SDS-PAGE and western blotting. Untreated cells (Mock) were used as control for dephosphorylated STAU2. All western blots are representatives of three independently performed experiments that showed similar profiles

    Techniques Used: shRNA, Clone Assay, Plasmid Preparation, Western Blot, Migration, Expressing, RNA Binding Assay, Binding Assay, Infection, SDS Page, Marker, Flow Cytometry, Incubation, In Vitro

    8) Product Images from "Regulation of the stability of RGF1 receptor by the ubiquitin-specific proteases UBP12/UBP13 is critical for root meristem maintenance"

    Article Title: Regulation of the stability of RGF1 receptor by the ubiquitin-specific proteases UBP12/UBP13 is critical for root meristem maintenance

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

    doi: 10.1073/pnas.1714177115

    UBP12 and UBP13 modulate RGFR1 stability. ( A ) The RGFR1 protein level decreased in ubp12,13 double-mutant plants. p35S:RGFR1-FLAG –expressing seedling s were treated with or without 20 μM RGF1 for 30 min. ( B ) RT-PCR analyses of the RGFR1 and RGFR2 transcript levels in the ubp12,13 mutant. ( C ) MG132 treatment stabilized RGFR1. Seedlings were treated with 50 μM MG132 for the indicated time. ( D and E ) UBP13 overexpression inhibited the RGF1-induced ubiquitination and degradation of RGFR1. Seedlings were treated with 20 μM RGF1 for 30 min ( D ) or with 20 μM RGF1 and 200 μM CHX for the indicated time ( E ). Asterisks in A , C , and D denote ubiquitinated RGFR1-FLAG. RGFR1/tubulin represents the signal ratio of RGFR1-FLAG to tubulin.
    Figure Legend Snippet: UBP12 and UBP13 modulate RGFR1 stability. ( A ) The RGFR1 protein level decreased in ubp12,13 double-mutant plants. p35S:RGFR1-FLAG –expressing seedling s were treated with or without 20 μM RGF1 for 30 min. ( B ) RT-PCR analyses of the RGFR1 and RGFR2 transcript levels in the ubp12,13 mutant. ( C ) MG132 treatment stabilized RGFR1. Seedlings were treated with 50 μM MG132 for the indicated time. ( D and E ) UBP13 overexpression inhibited the RGF1-induced ubiquitination and degradation of RGFR1. Seedlings were treated with 20 μM RGF1 for 30 min ( D ) or with 20 μM RGF1 and 200 μM CHX for the indicated time ( E ). Asterisks in A , C , and D denote ubiquitinated RGFR1-FLAG. RGFR1/tubulin represents the signal ratio of RGFR1-FLAG to tubulin.

    Techniques Used: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression

    UBP12 and UBP13 function in the RGF1-signaling pathway to regulate root meristem formation. ( A – D ) Wild-type ( A and C ) and ubp12,13 -mutant ( B and D ) plants were grown in the presence or absence of 100 nM RGF1 for 7 d ( A and B ); the roots were then stained with PI and observed by confocal microscopy ( C and D ). (Scale bars: 20 μm in C and D .) ( E ) Quantification of the cortical root meristem cell numbers for the plants shown in C and D . Error bars represent SD ( n > 20). ( F ) Seven-day-old wild-type, p35S:RGFR1-FLAG , ubp12,13 , and p35S:RGFR1-FLAG –expressing ubp12,13 seedlings. ( G ) Confocal microscopic images of PI-stained roots of plants shown in F . ( H ) Quantitation of root length and number of cortical root meristem cells in the plants shown in F and G . Error bars represent ± SD ( n > 20). White arrowheads in C , D , and G mark the junction between the meristematic and elongation zones.
    Figure Legend Snippet: UBP12 and UBP13 function in the RGF1-signaling pathway to regulate root meristem formation. ( A – D ) Wild-type ( A and C ) and ubp12,13 -mutant ( B and D ) plants were grown in the presence or absence of 100 nM RGF1 for 7 d ( A and B ); the roots were then stained with PI and observed by confocal microscopy ( C and D ). (Scale bars: 20 μm in C and D .) ( E ) Quantification of the cortical root meristem cell numbers for the plants shown in C and D . Error bars represent SD ( n > 20). ( F ) Seven-day-old wild-type, p35S:RGFR1-FLAG , ubp12,13 , and p35S:RGFR1-FLAG –expressing ubp12,13 seedlings. ( G ) Confocal microscopic images of PI-stained roots of plants shown in F . ( H ) Quantitation of root length and number of cortical root meristem cells in the plants shown in F and G . Error bars represent ± SD ( n > 20). White arrowheads in C , D , and G mark the junction between the meristematic and elongation zones.

    Techniques Used: Mutagenesis, Staining, Confocal Microscopy, Expressing, Quantitation Assay

    9) Product Images from "Nuclear translocation of 3?-phosphoinositide-dependent protein kinase 1 (PDK-1): A potential regulatory mechanism for PDK-1 function"

    Article Title: Nuclear translocation of 3?-phosphoinositide-dependent protein kinase 1 (PDK-1): A potential regulatory mechanism for PDK-1 function

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

    doi: 10.1073/pnas.2335486100

    Nuclear PDK-1 is phosphorylated in its activation loop and is kinaseactive. ( A ) Confocal images showing localization of Myc-mPDK-1, mPDK-1 Δ382–391 , or mPDK-1 S244A in murine hepatocytes treated with vehicle or 10 nM LMB for 2.5 h. (Bars = 20 μm.) ( B ) In vitro kinase activities of immunopurified Myc-tagged mPDK-1, mPDK-1 K114G , mPDK-1 Δ382–391 , or mPDK-1 K114G/Δ382–391 against a PKB-based peptide substrate. ( C ) Phosphorylation of immunopurified FLAG-p70 S6KβI by PDK-1 proteins from HeLa cells treated with 10 –6 M insulin for 10 mins. p70 S6KβI phosphorylation ( Top ), p70 S6KβI expression ( Middle ), and PDK-1 expression ( Bottom ) levels were detected by blotting with anti-phospho-Thr PDK-1 substrate, anti-FLAG, and anti-Myc antibodies, respectively. ( D ) Graphical representation of phosphorylation of p70 S6KβI by PDK-1. Bars represent the mean ± SEM from three independent experiments.
    Figure Legend Snippet: Nuclear PDK-1 is phosphorylated in its activation loop and is kinaseactive. ( A ) Confocal images showing localization of Myc-mPDK-1, mPDK-1 Δ382–391 , or mPDK-1 S244A in murine hepatocytes treated with vehicle or 10 nM LMB for 2.5 h. (Bars = 20 μm.) ( B ) In vitro kinase activities of immunopurified Myc-tagged mPDK-1, mPDK-1 K114G , mPDK-1 Δ382–391 , or mPDK-1 K114G/Δ382–391 against a PKB-based peptide substrate. ( C ) Phosphorylation of immunopurified FLAG-p70 S6KβI by PDK-1 proteins from HeLa cells treated with 10 –6 M insulin for 10 mins. p70 S6KβI phosphorylation ( Top ), p70 S6KβI expression ( Middle ), and PDK-1 expression ( Bottom ) levels were detected by blotting with anti-phospho-Thr PDK-1 substrate, anti-FLAG, and anti-Myc antibodies, respectively. ( D ) Graphical representation of phosphorylation of p70 S6KβI by PDK-1. Bars represent the mean ± SEM from three independent experiments.

    Techniques Used: Activation Assay, In Vitro, Expressing

    10) Product Images from "p53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1"

    Article Title: p53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1

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

    doi: 10.1073/pnas.2436329100

    Binding of wild-type p53 to NQO1 is inhibited by dicoumarol. ( A ) 293 human kidney cells were transiently transfected with pEFIRES FLAG p73β or pRc/CMV FLAG p53 with pEFIRES wild-type NQO1. Extracts were electrophoresed either before immunoprecipitation (TOTAL) or after immunoprecipitation of FLAG p73β or FLAG p53 with anti-FLAG agarose beads (IP α FLAG). Immunoblot analysis was carried out with mouse monoclonal anti-FLAG antibody, and the blots were then stripped and reprobed with goat anti-NQO1 antibody. ( B ) In vitro reticulocyte lysate translated [ 35 S]methionine-labeled wild-type p53 and NQO1 were incubated alone or mixed together without (-) or with (+) 300 μM dicoumarol (TOTAL). p53 was immunoprecipitated (IP α p53) with mouse monoclonal Pab 1801 anti-human p53 and NQO1 and p53 were detected by autoradiography. ( C ) In vitro reticulocyte lysate-translated, [ 35 S]methionine-labeled NQO1 FLAG was incubated for 1 h in Nonidet P-40 buffer without (-) or with (+) 300 μM dicoumarol (TOTAL). NQO1 FLAG was immunoprecipitated with anti-FLAG agarose beads (IP α FLAG), and NQO1 was detected by autoradiography.
    Figure Legend Snippet: Binding of wild-type p53 to NQO1 is inhibited by dicoumarol. ( A ) 293 human kidney cells were transiently transfected with pEFIRES FLAG p73β or pRc/CMV FLAG p53 with pEFIRES wild-type NQO1. Extracts were electrophoresed either before immunoprecipitation (TOTAL) or after immunoprecipitation of FLAG p73β or FLAG p53 with anti-FLAG agarose beads (IP α FLAG). Immunoblot analysis was carried out with mouse monoclonal anti-FLAG antibody, and the blots were then stripped and reprobed with goat anti-NQO1 antibody. ( B ) In vitro reticulocyte lysate translated [ 35 S]methionine-labeled wild-type p53 and NQO1 were incubated alone or mixed together without (-) or with (+) 300 μM dicoumarol (TOTAL). p53 was immunoprecipitated (IP α p53) with mouse monoclonal Pab 1801 anti-human p53 and NQO1 and p53 were detected by autoradiography. ( C ) In vitro reticulocyte lysate-translated, [ 35 S]methionine-labeled NQO1 FLAG was incubated for 1 h in Nonidet P-40 buffer without (-) or with (+) 300 μM dicoumarol (TOTAL). NQO1 FLAG was immunoprecipitated with anti-FLAG agarose beads (IP α FLAG), and NQO1 was detected by autoradiography.

    Techniques Used: Binding Assay, Transfection, Immunoprecipitation, In Vitro, Labeling, Incubation, Autoradiography

    p53 mutants R175H and R273H bind wild-type NQO1 in vivo with higher affinity than wild-type p53. 293 human kidney cells were transiently transfected with pRc/CMV FLAG wild-type p53 or with the p53 mutants R175H or R273H alone or cotransfected with pEFIRES NQO1. Extracts were electrophoresed either before immunoprecipitation (TOTAL) or after immunoprecipitation of FLAG p53 with anti-FLAG agarose beads (IP α FLAG). Immunoblot analysis was carried out with mouse monoclonal anti-FLAG antibody, and the blots were then stripped and reprobed with goat anti-NQO1 antibody. N.S, nonspecific band.
    Figure Legend Snippet: p53 mutants R175H and R273H bind wild-type NQO1 in vivo with higher affinity than wild-type p53. 293 human kidney cells were transiently transfected with pRc/CMV FLAG wild-type p53 or with the p53 mutants R175H or R273H alone or cotransfected with pEFIRES NQO1. Extracts were electrophoresed either before immunoprecipitation (TOTAL) or after immunoprecipitation of FLAG p53 with anti-FLAG agarose beads (IP α FLAG). Immunoblot analysis was carried out with mouse monoclonal anti-FLAG antibody, and the blots were then stripped and reprobed with goat anti-NQO1 antibody. N.S, nonspecific band.

    Techniques Used: In Vivo, Transfection, Immunoprecipitation

    11) Product Images from "Functional studies on the role of the C-terminal domain of mammalian polo-like kinase"

    Article Title: Functional studies on the role of the C-terminal domain of mammalian polo-like kinase

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

    doi: 10.1073/pnas.042689299

    The C-terminal domain of Plk binds to Plk, and does not bind to phosphorylated mitotic Plk. ( A ) GST-fused C-terminal protein C306–603 (GST-C) purified from High Five cells was incubated with lysates of HeLa cells that had been transfected with various FLAG-tagged Plk constructs. 1, FLAG-Plk WT protein bound to GST-C; 2, FLAG-ΔC1–401 protein bound to GST-C; 3, FLAG-ΔC1–401 protein (T210D) bound to GST-C. The Plk proteins associated with the GST-C-terminal domain were detected by Western blot analysis with anti-FLAG antibody (α-FLAG IB). The amount of GST or GST-C protein used was detected with anti-GST antibody (α-GST IB). ( B ) Binding of endogenous Plk to C-terminal domain (GST-C). GST-fused C306–603 protein was incubated with lysates of FT210 cells that were arrested in G 2 or mitosis as previously described, and was collected on glutathione-agarose. Endogenous Plks displayed phosphorylated M-phase and dephosphorylated G 2 forms ( Upper ) and endogenous Plk (filled arrowhead) associated with the C306–603 protein (GST-C, opened arrowhead) were detected by Western blot with anti-Plk antibody ( Lower ).
    Figure Legend Snippet: The C-terminal domain of Plk binds to Plk, and does not bind to phosphorylated mitotic Plk. ( A ) GST-fused C-terminal protein C306–603 (GST-C) purified from High Five cells was incubated with lysates of HeLa cells that had been transfected with various FLAG-tagged Plk constructs. 1, FLAG-Plk WT protein bound to GST-C; 2, FLAG-ΔC1–401 protein bound to GST-C; 3, FLAG-ΔC1–401 protein (T210D) bound to GST-C. The Plk proteins associated with the GST-C-terminal domain were detected by Western blot analysis with anti-FLAG antibody (α-FLAG IB). The amount of GST or GST-C protein used was detected with anti-GST antibody (α-GST IB). ( B ) Binding of endogenous Plk to C-terminal domain (GST-C). GST-fused C306–603 protein was incubated with lysates of FT210 cells that were arrested in G 2 or mitosis as previously described, and was collected on glutathione-agarose. Endogenous Plks displayed phosphorylated M-phase and dephosphorylated G 2 forms ( Upper ) and endogenous Plk (filled arrowhead) associated with the C306–603 protein (GST-C, opened arrowhead) were detected by Western blot with anti-Plk antibody ( Lower ).

    Techniques Used: Purification, Incubation, Transfection, Construct, Western Blot, Binding Assay

    Schematic representation of Plk1 proteins and kinase activity of immunocomplexes. ( A ) Plk wild-type and mutant proteins used in this study. The catalytic domain and polo-box are represented by deviant lined box and filled box, respectively. FLAG epitopes on the N-termini are shown as filled circles. From top to bottom: full-length wild-type Plk (WT); kinase-defective mutant Plk (K82M); Plk mutated Thr-210 to Asp (T210D); Plk deleted of the C-terminal amino acids 402–603 (ΔC1–401); N-terminal domain containing the mutation, T210D (ΔC TD); Plk deleted of the N-terminal amino acids 1–305 (C306–603). ( B ) Kinase activities of Plk proteins immunoprecipitated from transfected HeLa cell lysates. The amounts of immunoprecipitated FLAG-Plk proteins were detected with anti-FLAG antibody ( Upper ). The kinase activity of FLAG-Plk was measured by incubation with casein for 30 min as described in Materials and Methods ( Lower , CS). 1, FLAG-Plk K82M; 2, FLAG-Plk WT; 3, FLAG-Plk T210D; 4, FLAG-N terminus Plk (ΔC1–401); 5, FLAG-N terminus Plk containing T210D mutation (ΔC TD).
    Figure Legend Snippet: Schematic representation of Plk1 proteins and kinase activity of immunocomplexes. ( A ) Plk wild-type and mutant proteins used in this study. The catalytic domain and polo-box are represented by deviant lined box and filled box, respectively. FLAG epitopes on the N-termini are shown as filled circles. From top to bottom: full-length wild-type Plk (WT); kinase-defective mutant Plk (K82M); Plk mutated Thr-210 to Asp (T210D); Plk deleted of the C-terminal amino acids 402–603 (ΔC1–401); N-terminal domain containing the mutation, T210D (ΔC TD); Plk deleted of the N-terminal amino acids 1–305 (C306–603). ( B ) Kinase activities of Plk proteins immunoprecipitated from transfected HeLa cell lysates. The amounts of immunoprecipitated FLAG-Plk proteins were detected with anti-FLAG antibody ( Upper ). The kinase activity of FLAG-Plk was measured by incubation with casein for 30 min as described in Materials and Methods ( Lower , CS). 1, FLAG-Plk K82M; 2, FLAG-Plk WT; 3, FLAG-Plk T210D; 4, FLAG-N terminus Plk (ΔC1–401); 5, FLAG-N terminus Plk containing T210D mutation (ΔC TD).

    Techniques Used: Activity Assay, Mutagenesis, Immunoprecipitation, Transfection, Incubation

    The C-terminal domain of Plk inhibits its kinase activity. ( A ) Inhibitory effect of the C-terminal domain. In in vitro kinase assays, immunoprecipitated FLAG-Plk WT and FLAG-N-terminal domains from transfected HeLa cells were incubated with or without 1–2 μg of C-terminal domain protein C306–603 (GST-C), which was purified from High Five (Hi5) cells. The Plk activity was measured with casein as a substrate (CS). 1, 3, and 5, incubated without GST-C; 2, 4, and 6, incubated with GST-C. ( B ) Time course of the wild-type and N-terminal Plk kinase activity. Purified wild-type and N-terminal Plk proteins from Hi5 cells were preincubated with or without an equal molar level of purified C-terminal protein for 20 min at 30°C, and then assayed for protein kinase activity. The reactions were stopped by adding SDS/PAGE sample buffer. The samples were boiled and resolved by PAGE. The casein bands were excised from the gel and the radioactivity was quantitated by liquid scintillation spectrometry. Filled squares, ΔC1–401 protein; open squares, ΔC1–401 with C306–603; filled circles, Plk WT; open circles, Plk WT with C306–603. ( C ) Plk proteins associated with the GST-C-terminal domain were detected by Western blot analysis (α-FLAG IB). After stripping the FLAG signal, the membrane was incubated with GST antibody to quantify the GST-C protein (α-GST IB). The levels of wild-type and N-terminal Plk (WT and ΔC) in 10% of total lysates were analyzed by Western analysis (α-FLAG IB).
    Figure Legend Snippet: The C-terminal domain of Plk inhibits its kinase activity. ( A ) Inhibitory effect of the C-terminal domain. In in vitro kinase assays, immunoprecipitated FLAG-Plk WT and FLAG-N-terminal domains from transfected HeLa cells were incubated with or without 1–2 μg of C-terminal domain protein C306–603 (GST-C), which was purified from High Five (Hi5) cells. The Plk activity was measured with casein as a substrate (CS). 1, 3, and 5, incubated without GST-C; 2, 4, and 6, incubated with GST-C. ( B ) Time course of the wild-type and N-terminal Plk kinase activity. Purified wild-type and N-terminal Plk proteins from Hi5 cells were preincubated with or without an equal molar level of purified C-terminal protein for 20 min at 30°C, and then assayed for protein kinase activity. The reactions were stopped by adding SDS/PAGE sample buffer. The samples were boiled and resolved by PAGE. The casein bands were excised from the gel and the radioactivity was quantitated by liquid scintillation spectrometry. Filled squares, ΔC1–401 protein; open squares, ΔC1–401 with C306–603; filled circles, Plk WT; open circles, Plk WT with C306–603. ( C ) Plk proteins associated with the GST-C-terminal domain were detected by Western blot analysis (α-FLAG IB). After stripping the FLAG signal, the membrane was incubated with GST antibody to quantify the GST-C protein (α-GST IB). The levels of wild-type and N-terminal Plk (WT and ΔC) in 10% of total lysates were analyzed by Western analysis (α-FLAG IB).

    Techniques Used: Activity Assay, In Vitro, Immunoprecipitation, Transfection, Incubation, Purification, SDS Page, Polyacrylamide Gel Electrophoresis, Radioactivity, Western Blot, Stripping Membranes

    Transient expression of the C-terminal domain in HeLa cells induces mitotic arrest. ( A ) HeLa cells were cotransfected with pEGFP-F and various pCMV-FLAG-Plk constructs and fixed as described in Materials and Methods . DNA profiles of GFP-positive cells were determined by fluorescence-activated cell sorter (FACS) analysis. Left (ungated) and Right (GFP-positive) represent the DNA profiles from total cell populations and GFP-positive transfected cells after gating, respectively. 1, DNA profiles from transfectants with pCMV-FLAG vector; 2, with pCMV-FLAG-Plk K82M; 3, with pCMV-FLAG-Plk WT; 4, with pCMV-FLAG-Plk T210D; 5, with pCMV-FLAG-ΔC1–401; 6, with pCMV-FLAG-C306–603. ( B ) The percent of G 2 /M phase cells determined from the GFP-positive population by ModFit analysis. Data represent three independent experiments. ( C ) Western blot analysis of expressed proteins from transfected cell lysates with anti-FLAG antibody.
    Figure Legend Snippet: Transient expression of the C-terminal domain in HeLa cells induces mitotic arrest. ( A ) HeLa cells were cotransfected with pEGFP-F and various pCMV-FLAG-Plk constructs and fixed as described in Materials and Methods . DNA profiles of GFP-positive cells were determined by fluorescence-activated cell sorter (FACS) analysis. Left (ungated) and Right (GFP-positive) represent the DNA profiles from total cell populations and GFP-positive transfected cells after gating, respectively. 1, DNA profiles from transfectants with pCMV-FLAG vector; 2, with pCMV-FLAG-Plk K82M; 3, with pCMV-FLAG-Plk WT; 4, with pCMV-FLAG-Plk T210D; 5, with pCMV-FLAG-ΔC1–401; 6, with pCMV-FLAG-C306–603. ( B ) The percent of G 2 /M phase cells determined from the GFP-positive population by ModFit analysis. Data represent three independent experiments. ( C ) Western blot analysis of expressed proteins from transfected cell lysates with anti-FLAG antibody.

    Techniques Used: Expressing, Construct, Fluorescence, FACS, Transfection, Plasmid Preparation, Western Blot

    12) Product Images from "Defining the interaction of the protease CpaA with its type II secretion chaperone CpaB and its contribution to virulence in Acinetobacter species"

    Article Title: Defining the interaction of the protease CpaA with its type II secretion chaperone CpaB and its contribution to virulence in Acinetobacter species

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.808394

    CpaA and CpaB strongly interact physically. A and B , Western blot analysis of nickel affinity purifications from cell lysates of M2 Δ cpaAB expressing either pWH- cpaA-flag-cpaB-his ( A ) or pWH- cpaA-flag-cpaB ( B ). Note that, in B , CpaB does not contain a C-terminal hexahistidine tag. CpaA-FLAG was detected using an anti-FLAG monoclonal antibody, whereas CpaB was detected using anti-histidine polyclonal antibodies. The right panel in A shows Coomassie-stained SDS-PAGE of the elution from A and thus represents the CpaA–FLAG–CpaB–His complex. L , loaded cell lysate, FT , flow-through, W1–3 , washes one to three; E , elution.
    Figure Legend Snippet: CpaA and CpaB strongly interact physically. A and B , Western blot analysis of nickel affinity purifications from cell lysates of M2 Δ cpaAB expressing either pWH- cpaA-flag-cpaB-his ( A ) or pWH- cpaA-flag-cpaB ( B ). Note that, in B , CpaB does not contain a C-terminal hexahistidine tag. CpaA-FLAG was detected using an anti-FLAG monoclonal antibody, whereas CpaB was detected using anti-histidine polyclonal antibodies. The right panel in A shows Coomassie-stained SDS-PAGE of the elution from A and thus represents the CpaA–FLAG–CpaB–His complex. L , loaded cell lysate, FT , flow-through, W1–3 , washes one to three; E , elution.

    Techniques Used: Western Blot, Expressing, Staining, SDS Page, Flow Cytometry

    13) Product Images from "Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042"

    Article Title: Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042

    Journal: mSystems

    doi: 10.1128/mSystems.00220-17

    H-NS2-FLAG expression is upregulated in DMEM and M9 minimal medium and when cells enter the stationary growth phase. (A) Immunodetection of H-NS2-FLAG in cell extracts from E. coli strain 042 growing at 25°C and 37°C in LB, M9 minimal medium, and DMEM at the onset of the stationary phase (OD 600 of 2.0). (B) Immunodetection of H-NS2-FLAG in cell extracts from E. coli strain 042 growing in LB medium at 25°C and 37°C both at the exponential and early stationary growth phases (OD 600 of 0.4 and 2.0, respectively). Experiments were repeated three times. The results of a representative experiment are shown.
    Figure Legend Snippet: H-NS2-FLAG expression is upregulated in DMEM and M9 minimal medium and when cells enter the stationary growth phase. (A) Immunodetection of H-NS2-FLAG in cell extracts from E. coli strain 042 growing at 25°C and 37°C in LB, M9 minimal medium, and DMEM at the onset of the stationary phase (OD 600 of 2.0). (B) Immunodetection of H-NS2-FLAG in cell extracts from E. coli strain 042 growing in LB medium at 25°C and 37°C both at the exponential and early stationary growth phases (OD 600 of 0.4 and 2.0, respectively). Experiments were repeated three times. The results of a representative experiment are shown.

    Techniques Used: Expressing, Immunodetection

    H-NS2 is upregulated in an hns mutant derivative of the strain E. coli 042. Immunodetection of H-NS2-FLAG in cultures of the wild-type (WT) E. coli 042 strain and its derivatives containing a FLAG tag in the hns2 gene and an hns derivative containing a FLAG tag in the hns2 gene. The top panels show Coomassie blue-stained cell extracts used for the immunodetection of H-NS2-FLAG. Similar protein concentrations are apparent. The bottom panels show immunodetected H-NS2-FLAG. The optical density at 600 nm (O.D) values are shown above the brackets.
    Figure Legend Snippet: H-NS2 is upregulated in an hns mutant derivative of the strain E. coli 042. Immunodetection of H-NS2-FLAG in cultures of the wild-type (WT) E. coli 042 strain and its derivatives containing a FLAG tag in the hns2 gene and an hns derivative containing a FLAG tag in the hns2 gene. The top panels show Coomassie blue-stained cell extracts used for the immunodetection of H-NS2-FLAG. Similar protein concentrations are apparent. The bottom panels show immunodetected H-NS2-FLAG. The optical density at 600 nm (O.D) values are shown above the brackets.

    Techniques Used: Mutagenesis, Immunodetection, FLAG-tag, Staining

    14) Product Images from "CRMP5 Interacts with Tubulin to Inhibit Neurite Outgrowth, Thereby Modulating the Function of CRMP2"

    Article Title: CRMP5 Interacts with Tubulin to Inhibit Neurite Outgrowth, Thereby Modulating the Function of CRMP2

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0059-10.2010

    CRMP5 interacts with MAP2 and tubulin. A , Brain extract from P8 mice was immunoprecipitated by anti-MAP2 antibody, and Western blots were probed for CRMP5 and α-tubulin immunoreactivity. CRMP5 and tubulin were coimmunoprecipitated in a complex with MAP2 protein. Immunoprecipitation of MAP2 is shown as positive control. Note the absence of reactivity in the control IgG. B , Endogenous and overexpressed CRMP5 colocalized with tubulin in NGF-induced differentiated PC12 cells. Left panel, Cells transfected with FLAG-CRMP5 and immunostained with anti-α-tubulin and anti-FLAG antibodies show colocalization of CRMP5 and tubulin in the cytosol and in thin extensions. The inset is a magnification of this area. Middle panel, High magnification of one PC12 cell clearly shows colocalization of CRMP5 and tubulin. Right panel, Endogenous CRMP5 colocalized with tubulin at the cytosolic level and in thin extensions. The inset is a magnification of one PC12 cell. Scale bars, 20 μm. C , Immunoprecipitation of brain extract from P8 mice with anti-α- or anti-β-tubulin antibodies. Western blots were probed for CRMP5 immunoreactivity. CRMP5 was immunoprecipitated in a complex with α- and β-tubulin. As expected, α- and β-tubulin were also coimmunoprecipitated with anti-β- and anti-α-tubulin antibodies, respectively. Control IgG failed to precipitate any protein. D , Overlay assay using purified recombinant CRMP5 (15 μg) subjected to SDS-PAGE and transferred to nitrocellulose membrane. After an overnight incubation with brain extract from P8 mouse (+) or buffer (−), the membrane was probed for MAP2, α-tubulin, or βIII-tubulin immunoreactivity. CRMP5 interacts with MAP2, α-tubulin, or βIII-tubulin present in the brain extract. The intensity of interaction is higher for MAP2 and α-tubulin than for βIII-tubulin. E , Pull down of tubulin from mouse brain extract with GST or GST-CRMP5 (10 μg) conjugated to glutathione Sepharose 4B beads. Western blots of the pellet were probed for tubulin and MAP2 immunoreactivity. GST-CRMP5, but not GST, precipitated MAP2 and α-tubulin. Each result is representative of three to five separate experiments with similar results.
    Figure Legend Snippet: CRMP5 interacts with MAP2 and tubulin. A , Brain extract from P8 mice was immunoprecipitated by anti-MAP2 antibody, and Western blots were probed for CRMP5 and α-tubulin immunoreactivity. CRMP5 and tubulin were coimmunoprecipitated in a complex with MAP2 protein. Immunoprecipitation of MAP2 is shown as positive control. Note the absence of reactivity in the control IgG. B , Endogenous and overexpressed CRMP5 colocalized with tubulin in NGF-induced differentiated PC12 cells. Left panel, Cells transfected with FLAG-CRMP5 and immunostained with anti-α-tubulin and anti-FLAG antibodies show colocalization of CRMP5 and tubulin in the cytosol and in thin extensions. The inset is a magnification of this area. Middle panel, High magnification of one PC12 cell clearly shows colocalization of CRMP5 and tubulin. Right panel, Endogenous CRMP5 colocalized with tubulin at the cytosolic level and in thin extensions. The inset is a magnification of one PC12 cell. Scale bars, 20 μm. C , Immunoprecipitation of brain extract from P8 mice with anti-α- or anti-β-tubulin antibodies. Western blots were probed for CRMP5 immunoreactivity. CRMP5 was immunoprecipitated in a complex with α- and β-tubulin. As expected, α- and β-tubulin were also coimmunoprecipitated with anti-β- and anti-α-tubulin antibodies, respectively. Control IgG failed to precipitate any protein. D , Overlay assay using purified recombinant CRMP5 (15 μg) subjected to SDS-PAGE and transferred to nitrocellulose membrane. After an overnight incubation with brain extract from P8 mouse (+) or buffer (−), the membrane was probed for MAP2, α-tubulin, or βIII-tubulin immunoreactivity. CRMP5 interacts with MAP2, α-tubulin, or βIII-tubulin present in the brain extract. The intensity of interaction is higher for MAP2 and α-tubulin than for βIII-tubulin. E , Pull down of tubulin from mouse brain extract with GST or GST-CRMP5 (10 μg) conjugated to glutathione Sepharose 4B beads. Western blots of the pellet were probed for tubulin and MAP2 immunoreactivity. GST-CRMP5, but not GST, precipitated MAP2 and α-tubulin. Each result is representative of three to five separate experiments with similar results.

    Techniques Used: Mouse Assay, Immunoprecipitation, Western Blot, Positive Control, Transfection, Overlay Assay, Purification, Recombinant, SDS Page, Incubation

    CRMP5 neurite outgrowth inhibition abrogates the CRMP2 promotion function in N1E-115 cells. A , Cells were cotransfected with GFP-CRMP5 and FLAG-CRMP2 constructs, labeled with anti-FLAG antibody (red), and observed with fluorescent microscopy. Only the cells transfected with both vectors are yellow in the merge image. Note that the cells transfected with CRMP2 alone, which remained red, exhibited some neurite-like extensions. Cells cotransfected with full-length CRMP5, ΔN244, or ΔC518 and CRMP2 clearly show a round morphology. Cells double transfected with CRMP5 ΔC472 and CRMP2 show a polygonal shape. Scale bars, 50 μm. B , Quantification of cells from A exhibiting neurite lengths > 25 μm. CRMP5 decreases, but CRMP2 increases the percentage of cells bearing longer neurite lengths. In the presence of both CRMPs, the cells ( n = 79) show rather a low percentage of longer neurites, indicating the dominant effect of CRMP5 when bearing the tubulin-binding site. However, when CRMP5 ΔC472 and CRMP2 are overexpressed, the percentage of cells with longer neurite lengths reaches the value of CRMPP2 expression alone, suggesting that the antagonizing effect of CRMP5 is exerted through a tubulin-based mechanism (** p
    Figure Legend Snippet: CRMP5 neurite outgrowth inhibition abrogates the CRMP2 promotion function in N1E-115 cells. A , Cells were cotransfected with GFP-CRMP5 and FLAG-CRMP2 constructs, labeled with anti-FLAG antibody (red), and observed with fluorescent microscopy. Only the cells transfected with both vectors are yellow in the merge image. Note that the cells transfected with CRMP2 alone, which remained red, exhibited some neurite-like extensions. Cells cotransfected with full-length CRMP5, ΔN244, or ΔC518 and CRMP2 clearly show a round morphology. Cells double transfected with CRMP5 ΔC472 and CRMP2 show a polygonal shape. Scale bars, 50 μm. B , Quantification of cells from A exhibiting neurite lengths > 25 μm. CRMP5 decreases, but CRMP2 increases the percentage of cells bearing longer neurite lengths. In the presence of both CRMPs, the cells ( n = 79) show rather a low percentage of longer neurites, indicating the dominant effect of CRMP5 when bearing the tubulin-binding site. However, when CRMP5 ΔC472 and CRMP2 are overexpressed, the percentage of cells with longer neurite lengths reaches the value of CRMPP2 expression alone, suggesting that the antagonizing effect of CRMP5 is exerted through a tubulin-based mechanism (** p

    Techniques Used: Inhibition, Construct, Labeling, Microscopy, Transfection, Binding Assay, Expressing

    CRMP5 induces inhibition of neurite outgrowth and formation in N1E-115 cells. A , Cells were transfected with EGFP alone (top panel) or with FLAG-CRMP5 (middle panel) and FLAG-CRMP2 (bottom panel), and then labeled by anti-FLAG antibody and observed by fluorescent microscopy. The nucleus is stained blue with DAPI. Although N1E-115 cells transfected with EGFP present a polygonal shape and exhibit some neurite extensions, cells expressing CRMP5 present round cell morphologies. As reported, cells expressing CRMP2 clearly exhibit longer neurite extensions. The typical pattern of immunostaining is shown in high-magnification images in three right panels. Scale bar, 50 μm. B , Knockdown of CRMP5 expression by siCRMP5. N1E-115 cells were transfected or not with scrambled siRNA (SC) or siCRMP5, and CRMP5 expression was probed by anti-CRMP5 antibody. The GAPDH antibody is used as loading control. Note the complete depletion of CRMP5 expression after siCRMP5 treatment. C , CRMP5 depletion induces long neurite. Endogenous CRMP5 was detected by immunostaining with anti-CRMP5 antibody in SC-treated cells, whereas CRMP5 labeling was absent from siCRMP5-treated cells (top panels), detected by DAPI staining. A double staining with phalloidin, shows unambiguously the presence of longer neurite in siCRMP5-treated cells compared with SC cells (bottom panel). The CRMP5 inhibition of the neurite outgrowth is rescued by overexpression of CRMP5 in siCRMP5-treated cells (right panel). Scale bar, 50 μm. D , E , Quantification of neurite length and number after overexpression or knockdown of CRMP5. D , Comparison of neurite outgrowth between N1E-115 cells expressing EGFP ( n = 77), overexpressing CRMP2 ( n = 248) or CRMP5 ( n = 206), or transfected with siCRMP5 ( n = 100) or SC ( n = 157). Overexpression of CRMP5 leads to a significant decrease in neurite outgrowth initiation when compared with controls, whereas CRMP2 promotes neurite extension; however, knockdown of CRMP5 induces an increase in neurite length when compared with cells treated with the control SC. E , Comparison of neurite numbers per cell after overexpression or knockdown of CRMP5 as for D . Overexpression or knockdown of CRMP5 generates a decrease or increase in the neurite number per cell, respectively. Data show the mean values of four individual experiments, expressed as cells bearing neurite lengths > 25 μm (** p
    Figure Legend Snippet: CRMP5 induces inhibition of neurite outgrowth and formation in N1E-115 cells. A , Cells were transfected with EGFP alone (top panel) or with FLAG-CRMP5 (middle panel) and FLAG-CRMP2 (bottom panel), and then labeled by anti-FLAG antibody and observed by fluorescent microscopy. The nucleus is stained blue with DAPI. Although N1E-115 cells transfected with EGFP present a polygonal shape and exhibit some neurite extensions, cells expressing CRMP5 present round cell morphologies. As reported, cells expressing CRMP2 clearly exhibit longer neurite extensions. The typical pattern of immunostaining is shown in high-magnification images in three right panels. Scale bar, 50 μm. B , Knockdown of CRMP5 expression by siCRMP5. N1E-115 cells were transfected or not with scrambled siRNA (SC) or siCRMP5, and CRMP5 expression was probed by anti-CRMP5 antibody. The GAPDH antibody is used as loading control. Note the complete depletion of CRMP5 expression after siCRMP5 treatment. C , CRMP5 depletion induces long neurite. Endogenous CRMP5 was detected by immunostaining with anti-CRMP5 antibody in SC-treated cells, whereas CRMP5 labeling was absent from siCRMP5-treated cells (top panels), detected by DAPI staining. A double staining with phalloidin, shows unambiguously the presence of longer neurite in siCRMP5-treated cells compared with SC cells (bottom panel). The CRMP5 inhibition of the neurite outgrowth is rescued by overexpression of CRMP5 in siCRMP5-treated cells (right panel). Scale bar, 50 μm. D , E , Quantification of neurite length and number after overexpression or knockdown of CRMP5. D , Comparison of neurite outgrowth between N1E-115 cells expressing EGFP ( n = 77), overexpressing CRMP2 ( n = 248) or CRMP5 ( n = 206), or transfected with siCRMP5 ( n = 100) or SC ( n = 157). Overexpression of CRMP5 leads to a significant decrease in neurite outgrowth initiation when compared with controls, whereas CRMP2 promotes neurite extension; however, knockdown of CRMP5 induces an increase in neurite length when compared with cells treated with the control SC. E , Comparison of neurite numbers per cell after overexpression or knockdown of CRMP5 as for D . Overexpression or knockdown of CRMP5 generates a decrease or increase in the neurite number per cell, respectively. Data show the mean values of four individual experiments, expressed as cells bearing neurite lengths > 25 μm (** p

    Techniques Used: Inhibition, Transfection, Labeling, Microscopy, Staining, Expressing, Immunostaining, Double Staining, Over Expression

    15) Product Images from "Alcadein Cleavages by Amyloid ?-Precursor Protein (APP) ?- and ?-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related ?-Secretase Dysfunction *"

    Article Title: Alcadein Cleavages by Amyloid ?-Precursor Protein (APP) ?- and ?-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related ?-Secretase Dysfunction *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.057497

    Evidence that Alc family proteins and APP undergo identical processing by ADAM 10 α-secretase. A–D , generation of sAlc and sAPP in ADAM 10-deficient cells. ADAM 10 homozygous (−/−, lanes 2 and 4 ) and heterozygous (+/−, lanes 1 and 3 ) deficient MEFs were transiently transfected with 1.5 μg of pcDNA3-FLAG-hAlc α1 (+ in A ), pcDNA3.1-FLAG-hAlc β (+ in B ), pcDNA3.1-FLAG-hAlc γ (+ in C ), and pcDNA3-FLAG-APP 695 (+ in D ), or vector alone (− in A–D ). Culture medium (1 ml) was immunoprecipitated with an anti-FLAG M2 antibody. The immunoprecipitates of conditioned medium ( Medium ) and cell lysate ( Cell ; 20 μg of protein) were analyzed by Western blotting with M2 ( left panels ). The levels of sAlc and sAPP in lane 2 are indicated as ratios relative to the levels shown in lane 1 , which was assigned a reference value of 1.0 (values represent means ± S.E.). The asterisks indicate statistical significance as determined by Student's t test ( n = 3; *, p
    Figure Legend Snippet: Evidence that Alc family proteins and APP undergo identical processing by ADAM 10 α-secretase. A–D , generation of sAlc and sAPP in ADAM 10-deficient cells. ADAM 10 homozygous (−/−, lanes 2 and 4 ) and heterozygous (+/−, lanes 1 and 3 ) deficient MEFs were transiently transfected with 1.5 μg of pcDNA3-FLAG-hAlc α1 (+ in A ), pcDNA3.1-FLAG-hAlc β (+ in B ), pcDNA3.1-FLAG-hAlc γ (+ in C ), and pcDNA3-FLAG-APP 695 (+ in D ), or vector alone (− in A–D ). Culture medium (1 ml) was immunoprecipitated with an anti-FLAG M2 antibody. The immunoprecipitates of conditioned medium ( Medium ) and cell lysate ( Cell ; 20 μg of protein) were analyzed by Western blotting with M2 ( left panels ). The levels of sAlc and sAPP in lane 2 are indicated as ratios relative to the levels shown in lane 1 , which was assigned a reference value of 1.0 (values represent means ± S.E.). The asterisks indicate statistical significance as determined by Student's t test ( n = 3; *, p

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

    16) Product Images from "The Transcription Factors TBX2 and TBX3 Interact with Human Papillomavirus 16 (HPV16) L2 and Repress the Long Control Region of HPVs"

    Article Title: The Transcription Factors TBX2 and TBX3 Interact with Human Papillomavirus 16 (HPV16) L2 and Repress the Long Control Region of HPVs

    Journal: Journal of Virology

    doi: 10.1128/JVI.01803-12

    L2 colocalizes with TBX2 and TBX3 in the cell nucleus, but not in ND10. (A) TBX2-3×FLAG and TBX3-3×FLAG were either expressed alone (−L2) or coexpressed with HPV16 L2 (+L2) in HeLa cells for 24 h. The cells were fixed and permeabilized with methanol. Transiently expressed TBX2 or TBX3 was detected with rabbit polyclonal anti-FLAG antibody (green), and L2 was stained with mouse monoclonal anti-L2 antibody (red). DNA was stained with Hoechst and is shown in blue. (B) YFP-L2 was coexpressed with TBX2-3×FLAG in HeLa cells. TBX2 was stained with rabbit polyclonal anti-FLAG antibody (red) and endogenous PML with a specific mouse monoclonal antibody (blue).
    Figure Legend Snippet: L2 colocalizes with TBX2 and TBX3 in the cell nucleus, but not in ND10. (A) TBX2-3×FLAG and TBX3-3×FLAG were either expressed alone (−L2) or coexpressed with HPV16 L2 (+L2) in HeLa cells for 24 h. The cells were fixed and permeabilized with methanol. Transiently expressed TBX2 or TBX3 was detected with rabbit polyclonal anti-FLAG antibody (green), and L2 was stained with mouse monoclonal anti-L2 antibody (red). DNA was stained with Hoechst and is shown in blue. (B) YFP-L2 was coexpressed with TBX2-3×FLAG in HeLa cells. TBX2 was stained with rabbit polyclonal anti-FLAG antibody (red) and endogenous PML with a specific mouse monoclonal antibody (blue).

    Techniques Used: Staining

    17) Product Images from "Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer"

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    Journal: Nature

    doi: 10.1038/s41586-018-0018-1

    PFKFB4 phosphorylates SRC-3 by functioning as a protein kinase a , Upper panel-Recombinant GST-fused PFKFB4 incubated with full-length SRC-3 (SRC-3 FL) in presence of [ 32 P]ATP in an in vitro kinase assay. Lower panels- SRC-3 and PFKFB4 protein levels were analyzed by immunoblotting. b , In vitro kinase assay of PFKFB4 in the presence of SRC-3 fragments expressing different domains or full length SRC-3-FL. c , HEK293T cells expressing Flag-tagged-SRC-3 and PFKFB4 cultured in different concentrations of glucose and immunoprecipitated by Flag or p-Ser/Thr antibodies followed by immunoblotting. d , MDA-MB-231 cells stably expressing shRNAs targeting PFKFB4 (sh-PFK#09 and sh-PFK#20) or control NT-shRNA grown in presence of 5mM, 25mM glucose or glucose withdrawn from cells grown in 25mM of glucose and replaced with 5mM (WD) for 6 hours. Protein levels of pSRC-3-S857, PFKFB4 and β-actin were detected by immunoblotting. e , HEK293T cells expressing pBIND, pBIND-SRC-3 or pBIND-SRC-3-S857A were transduced with Adv. GFP or PFKFB4, and cultured in 5mM or 25mM glucose followed by luciferase assay. [Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points, + indicates mean, n =6 biologically independent experiments; Two-way ANOVA with Tukey’s Multiple comparisons test]. Data shown in (a–e) are representative of 3 biologically independent experiments with similar results. For exact P -values please refer to source data.
    Figure Legend Snippet: PFKFB4 phosphorylates SRC-3 by functioning as a protein kinase a , Upper panel-Recombinant GST-fused PFKFB4 incubated with full-length SRC-3 (SRC-3 FL) in presence of [ 32 P]ATP in an in vitro kinase assay. Lower panels- SRC-3 and PFKFB4 protein levels were analyzed by immunoblotting. b , In vitro kinase assay of PFKFB4 in the presence of SRC-3 fragments expressing different domains or full length SRC-3-FL. c , HEK293T cells expressing Flag-tagged-SRC-3 and PFKFB4 cultured in different concentrations of glucose and immunoprecipitated by Flag or p-Ser/Thr antibodies followed by immunoblotting. d , MDA-MB-231 cells stably expressing shRNAs targeting PFKFB4 (sh-PFK#09 and sh-PFK#20) or control NT-shRNA grown in presence of 5mM, 25mM glucose or glucose withdrawn from cells grown in 25mM of glucose and replaced with 5mM (WD) for 6 hours. Protein levels of pSRC-3-S857, PFKFB4 and β-actin were detected by immunoblotting. e , HEK293T cells expressing pBIND, pBIND-SRC-3 or pBIND-SRC-3-S857A were transduced with Adv. GFP or PFKFB4, and cultured in 5mM or 25mM glucose followed by luciferase assay. [Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points, + indicates mean, n =6 biologically independent experiments; Two-way ANOVA with Tukey’s Multiple comparisons test]. Data shown in (a–e) are representative of 3 biologically independent experiments with similar results. For exact P -values please refer to source data.

    Techniques Used: Recombinant, Incubation, In Vitro, Kinase Assay, Expressing, Cell Culture, Immunoprecipitation, Multiple Displacement Amplification, Stable Transfection, shRNA, Transduction, Luciferase

    18) Product Images from "NFBD1/MDC1 Is Phosphorylated by PLK1 and Controls G2/M Transition through the Regulation of a TOPOII?-Mediated Decatenation Checkpoint"

    Article Title: NFBD1/MDC1 Is Phosphorylated by PLK1 and Controls G2/M Transition through the Regulation of a TOPOII?-Mediated Decatenation Checkpoint

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082744

    Mapping of the binding regions between NFBD1 and PLK1. (A) A schematic drawing of the structure of NFBD1 is shown. Numbers indicate the amino acid positions relative to the first Met (+1). FHA, forkhead-associated; PST, proline/serine/threonine-rich; BRCT, BRCA1 carboxyl terminus. (B) In vitro binding assay. Whole cell lysates prepared from COS7 cells transfected with FLAG-Plk1 expression plasmid were incubated with 35 S-labeled FHA, PST, or the BRCT domain of NFBD1. Bound materials were recovered by immunoprecipitation with an anti-PLK1 antibody and analyzed by SDS-PAGE followed by autoradiography (left panel). The right panel shows 1/10 loading. (C) The structure of wild-type Plk1 and various COOH-terminal deletion mutants of PLK1. KD, kinase domain; polo, polo box domain. (D) GST pull-down assay. 35 S-labeled wild-type PLK1 or the indicated deletion mutants of PLK1 were incubated with GST-BRCT fusion protein. After the incubation, bound proteins were recovered on glutathione Sepharose beads and separated by SDS-PAGE followed by autoradiography (left panel). The right panel shows 1/10 loading.
    Figure Legend Snippet: Mapping of the binding regions between NFBD1 and PLK1. (A) A schematic drawing of the structure of NFBD1 is shown. Numbers indicate the amino acid positions relative to the first Met (+1). FHA, forkhead-associated; PST, proline/serine/threonine-rich; BRCT, BRCA1 carboxyl terminus. (B) In vitro binding assay. Whole cell lysates prepared from COS7 cells transfected with FLAG-Plk1 expression plasmid were incubated with 35 S-labeled FHA, PST, or the BRCT domain of NFBD1. Bound materials were recovered by immunoprecipitation with an anti-PLK1 antibody and analyzed by SDS-PAGE followed by autoradiography (left panel). The right panel shows 1/10 loading. (C) The structure of wild-type Plk1 and various COOH-terminal deletion mutants of PLK1. KD, kinase domain; polo, polo box domain. (D) GST pull-down assay. 35 S-labeled wild-type PLK1 or the indicated deletion mutants of PLK1 were incubated with GST-BRCT fusion protein. After the incubation, bound proteins were recovered on glutathione Sepharose beads and separated by SDS-PAGE followed by autoradiography (left panel). The right panel shows 1/10 loading.

    Techniques Used: Binding Assay, In Vitro, Transfection, Expressing, Plasmid Preparation, Incubation, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Pull Down Assay

    PLK1-mediated phosphorylation of NFBD1 is essential for the M phase entry. (A) Schematic representation of PST domains of wild-type NFBD1 and mutant form of NFBD1 termed GST-PST(T847A). (B) In vitro kinase reaction. GST, GST-PST, or GST-PST(T847A) were purified using glutathione Sepharose beads (right panel) and incubated with purified PLK1 in the presence of [γ- 32 P]ATP. The reaction mixtures were analyzed by SDS-PAGE followed by autoradiography (left panel). (C) Enforced expression of PLK1 but not the kinase-deficient mutant form of PLK1 [PLK1(K28M)] induces phospho-histone H3. HeLa cells were transiently transfected with the indicated expression plasmids. Forty-eight hours after transfection, whole cell lysates were prepared and immunoblotted with the indicated antibodies. (D) Expression of GFP-PST and GFP-PST(T847A). HeLa cells were transiently transfected with the expression plasmids. Forty-eight hours after transfection, whole cell lysates were prepared and immunoblotted with an anti-GFP antibody. (E and F) GFP-PST but not GFP-PST(T847A) inhibits the phosphorylation of histone H3. HeLa cells were transiently transfected with the expression plasmid for FLAG-PLK1 alone or FLAG-PLK1 and increasing amounts of GFP-PST (E) or GFP-PST(T847A) (F). Forty-eight hours after transfection, whole cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies.
    Figure Legend Snippet: PLK1-mediated phosphorylation of NFBD1 is essential for the M phase entry. (A) Schematic representation of PST domains of wild-type NFBD1 and mutant form of NFBD1 termed GST-PST(T847A). (B) In vitro kinase reaction. GST, GST-PST, or GST-PST(T847A) were purified using glutathione Sepharose beads (right panel) and incubated with purified PLK1 in the presence of [γ- 32 P]ATP. The reaction mixtures were analyzed by SDS-PAGE followed by autoradiography (left panel). (C) Enforced expression of PLK1 but not the kinase-deficient mutant form of PLK1 [PLK1(K28M)] induces phospho-histone H3. HeLa cells were transiently transfected with the indicated expression plasmids. Forty-eight hours after transfection, whole cell lysates were prepared and immunoblotted with the indicated antibodies. (D) Expression of GFP-PST and GFP-PST(T847A). HeLa cells were transiently transfected with the expression plasmids. Forty-eight hours after transfection, whole cell lysates were prepared and immunoblotted with an anti-GFP antibody. (E and F) GFP-PST but not GFP-PST(T847A) inhibits the phosphorylation of histone H3. HeLa cells were transiently transfected with the expression plasmid for FLAG-PLK1 alone or FLAG-PLK1 and increasing amounts of GFP-PST (E) or GFP-PST(T847A) (F). Forty-eight hours after transfection, whole cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies.

    Techniques Used: Mutagenesis, In Vitro, Purification, Incubation, SDS Page, Autoradiography, Expressing, Transfection, Plasmid Preparation

    19) Product Images from "Gre factors-mediated control of hilD transcription is essential for the invasion of epithelial cells by Salmonella enterica serovar Typhimurium"

    Article Title: Gre factors-mediated control of hilD transcription is essential for the invasion of epithelial cells by Salmonella enterica serovar Typhimurium

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006312

    Gre factors-mediated regulation of S . Typhimurium virulence is focused in the regulation of the master regulator HilD. (A) Relative hilC , hilD and rtsA mRNA quantification by qPCR in WT and Δ greA Δ greB derivative strains. Results are normalized after detection of gapA (GAPDH) that was used as an endogenous control. Same RNA samples as in Fig 3B . (B) Transcriptional expression of hilA in WT, hilC , rtsA and hilD derivative strains either proficient (grey bars) or deficient (black bars) in the Gre factors was monitored by β-galactosidase activity determination from a hilA :: lacZ fusion. In A and B, a bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. Immunodetection (lower panels) of HilA-FLAG protein (C) and the SPI-2 encoded SsrA-FLAG protein (D) in whole cell extracts from cultures of WT and Δ greA Δ greB derivative strains in a hilD + and hilD - genetic backgrounds. The upper panels are sections of Coomassie stained gels as loading controls. In all cases bacterial cultures were grown in LB at 37°C up to an OD 600nm of 2.0.
    Figure Legend Snippet: Gre factors-mediated regulation of S . Typhimurium virulence is focused in the regulation of the master regulator HilD. (A) Relative hilC , hilD and rtsA mRNA quantification by qPCR in WT and Δ greA Δ greB derivative strains. Results are normalized after detection of gapA (GAPDH) that was used as an endogenous control. Same RNA samples as in Fig 3B . (B) Transcriptional expression of hilA in WT, hilC , rtsA and hilD derivative strains either proficient (grey bars) or deficient (black bars) in the Gre factors was monitored by β-galactosidase activity determination from a hilA :: lacZ fusion. In A and B, a bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. Immunodetection (lower panels) of HilA-FLAG protein (C) and the SPI-2 encoded SsrA-FLAG protein (D) in whole cell extracts from cultures of WT and Δ greA Δ greB derivative strains in a hilD + and hilD - genetic backgrounds. The upper panels are sections of Coomassie stained gels as loading controls. In all cases bacterial cultures were grown in LB at 37°C up to an OD 600nm of 2.0.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Standard Deviation, Immunodetection, Staining

    The Gre factors affect the expression of SPI-1 effector proteins. (A) Contact haemolysis assays with cell-free supernatants from cultures of the WT and Δ greA Δ greB derivative strains. The haemolytic activity of LB bacterial cultures of WT and Δ greA Δ greB strains was monitored as an increase in the OD 550nm of the supernatant of a blood suspension with serial dilutions of cell-free supernatants. U: undiluted supernatant. Three independent bacterial cultures were tested. (B) Cell-free supernatants of two independent LB cultures of WT (SV5015) and its Δ greA , Δ greB and Δ greA Δ greB derivatives. Extracts were analyzed by Coomassie blue stained 12.5% SDS-PAGE. Lane M: molecular mass markers (size in kDa indicated). The bands labelled were identified as SipA (1), FliD (2) and SipC (3) by LC-MS/MS. (C) Immunodetection (lower panel) of the SPI-1 encoded SipA-FLAG protein in whole culture extracts from two independent cultures of WT and Δ greA Δ greB derivative strains. The upper panel is a section of a Coomassie stained gel as a loading control. (D) Immunodetection of SopE protein was performed in extracts from WT and Δ greA Δ greB strains obtained from cell-free supernatants of two independent LB cultures. (E) Semiquantitative RT-PCR of sipA in total RNA samples from LB cultures of the WT (SV5015) and Δ greA Δ greB strains. 16S RNA was used as endogenous control to confirm that equivalent quantities of templates were used. (F) sipC transcriptional expression was tested in cultures of WT and Δ greA Δ greB derivative strains carrying a chromosomal sipC :: lacZ fusion and either pBR322 or pBR greAB . A bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. All cultures were grown in LB at 37°C with vigorous shaking (200 rpm) up to an OD 600nm of 2.0.
    Figure Legend Snippet: The Gre factors affect the expression of SPI-1 effector proteins. (A) Contact haemolysis assays with cell-free supernatants from cultures of the WT and Δ greA Δ greB derivative strains. The haemolytic activity of LB bacterial cultures of WT and Δ greA Δ greB strains was monitored as an increase in the OD 550nm of the supernatant of a blood suspension with serial dilutions of cell-free supernatants. U: undiluted supernatant. Three independent bacterial cultures were tested. (B) Cell-free supernatants of two independent LB cultures of WT (SV5015) and its Δ greA , Δ greB and Δ greA Δ greB derivatives. Extracts were analyzed by Coomassie blue stained 12.5% SDS-PAGE. Lane M: molecular mass markers (size in kDa indicated). The bands labelled were identified as SipA (1), FliD (2) and SipC (3) by LC-MS/MS. (C) Immunodetection (lower panel) of the SPI-1 encoded SipA-FLAG protein in whole culture extracts from two independent cultures of WT and Δ greA Δ greB derivative strains. The upper panel is a section of a Coomassie stained gel as a loading control. (D) Immunodetection of SopE protein was performed in extracts from WT and Δ greA Δ greB strains obtained from cell-free supernatants of two independent LB cultures. (E) Semiquantitative RT-PCR of sipA in total RNA samples from LB cultures of the WT (SV5015) and Δ greA Δ greB strains. 16S RNA was used as endogenous control to confirm that equivalent quantities of templates were used. (F) sipC transcriptional expression was tested in cultures of WT and Δ greA Δ greB derivative strains carrying a chromosomal sipC :: lacZ fusion and either pBR322 or pBR greAB . A bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. All cultures were grown in LB at 37°C with vigorous shaking (200 rpm) up to an OD 600nm of 2.0.

    Techniques Used: Expressing, Activity Assay, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunodetection, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Gre factors are essential for HilA expression. (A) Transcriptional expression of hilA in WT and Δ greA Δ greB derivative strains. β-galactosidase activity from a hilA :: lacZ fusion was assessed in LB cultures grown at 37°C up to logarithmic (OD 600nm 0.4) and stationary growth phase (OD 600nm 2.0). (B) Relative hilA mRNA quantification by qPCR in WT and Δ greA Δ greB derivative strains. Results are normalized with gapA (GAPDH) as an endogenous control. RNA samples were extracted from cultures of WT and Δ greA Δ greB derivative strains grown in LB at 37°C up to an OD 600nm 2.0. In A and B, a bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. (C) Immunodetection of HilA-FLAG (lower left panel) and InvF-FLAG (lower right panel) proteins in whole cell extracts from cultures of WT and Δ greA Δ greB derivative strains grown as in B. The upper panels are sections of Coomassie stained gels as loading controls. (D) Cell-free supernatants of LB cultures, grown at 37°C up to an OD 600nm of 2.0, of WT and Δ greA Δ greB derivative strains carrying either pBAD18 or pBADHilA. Arabinose (0.02%) was added in all cultures. Extracts were analyzed by Coomassie blue stained 12.5% SDS-PAGE.
    Figure Legend Snippet: Gre factors are essential for HilA expression. (A) Transcriptional expression of hilA in WT and Δ greA Δ greB derivative strains. β-galactosidase activity from a hilA :: lacZ fusion was assessed in LB cultures grown at 37°C up to logarithmic (OD 600nm 0.4) and stationary growth phase (OD 600nm 2.0). (B) Relative hilA mRNA quantification by qPCR in WT and Δ greA Δ greB derivative strains. Results are normalized with gapA (GAPDH) as an endogenous control. RNA samples were extracted from cultures of WT and Δ greA Δ greB derivative strains grown in LB at 37°C up to an OD 600nm 2.0. In A and B, a bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. (C) Immunodetection of HilA-FLAG (lower left panel) and InvF-FLAG (lower right panel) proteins in whole cell extracts from cultures of WT and Δ greA Δ greB derivative strains grown as in B. The upper panels are sections of Coomassie stained gels as loading controls. (D) Cell-free supernatants of LB cultures, grown at 37°C up to an OD 600nm of 2.0, of WT and Δ greA Δ greB derivative strains carrying either pBAD18 or pBADHilA. Arabinose (0.02%) was added in all cultures. Extracts were analyzed by Coomassie blue stained 12.5% SDS-PAGE.

    Techniques Used: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Immunodetection, Staining, SDS Page

    Overexpression of HilD restores HilA expression and epithelial cell invasiveness in Δ greA Δ greB strains. (A) In lower panels, immunodetection of the HilA-FLAG protein in whole cell extracts from cultures of WT and Δ greA Δ greB derivative strains carrying either pBAD18 or pBADHilD grown in LB at 37°C up to an OD 600nm of 2.0, arabinose (0.02%) was added in all cultures. The upper panels are sections of Coomassie stained gels as loading controls. (B) Invasion assays using WT, Δ motA and Δ greA Δ greB strains carrying the indicated plasmids. Bacterial cultures were grown as in Fig 1 . A bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation.
    Figure Legend Snippet: Overexpression of HilD restores HilA expression and epithelial cell invasiveness in Δ greA Δ greB strains. (A) In lower panels, immunodetection of the HilA-FLAG protein in whole cell extracts from cultures of WT and Δ greA Δ greB derivative strains carrying either pBAD18 or pBADHilD grown in LB at 37°C up to an OD 600nm of 2.0, arabinose (0.02%) was added in all cultures. The upper panels are sections of Coomassie stained gels as loading controls. (B) Invasion assays using WT, Δ motA and Δ greA Δ greB strains carrying the indicated plasmids. Bacterial cultures were grown as in Fig 1 . A bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation.

    Techniques Used: Over Expression, Expressing, Immunodetection, Staining, Standard Deviation

    20) Product Images from "Coordinate Control of the Locus of Enterocyte Effacement and Enterohemolysin Genes by Multiple Common Virulence Regulators in Enterohemorrhagic Escherichia coli ▿"

    Article Title: Coordinate Control of the Locus of Enterocyte Effacement and Enterohemolysin Genes by Multiple Common Virulence Regulators in Enterohemorrhagic Escherichia coli ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.05023-11

    Expression levels of EhxC. The amounts of EhxC-FLAG and DnaK (loading control) in whole-cell lysates were examined by Western blotting with monoclonal anti-FLAG and anti-DnaK. (A) grlA (SKI-5223) or ler (SKI-5222) with vector (pMW118/119), ler + (pMW118LER)
    Figure Legend Snippet: Expression levels of EhxC. The amounts of EhxC-FLAG and DnaK (loading control) in whole-cell lysates were examined by Western blotting with monoclonal anti-FLAG and anti-DnaK. (A) grlA (SKI-5223) or ler (SKI-5222) with vector (pMW118/119), ler + (pMW118LER)

    Techniques Used: Expressing, Western Blot, Plasmid Preparation

    21) Product Images from "Tomato yellow leaf curl virus V2 protein plays a critical role in the nuclear export of V1 protein and viral systemic infection"

    Article Title: Tomato yellow leaf curl virus V2 protein plays a critical role in the nuclear export of V1 protein and viral systemic infection

    Journal: bioRxiv

    doi: 10.1101/669754

    Identification of the interaction between V2 and V1 proteins. (a) Co-immunoprecipitation (Co-IP) analysis of the interaction between FLAG-V2 and V1-YFP. N. benthamiana leaves were co-infiltrated with A. tumefaciens cells harbouring expression vectors to express FLAG-V2 and V1-YFP (Lane 1), FLAG-V2 and YFP (Lane 2), or V1-YFP alone (Lane 3). Cell lysates were incubated with FLAG-trap beads (Sigma, USA). Samples before (Input) and after (IP) immunoprecipitation were analyzed by immunoblotting using anti-GFP or -FLAG antibody. (b) BiFC assays between V1 and V2 proteins in the leaves of N. benthamiana . Confocal imaging was performed at 48 hpi. V1 and V2 were fused to the N (nYFP) and C-terminal (cYFP) fragments of YFP, respectively. The V1-V2 interaction will lead to a reconstituted fluorescence signal. DAPI stains DNA in the nucleus. Bars: 50 µm.
    Figure Legend Snippet: Identification of the interaction between V2 and V1 proteins. (a) Co-immunoprecipitation (Co-IP) analysis of the interaction between FLAG-V2 and V1-YFP. N. benthamiana leaves were co-infiltrated with A. tumefaciens cells harbouring expression vectors to express FLAG-V2 and V1-YFP (Lane 1), FLAG-V2 and YFP (Lane 2), or V1-YFP alone (Lane 3). Cell lysates were incubated with FLAG-trap beads (Sigma, USA). Samples before (Input) and after (IP) immunoprecipitation were analyzed by immunoblotting using anti-GFP or -FLAG antibody. (b) BiFC assays between V1 and V2 proteins in the leaves of N. benthamiana . Confocal imaging was performed at 48 hpi. V1 and V2 were fused to the N (nYFP) and C-terminal (cYFP) fragments of YFP, respectively. The V1-V2 interaction will lead to a reconstituted fluorescence signal. DAPI stains DNA in the nucleus. Bars: 50 µm.

    Techniques Used: Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Incubation, Bimolecular Fluorescence Complementation Assay, Imaging, Fluorescence

    The effect of V2 protein on the nuclear distribution of V1 protein. (a) The localization of V1 protein in the absence or presence of V2 protein in N. benthamiana cells. V1-YFP was expressed in the absence or presence of FLAG-V2 and was detected either by confocal microscopy (left panel) or by Western blotting using an anti-GFP polyclonal antibody (right panel). DAPI stains DNA in the nucleus. Actin serves as a control for equal loading of total lysates. Bars: 50 µm. (b) The localization of V2 in N. benthamiana cells. The expressed YFP or YFP-V2 in epidermal cells of N. benthamiana leaves was detected either by confocal microscopy (left panel) or by Western blotting using an anti-GFP polyclonal antibody (right panel). DAPI stains DNA in the nucleus. Bars: 50 µm. (c) Nuclear-cytoplasmic fractionation assay of the distribution of V1 protein in the absence or presence of FLAG-V2 in H2B-RFP transgenic N. benthamiana plants. Nuclei were purified from plant tissues expressing V1-YFP in the absence or presence of FLAG-V2 using percoll density gradient centrifugation. Western blot analysis was conducted with antibodies specific to the indicated proteins using an anti-GFP polyclonal antibody or an anti-FLAG monoclonal antibody. PEPC was used as a marker for the cytoplasmic fraction and H2B-RFP was used as a marker for the nuclear fraction. Protein signal intensity was measured by using Adobe Photoshop CS6, with the cytoplasm plus the nucleus levels totaling as 100%.
    Figure Legend Snippet: The effect of V2 protein on the nuclear distribution of V1 protein. (a) The localization of V1 protein in the absence or presence of V2 protein in N. benthamiana cells. V1-YFP was expressed in the absence or presence of FLAG-V2 and was detected either by confocal microscopy (left panel) or by Western blotting using an anti-GFP polyclonal antibody (right panel). DAPI stains DNA in the nucleus. Actin serves as a control for equal loading of total lysates. Bars: 50 µm. (b) The localization of V2 in N. benthamiana cells. The expressed YFP or YFP-V2 in epidermal cells of N. benthamiana leaves was detected either by confocal microscopy (left panel) or by Western blotting using an anti-GFP polyclonal antibody (right panel). DAPI stains DNA in the nucleus. Bars: 50 µm. (c) Nuclear-cytoplasmic fractionation assay of the distribution of V1 protein in the absence or presence of FLAG-V2 in H2B-RFP transgenic N. benthamiana plants. Nuclei were purified from plant tissues expressing V1-YFP in the absence or presence of FLAG-V2 using percoll density gradient centrifugation. Western blot analysis was conducted with antibodies specific to the indicated proteins using an anti-GFP polyclonal antibody or an anti-FLAG monoclonal antibody. PEPC was used as a marker for the cytoplasmic fraction and H2B-RFP was used as a marker for the nuclear fraction. Protein signal intensity was measured by using Adobe Photoshop CS6, with the cytoplasm plus the nucleus levels totaling as 100%.

    Techniques Used: Confocal Microscopy, Western Blot, Fractionation, Transgenic Assay, Purification, Expressing, Gradient Centrifugation, Marker

    22) Product Images from "Deficient Sumoylation of Yeast 2-Micron Plasmid Proteins Rep1 and Rep2 Associated with Their Loss from the Plasmid-Partitioning Locus and Impaired Plasmid Inheritance"

    Article Title: Deficient Sumoylation of Yeast 2-Micron Plasmid Proteins Rep1 and Rep2 Associated with Their Loss from the Plasmid-Partitioning Locus and Impaired Plasmid Inheritance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060384

    Lysine mutations in Rep1 3R and Rep2 13R impair Rep- STB association but do not reduce levels of Rep fusion proteins. ( A ) Five-fold serial dilutions of a cir + or cir 0 yeast one-hybrid reporter strain encoding STB upstream of a HIS3 reporter gene and transformed with plasmids encoding the indicated Rep1 and Rep2 proteins fused to B42 AD -HA were spotted onto galactose media to induce expression of fusion proteins. Recruitment of the Rep proteins to STB was monitored by growth on medium lacking histidine supplemented with 5 mM 3-aminotriazole. (B) Levels of the B42 AD -HA fusion proteins were monitored by western blot analysis of total protein extracted from the yeast transformants 24 h after galactose induction. (C) ChIP assays were performed with anti-Rep1, anti-Rep2, or anti-FLAG antibodies and the precipitated DNA amplified using primers specific for STB . ChIP efficiency is indicated by the percent of input DNA immunoprecipitated (avg ± sd from triplicate assays) (left) and ethidium-stained agarose gels of PCR products from a representative assay are shown (right). Template DNA amplified in “input” PCR reactions represented 40% of the DNA that was immunoprecipitated and used as template in “ChIP” PCR reactions.
    Figure Legend Snippet: Lysine mutations in Rep1 3R and Rep2 13R impair Rep- STB association but do not reduce levels of Rep fusion proteins. ( A ) Five-fold serial dilutions of a cir + or cir 0 yeast one-hybrid reporter strain encoding STB upstream of a HIS3 reporter gene and transformed with plasmids encoding the indicated Rep1 and Rep2 proteins fused to B42 AD -HA were spotted onto galactose media to induce expression of fusion proteins. Recruitment of the Rep proteins to STB was monitored by growth on medium lacking histidine supplemented with 5 mM 3-aminotriazole. (B) Levels of the B42 AD -HA fusion proteins were monitored by western blot analysis of total protein extracted from the yeast transformants 24 h after galactose induction. (C) ChIP assays were performed with anti-Rep1, anti-Rep2, or anti-FLAG antibodies and the precipitated DNA amplified using primers specific for STB . ChIP efficiency is indicated by the percent of input DNA immunoprecipitated (avg ± sd from triplicate assays) (left) and ethidium-stained agarose gels of PCR products from a representative assay are shown (right). Template DNA amplified in “input” PCR reactions represented 40% of the DNA that was immunoprecipitated and used as template in “ChIP” PCR reactions.

    Techniques Used: Transformation Assay, Expressing, Western Blot, Chromatin Immunoprecipitation, Amplification, Immunoprecipitation, Staining, Polymerase Chain Reaction

    23) Product Images from "PTEN inhibits BMI1 function independently of its phosphatase activity"

    Article Title: PTEN inhibits BMI1 function independently of its phosphatase activity

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-8-98

    Characterization of the interaction between PTEN and BMI1 proteins . ( A ) PTEN binds to BMI1 independently of its phosphatase activity. 293T cells were transiently transfected with BMI1 , PTEN, PTEN(C124S) (C124S), PTEN(G129E) (G129E) , a C-terminal PTEN fragment (residues 186-403) (C-PTEN) for 48 hours. Cell lysates were prepared and immunoprecipitated with anti-PTEN and anti-FLAG (M2) (for ectopic BMI1) antibodies. The precipitates and lysates were analyzed by western blot using the indicated antibodies. The # and * symbols indicate endogenous PTEN and a possible oligomer of C-PTEN, respectively. ( B ) Mapping the BMI1 binding motif of the PTEN protein. A set of PTEN truncation mutants were constructed. Their interaction with BMI1 was examined. C2: C2 domain. The + and - symbols indicate binding or not-binding of individual PTEN proteins to BMI1. ( C ) C2 binds to BMI1. FLAG-tagged BMI1 and HA-tagged C2 were transfected into 293T cells as indicated. BMI1 was immunoprecipitated with an anti-FLAG antibody (M2) or a control IgG (IgG), followed by western blot examination for BMI1 and C2. 20% of the cell lysates that were used for immunoprecipitations were also analyzed. The * symbols indicate background bands. ( D ) C2N binds to BMI1. C2N, C2C , and C-tail (left panel) and their GFP fusion counterparts (right panel) were co-transfected with either an empty vector (-) or FLAG-tagged BMI1 as indicated, followed by immunoprecipitation with M2 or control IgG (IgG) and then western blot (WB) with the indicated antibodies. The respective cell lysates were shown at the bottom panels.
    Figure Legend Snippet: Characterization of the interaction between PTEN and BMI1 proteins . ( A ) PTEN binds to BMI1 independently of its phosphatase activity. 293T cells were transiently transfected with BMI1 , PTEN, PTEN(C124S) (C124S), PTEN(G129E) (G129E) , a C-terminal PTEN fragment (residues 186-403) (C-PTEN) for 48 hours. Cell lysates were prepared and immunoprecipitated with anti-PTEN and anti-FLAG (M2) (for ectopic BMI1) antibodies. The precipitates and lysates were analyzed by western blot using the indicated antibodies. The # and * symbols indicate endogenous PTEN and a possible oligomer of C-PTEN, respectively. ( B ) Mapping the BMI1 binding motif of the PTEN protein. A set of PTEN truncation mutants were constructed. Their interaction with BMI1 was examined. C2: C2 domain. The + and - symbols indicate binding or not-binding of individual PTEN proteins to BMI1. ( C ) C2 binds to BMI1. FLAG-tagged BMI1 and HA-tagged C2 were transfected into 293T cells as indicated. BMI1 was immunoprecipitated with an anti-FLAG antibody (M2) or a control IgG (IgG), followed by western blot examination for BMI1 and C2. 20% of the cell lysates that were used for immunoprecipitations were also analyzed. The * symbols indicate background bands. ( D ) C2N binds to BMI1. C2N, C2C , and C-tail (left panel) and their GFP fusion counterparts (right panel) were co-transfected with either an empty vector (-) or FLAG-tagged BMI1 as indicated, followed by immunoprecipitation with M2 or control IgG (IgG) and then western blot (WB) with the indicated antibodies. The respective cell lysates were shown at the bottom panels.

    Techniques Used: Activity Assay, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Construct, Plasmid Preparation

    PTEN inhibits BMI1 function . ( A ) DU145 cells were stably transfected with pBabe or pBabe-BMI1 retrovirus, followed by transiently transfected with pLHCX (empty vector) and pLHCX-PTEN retrovirus for 48 hours. The expression of FLAG-tagged BMI1, HA-tagged PTEN, p16 INK4A , p14 ARF , and actin was examined by western blot using specific antibodies. The relative p14 ARF and p16 INK4A expression was normalized against the respective actin and then expressed as fold changes of p14 ARF and p16 INK4A in DU145 cells co-infected with pBabe and pLHCX. The experiment was repeated at least three times by three individuals with identical results and representatives are shown. This information was presented under the p14 and p16 panels. Symbols * and ** show statistical significance ( p
    Figure Legend Snippet: PTEN inhibits BMI1 function . ( A ) DU145 cells were stably transfected with pBabe or pBabe-BMI1 retrovirus, followed by transiently transfected with pLHCX (empty vector) and pLHCX-PTEN retrovirus for 48 hours. The expression of FLAG-tagged BMI1, HA-tagged PTEN, p16 INK4A , p14 ARF , and actin was examined by western blot using specific antibodies. The relative p14 ARF and p16 INK4A expression was normalized against the respective actin and then expressed as fold changes of p14 ARF and p16 INK4A in DU145 cells co-infected with pBabe and pLHCX. The experiment was repeated at least three times by three individuals with identical results and representatives are shown. This information was presented under the p14 and p16 panels. Symbols * and ** show statistical significance ( p

    Techniques Used: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Western Blot, Infection

    Nuclear PTEN reduces BMI1 function . ( A ) Interaction of nuclear PTEN with BMI1. Chimpanzee PTEN and the indicated mutants PTEN/1-375 and PTEN/1-375(K13A) were transfected without and with FLAG-tagged BMI1 in 293T cells, followed by immunoprecipitation of BMI1 using an anti-FLAG (α-FLAG) and then immunobloted (IB) with the indicated antibodies. Control IgG did not precipitate either BMI1 or PTEN (data not shown). ( B ) Nuclear PTEN inhibits BMI1 function. DU145 cells were transiently expressed with PTEN/1-375 (top panel) or PTEN/1-375(K13A) (bottom panel). Cells were then double IF stained for ectopic PTEN mutants using an anti-HA antibody (red) or endogenous p14 ARF (green). Nuclei were counter-stained with DAPI (blue). More than 200 transfected cells were randomly counted. Typical images of 1-375 and 1-375(K13A) were shown and the related quantification was discussed (see Discussion for details).
    Figure Legend Snippet: Nuclear PTEN reduces BMI1 function . ( A ) Interaction of nuclear PTEN with BMI1. Chimpanzee PTEN and the indicated mutants PTEN/1-375 and PTEN/1-375(K13A) were transfected without and with FLAG-tagged BMI1 in 293T cells, followed by immunoprecipitation of BMI1 using an anti-FLAG (α-FLAG) and then immunobloted (IB) with the indicated antibodies. Control IgG did not precipitate either BMI1 or PTEN (data not shown). ( B ) Nuclear PTEN inhibits BMI1 function. DU145 cells were transiently expressed with PTEN/1-375 (top panel) or PTEN/1-375(K13A) (bottom panel). Cells were then double IF stained for ectopic PTEN mutants using an anti-HA antibody (red) or endogenous p14 ARF (green). Nuclei were counter-stained with DAPI (blue). More than 200 transfected cells were randomly counted. Typical images of 1-375 and 1-375(K13A) were shown and the related quantification was discussed (see Discussion for details).

    Techniques Used: Transfection, Immunoprecipitation, Staining

    PTEN binds to BMI1 . ( A ) 293T cells were transiently transfected with FLAG-tagged BMI1 and HA-tagged PTEN . Cell lysates were prepared and immunoprecipitated with anti-PTEN (top panel) and anti-FLAG (M2) (middle panel) antibodies. The precipitates and lysates (bottom panel) were analyzed by western blot using the indicated antibodies. The * symbol indicates endogenous PTEN. (Note: the reason why endogenous PTEN was not detected in the lysate panel was attributable to a low level of endogenous PTEN in 293T cells). ( B ) DU145 cell lysates were cross-linked with DSP, immunoprecipitated with anti-BMI1 antibody or control IgG, and analyzed by western blot for PTEN and BMI1. Twenty percent of cell lysate used for immunoprecipitation was also analyzed by western blot. ( C ) Co-localization between PTEN and BMI1. Ectopic PTEN and ectopic BMI1 in 293T cells and their respective endogenous proteins in MCF-7 and DU145 cells were examined by double immunofluorescent (IF) staining. Nuclei were counter-stained with DAPI (blue). Scale bar represents 10 μM.
    Figure Legend Snippet: PTEN binds to BMI1 . ( A ) 293T cells were transiently transfected with FLAG-tagged BMI1 and HA-tagged PTEN . Cell lysates were prepared and immunoprecipitated with anti-PTEN (top panel) and anti-FLAG (M2) (middle panel) antibodies. The precipitates and lysates (bottom panel) were analyzed by western blot using the indicated antibodies. The * symbol indicates endogenous PTEN. (Note: the reason why endogenous PTEN was not detected in the lysate panel was attributable to a low level of endogenous PTEN in 293T cells). ( B ) DU145 cell lysates were cross-linked with DSP, immunoprecipitated with anti-BMI1 antibody or control IgG, and analyzed by western blot for PTEN and BMI1. Twenty percent of cell lysate used for immunoprecipitation was also analyzed by western blot. ( C ) Co-localization between PTEN and BMI1. Ectopic PTEN and ectopic BMI1 in 293T cells and their respective endogenous proteins in MCF-7 and DU145 cells were examined by double immunofluorescent (IF) staining. Nuclei were counter-stained with DAPI (blue). Scale bar represents 10 μM.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Staining

    24) Product Images from "Cell-cycle dependent localization of MELK and its new partner RACK1 in epithelial versus mesenchyme-like cells in Xenopus embryo"

    Article Title: Cell-cycle dependent localization of MELK and its new partner RACK1 in epithelial versus mesenchyme-like cells in Xenopus embryo

    Journal: Biology Open

    doi: 10.1242/bio.20136080

    RACK1 regulates localization of iMELK. (A) Gastrula embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were fixed and processed for indirect immunofluorescence with anti-FLAG (a,d,g) and anti-xMELK antibodies (b,e,h). Pictures were merged (merge, c,f,i) together with pictures of DNA (blue) at the same confocal planes to visualize co-localization of xMELK (red) with FLAG-RACK1 constructs (green). Embryos expressing FLAG-RACK1 WD5–7 were incubated with a rabbit polyclonal anti-FLAG (j) and a mouse monoclonal anti-tubulin (k) antibody. Pictures were merged together with pictures of DNA (blue) to visualize FLAG-RACK1 WD5–7 and microtubules. White dashed arrows in panels c, f and i indicate the plane used for orthogonal projections of confocal planes shown on the right. Asterisks indicate cytokinetic cells. Arrowheads point on xMELK concentrated at the tight junctions. Scale bars: 20 µm (a–i), 10 µm (j–l). Intensity of the xMELK fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were quantified in interphase and mitotic cells for each 0.5 µm confocal plane. (B) Embryos were coinjected with FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 mRNAs with GFP-xMELK KR or GFP-gpi mRNAs. White dashed lines mark the plane used for orthogonal projections of confocal planes shown in the center. Arrows points to the apical junctional complex. Black and white arrows points to GFP-xMELK and GFP-gpi, respectively. Scale bars: 20 µm. The intensity of the GFP-xMELK KR and GFP-gpi fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 was quantified in interphase and mitotic cells for each 0.5 µm confocal plane. Statistical analysis was performed. *GFP-xMELK KR+ FLAG-RACK1 WD5–7 is significantly different from GFP-xMELK KR+ FLAG-RACK1 FL at p
    Figure Legend Snippet: RACK1 regulates localization of iMELK. (A) Gastrula embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were fixed and processed for indirect immunofluorescence with anti-FLAG (a,d,g) and anti-xMELK antibodies (b,e,h). Pictures were merged (merge, c,f,i) together with pictures of DNA (blue) at the same confocal planes to visualize co-localization of xMELK (red) with FLAG-RACK1 constructs (green). Embryos expressing FLAG-RACK1 WD5–7 were incubated with a rabbit polyclonal anti-FLAG (j) and a mouse monoclonal anti-tubulin (k) antibody. Pictures were merged together with pictures of DNA (blue) to visualize FLAG-RACK1 WD5–7 and microtubules. White dashed arrows in panels c, f and i indicate the plane used for orthogonal projections of confocal planes shown on the right. Asterisks indicate cytokinetic cells. Arrowheads point on xMELK concentrated at the tight junctions. Scale bars: 20 µm (a–i), 10 µm (j–l). Intensity of the xMELK fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were quantified in interphase and mitotic cells for each 0.5 µm confocal plane. (B) Embryos were coinjected with FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 mRNAs with GFP-xMELK KR or GFP-gpi mRNAs. White dashed lines mark the plane used for orthogonal projections of confocal planes shown in the center. Arrows points to the apical junctional complex. Black and white arrows points to GFP-xMELK and GFP-gpi, respectively. Scale bars: 20 µm. The intensity of the GFP-xMELK KR and GFP-gpi fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 was quantified in interphase and mitotic cells for each 0.5 µm confocal plane. Statistical analysis was performed. *GFP-xMELK KR+ FLAG-RACK1 WD5–7 is significantly different from GFP-xMELK KR+ FLAG-RACK1 FL at p

    Techniques Used: Expressing, Immunofluorescence, Construct, Incubation

    xMELK and RACK1 are in the same complex. (A) Identification of RACK1 as a potential xMELK partner. Proteins extracted from FLAG-xMELK expressing or uninjected control (U.) embryos were immunoprecipitated with anti-FLAG antibodies, separated by SDS-PAGE and silver stained. The 35 kDa band present in the FLAG-xMELK but not in the control immunoprecipitate was cut out from the gel and analyzed by mass spectrometry. Two peptides matching RACK1 protein sequence (underlined) were identified. Two additional peptides were identified in an independent experiment (dashed underline). Ig HC and Ig LC: immunoglobulins heavy and light chains, respectively. (B,C) Validation of xMELK and RACK1 interaction. (B) Proteins were extracted from FLAG-xMELK (F-MELK) expressing or uninjected (U.) embryos (inputs). Proteins were immunoprecipitated with anti-FLAG antibodies (IP FLAG) and Western blots were incubated with anti-xMELK and anti-RACK1 antibodies. (C) Protein extracts (inputs) were prepared from embryos co-expressing FLAG-RACK1 (F-RACK1) and myc-GFP (m-GFP), FLAG-RACK1 and myc-xMELK (m-MELK) or uninjected control embryos. Proteins were immunoprecipitated with anti-FLAG antibodies (IP FLAG) and Western blots were incubated with anti-myc and anti-RACK1 antibodies. (D) xMELK preferentially associates with RACK1 N-terminal domain. Protein extracts (inputs) were prepared from embryos co-expressing myc-xMELK with full length FLAG-RACK1 (F-RACK1 FL), FLAG-RACK1 WD1–4 (F-WD1–4), and FLAG-RACK1 WD5–7 (F-WD5–7) or uninjected (U.) embryos. Proteins were immunoprecipitated with anti-Flag antibodies (IP FLAG) and Western blots were incubated with anti-FLAG and anti-myc antibodies. The histogram on the right represents quantifications of the myc signal obtained in 3 independent immunoprecipitation experiments normalized with the corresponding FLAG signals (myc/FLAG ratio). Error bars denote s.e.m., a t -test was performed and p values are indicated above bars. Schematic representation of the RACK1 constructs is shown. The grey box indicates the FLAG tag.
    Figure Legend Snippet: xMELK and RACK1 are in the same complex. (A) Identification of RACK1 as a potential xMELK partner. Proteins extracted from FLAG-xMELK expressing or uninjected control (U.) embryos were immunoprecipitated with anti-FLAG antibodies, separated by SDS-PAGE and silver stained. The 35 kDa band present in the FLAG-xMELK but not in the control immunoprecipitate was cut out from the gel and analyzed by mass spectrometry. Two peptides matching RACK1 protein sequence (underlined) were identified. Two additional peptides were identified in an independent experiment (dashed underline). Ig HC and Ig LC: immunoglobulins heavy and light chains, respectively. (B,C) Validation of xMELK and RACK1 interaction. (B) Proteins were extracted from FLAG-xMELK (F-MELK) expressing or uninjected (U.) embryos (inputs). Proteins were immunoprecipitated with anti-FLAG antibodies (IP FLAG) and Western blots were incubated with anti-xMELK and anti-RACK1 antibodies. (C) Protein extracts (inputs) were prepared from embryos co-expressing FLAG-RACK1 (F-RACK1) and myc-GFP (m-GFP), FLAG-RACK1 and myc-xMELK (m-MELK) or uninjected control embryos. Proteins were immunoprecipitated with anti-FLAG antibodies (IP FLAG) and Western blots were incubated with anti-myc and anti-RACK1 antibodies. (D) xMELK preferentially associates with RACK1 N-terminal domain. Protein extracts (inputs) were prepared from embryos co-expressing myc-xMELK with full length FLAG-RACK1 (F-RACK1 FL), FLAG-RACK1 WD1–4 (F-WD1–4), and FLAG-RACK1 WD5–7 (F-WD5–7) or uninjected (U.) embryos. Proteins were immunoprecipitated with anti-Flag antibodies (IP FLAG) and Western blots were incubated with anti-FLAG and anti-myc antibodies. The histogram on the right represents quantifications of the myc signal obtained in 3 independent immunoprecipitation experiments normalized with the corresponding FLAG signals (myc/FLAG ratio). Error bars denote s.e.m., a t -test was performed and p values are indicated above bars. Schematic representation of the RACK1 constructs is shown. The grey box indicates the FLAG tag.

    Techniques Used: Expressing, Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Sequencing, Western Blot, Incubation, Construct, FLAG-tag

    25) Product Images from "The RING Domain and First Zinc Finger of TRAF6 Coordinate Signaling by Interleukin-1, Lipopolysaccharide, and RANKL *"

    Article Title: The RING Domain and First Zinc Finger of TRAF6 Coordinate Signaling by Interleukin-1, Lipopolysaccharide, and RANKL *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M802749200

    An intact RING and ZF1 domain of TRAF6 is required for LPS signaling. A and B , TRAF6-deficient MEFs stably expressing empty vector (pMX) or the indicated FLAG-tagged TRAF6 construct were left unstimulated (time 0) or stimulated with LPS (5 μg/ml)
    Figure Legend Snippet: An intact RING and ZF1 domain of TRAF6 is required for LPS signaling. A and B , TRAF6-deficient MEFs stably expressing empty vector (pMX) or the indicated FLAG-tagged TRAF6 construct were left unstimulated (time 0) or stimulated with LPS (5 μg/ml)

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Construct

    26) Product Images from "SOAR and the polybasic STIM1 domains gate and regulate the Orai channels"

    Article Title: SOAR and the polybasic STIM1 domains gate and regulate the Orai channels

    Journal: Nature cell biology

    doi: 10.1038/ncb1842

    SOAR gates Orai1 but does not mediate clustering of STIM1 or co-clustering of STIM1-Orai1 (a–b) CRAC current was measured in cells transfected with Orai1 and either SOAR (black), SOAR(LQ/AA) (red), STIM1 (blue) or STIM1(LQ/AA) (green). (b) is the mean±s.e.m. of 4 experiments. In (c), cells were co-transfected with mCherry-Orai1 and EGFP-SOAR(LQ/AA). In (d), cells were transfected with STIM1(LQ/AA) alone and were untreated (upper image) or treated with 25 µM CPA in Ca 2+ - free media for 10 min. (e) shows two examples of cells co-transfected with mCherry-Orai1 and EGFP-STIM1(LQ/AA) and treated with CPA for 10 min. Arrows in (e) point to co-localized Orai1-STIM1 in puncta. Each set of images represent at least 50 analyzed cells from 2 transfections. In (f, g), cells were transfected with Orai1 and SOAR or SOAR(LQ/AA) (f) or Orai1 and STIM1 or STIM1(LQ/AA) (g) and used to IP Orai1 with anti FLAG and probe for co-IP of SOAR or STIM1 with anti-GFP. Fig. S7b shows full blot of Orai1 and Fig. S7c shows full blot of EGFP-STIM1 and EGFP-SOAR. In addition, Fig. S7c is the control for the co-IP in panels (f, g). In (h), TRPC1 was expressed in control cells alone (condition 1, black) or together with STIM1 (condition 2, magenta), STIM1(LQ/AA) (condition 3, red), or in siSTIM1-treated cells alone (condition 4, green) or together with STIM1(LQ/AA) (condition 5, blue), and the carbachol-activated TRPC1-mediated Na + current was measured. The columns show the mean±s.e.m. of the % current in 4 experiments.
    Figure Legend Snippet: SOAR gates Orai1 but does not mediate clustering of STIM1 or co-clustering of STIM1-Orai1 (a–b) CRAC current was measured in cells transfected with Orai1 and either SOAR (black), SOAR(LQ/AA) (red), STIM1 (blue) or STIM1(LQ/AA) (green). (b) is the mean±s.e.m. of 4 experiments. In (c), cells were co-transfected with mCherry-Orai1 and EGFP-SOAR(LQ/AA). In (d), cells were transfected with STIM1(LQ/AA) alone and were untreated (upper image) or treated with 25 µM CPA in Ca 2+ - free media for 10 min. (e) shows two examples of cells co-transfected with mCherry-Orai1 and EGFP-STIM1(LQ/AA) and treated with CPA for 10 min. Arrows in (e) point to co-localized Orai1-STIM1 in puncta. Each set of images represent at least 50 analyzed cells from 2 transfections. In (f, g), cells were transfected with Orai1 and SOAR or SOAR(LQ/AA) (f) or Orai1 and STIM1 or STIM1(LQ/AA) (g) and used to IP Orai1 with anti FLAG and probe for co-IP of SOAR or STIM1 with anti-GFP. Fig. S7b shows full blot of Orai1 and Fig. S7c shows full blot of EGFP-STIM1 and EGFP-SOAR. In addition, Fig. S7c is the control for the co-IP in panels (f, g). In (h), TRPC1 was expressed in control cells alone (condition 1, black) or together with STIM1 (condition 2, magenta), STIM1(LQ/AA) (condition 3, red), or in siSTIM1-treated cells alone (condition 4, green) or together with STIM1(LQ/AA) (condition 5, blue), and the carbachol-activated TRPC1-mediated Na + current was measured. The columns show the mean±s.e.m. of the % current in 4 experiments.

    Techniques Used: Transfection, Co-Immunoprecipitation Assay

    27) Product Images from "Arf GTPase-activating Protein AGAP2 Regulates Focal Adhesion Kinase Activity and Focal Adhesion Remodeling *"

    Article Title: Arf GTPase-activating Protein AGAP2 Regulates Focal Adhesion Kinase Activity and Focal Adhesion Remodeling *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M900469200

    AGAP2 regulates FAK phosphorylation. A, overexpression of AGAP2 increases FAK phosphorylation. The cDNAs encoding empty vector or FLAG-AGAP2 were transfected into HEK293 cells for 48 h. Cleared cell lysates were resolved by SDS-PAGE and followed by
    Figure Legend Snippet: AGAP2 regulates FAK phosphorylation. A, overexpression of AGAP2 increases FAK phosphorylation. The cDNAs encoding empty vector or FLAG-AGAP2 were transfected into HEK293 cells for 48 h. Cleared cell lysates were resolved by SDS-PAGE and followed by

    Techniques Used: Over Expression, Plasmid Preparation, Transfection, SDS Page

    AGAP2 co-localizes with FAK and paxillin. U87 cells stably expressing empty vector or FLAG-AGAP2 were plated on fibronectin-coated glass coverslips for 5 h, fixed in 4% formaldehyde, processed for immunofluorescence staining, and examined by confocal
    Figure Legend Snippet: AGAP2 co-localizes with FAK and paxillin. U87 cells stably expressing empty vector or FLAG-AGAP2 were plated on fibronectin-coated glass coverslips for 5 h, fixed in 4% formaldehyde, processed for immunofluorescence staining, and examined by confocal

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Staining

    AGAP2 disrupts focal adhesions. U87 cells were transfected with cDNAs encoding empty vector or FLAG-AGAP2 for 48 h and replated on fibronectin-coated glass coverslips for 5 h. Cells were stained with anti-paxillin ( A and B ) or anti-vinculin ( D and
    Figure Legend Snippet: AGAP2 disrupts focal adhesions. U87 cells were transfected with cDNAs encoding empty vector or FLAG-AGAP2 for 48 h and replated on fibronectin-coated glass coverslips for 5 h. Cells were stained with anti-paxillin ( A and B ) or anti-vinculin ( D and

    Techniques Used: Transfection, Plasmid Preparation, Staining

    AGAP2 forms a complex with focal adhesion kinase. A, co-immunoprecipitation of FAK with AGAP2. HEK293 cells were transfected with cDNAs encoding empty vector or FLAG-AGAP2. The AGAP2 was immunoprecipitated ( I.P. ) with anti-FLAG antibody and eluted
    Figure Legend Snippet: AGAP2 forms a complex with focal adhesion kinase. A, co-immunoprecipitation of FAK with AGAP2. HEK293 cells were transfected with cDNAs encoding empty vector or FLAG-AGAP2. The AGAP2 was immunoprecipitated ( I.P. ) with anti-FLAG antibody and eluted

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation

    28) Product Images from "The Interferon Stimulated Gene 54 Promotes Apoptosis *"

    Article Title: The Interferon Stimulated Gene 54 Promotes Apoptosis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.207068

    Glycerol gradient sedimentation of ISG protein complexes. HeLa cells were co-transfected with ISG54-V5, FLAG-ISG56, and FLAG-ISG60 plasmids and treated with IFN-α for 18 h. Lysates were sedimented through 25–40% glycerol gradients, and
    Figure Legend Snippet: Glycerol gradient sedimentation of ISG protein complexes. HeLa cells were co-transfected with ISG54-V5, FLAG-ISG56, and FLAG-ISG60 plasmids and treated with IFN-α for 18 h. Lysates were sedimented through 25–40% glycerol gradients, and

    Techniques Used: Sedimentation, Transfection

    29) Product Images from "The Latency-Associated Nuclear Antigen of Rhesus Monkey Rhadinovirus Inhibits Viral Replication through Repression of Orf50/Rta Transcriptional Activation"

    Article Title: The Latency-Associated Nuclear Antigen of Rhesus Monkey Rhadinovirus Inhibits Viral Replication through Repression of Orf50/Rta Transcriptional Activation

    Journal:

    doi: 10.1128/JVI.79.5.3127-3138.2005

    R-LANA inhibits RRV lytic replication. RhFs were transfected with either pEF or pEF-R-LANA FLAG . Twenty-four hours posttransfection, the cells were subsequently infected with RRV-GFP at an MOI of ≥1. Seventy-two hours postinfection, cell-free supernatants
    Figure Legend Snippet: R-LANA inhibits RRV lytic replication. RhFs were transfected with either pEF or pEF-R-LANA FLAG . Twenty-four hours posttransfection, the cells were subsequently infected with RRV-GFP at an MOI of ≥1. Seventy-two hours postinfection, cell-free supernatants

    Techniques Used: Transfection, Infection

    30) Product Images from "Shank-interacting protein-like 1 promotes tumorigenesis via PTEN inhibition in human tumor cells"

    Article Title: Shank-interacting protein-like 1 promotes tumorigenesis via PTEN inhibition in human tumor cells

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI40778

    SIPL1 reduces PTEN’s PIP 3 phosphatase activity in the cell. ( A ) 0, 5, 10, 20 μg SIPL1 and 5 μg PTEN (S0P5, S5P5, S10P5 and S20P5, where S represents SIPL1) were cotransfected into 293T cells. 5 μg PTEN C124S mutant (C124S) served as a negative control. IPs were carried out using an anti-PTEN antibody, followed by assaying for PTEN’s PIP 3 phosphatase activity. S20P0, 20 μg SIPL1 (FLAG-tagged) was used (negative control). Other negative controls (a reaction without the addition of PTEN and a reaction without the addition of PIP 3 substrate) showed no detectable activities (data not shown). Western blot shows the immunoprecipitated PTEN and SIPL1. ( B ) DU145 cells were treated with control siRNA or SIPL1 siRNA retrovirus as indicated. Knockdown of SIPL1 was demonstrated (right panel). PTEN was immunoprecipitated from both cells, followed by assaying for PTEN’s PIP 3 phosphatase activity in vitro. Comparable levels of PTEN were immunoprecipitated from both cell lines (data not shown). Experiments were repeated 3 times. ( C ) PTEN (5 μg) was transiently coexpressed in 293T cells with the indicated SIPL1 mutants (20 μg), followed by IP with anti-PTEN or M2 anti-FLAG (for SIPL1 mutants), and then assayed for PTEN’s PIP 3 phosphatase activity. PTEN C124S (C124S, 5 μg) was used as a negative control. Under this assay system, 20 μg of the SIPL1 construct completely inhibited the PIP 3 phosphatase activity produced by cotransfection of cells with 5 μg of the PTEN construct (data not shown; also see A ).
    Figure Legend Snippet: SIPL1 reduces PTEN’s PIP 3 phosphatase activity in the cell. ( A ) 0, 5, 10, 20 μg SIPL1 and 5 μg PTEN (S0P5, S5P5, S10P5 and S20P5, where S represents SIPL1) were cotransfected into 293T cells. 5 μg PTEN C124S mutant (C124S) served as a negative control. IPs were carried out using an anti-PTEN antibody, followed by assaying for PTEN’s PIP 3 phosphatase activity. S20P0, 20 μg SIPL1 (FLAG-tagged) was used (negative control). Other negative controls (a reaction without the addition of PTEN and a reaction without the addition of PIP 3 substrate) showed no detectable activities (data not shown). Western blot shows the immunoprecipitated PTEN and SIPL1. ( B ) DU145 cells were treated with control siRNA or SIPL1 siRNA retrovirus as indicated. Knockdown of SIPL1 was demonstrated (right panel). PTEN was immunoprecipitated from both cells, followed by assaying for PTEN’s PIP 3 phosphatase activity in vitro. Comparable levels of PTEN were immunoprecipitated from both cell lines (data not shown). Experiments were repeated 3 times. ( C ) PTEN (5 μg) was transiently coexpressed in 293T cells with the indicated SIPL1 mutants (20 μg), followed by IP with anti-PTEN or M2 anti-FLAG (for SIPL1 mutants), and then assayed for PTEN’s PIP 3 phosphatase activity. PTEN C124S (C124S, 5 μg) was used as a negative control. Under this assay system, 20 μg of the SIPL1 construct completely inhibited the PIP 3 phosphatase activity produced by cotransfection of cells with 5 μg of the PTEN construct (data not shown; also see A ).

    Techniques Used: Activity Assay, Mutagenesis, Negative Control, Western Blot, Immunoprecipitation, In Vitro, Construct, Produced, Cotransfection

    SIPL1 binds to PTEN. ( A ) SIPL1 was coexpressed with the indicated cDNAs in 293T cells, followed by IP with the indicated antibodies and Western blot (WB) analysis using anti-PTEN or anti-FLAG antibodies. ( B ) IP of endogenous SIPL1 with the indicated antibodies, followed by Western blot analysis for PTEN and SIPL1 using specific antibodies. “PTEN siRNA” indicates PTEN-knockdown DU145 cells. ( C ) Colocalization of PTEN and SIPL1 in MCF7 cells. PTEN and SIPL1 were immunofluorescently stained as red and green, respectively. Typical z-stack images captured using a confocal microscope (MP Leica TCS SP5) are shown. Nuclei were counterstained with DAPI (blue). Scale bars: 10 μm. Experiments in A , B , and C were repeated 3 times.
    Figure Legend Snippet: SIPL1 binds to PTEN. ( A ) SIPL1 was coexpressed with the indicated cDNAs in 293T cells, followed by IP with the indicated antibodies and Western blot (WB) analysis using anti-PTEN or anti-FLAG antibodies. ( B ) IP of endogenous SIPL1 with the indicated antibodies, followed by Western blot analysis for PTEN and SIPL1 using specific antibodies. “PTEN siRNA” indicates PTEN-knockdown DU145 cells. ( C ) Colocalization of PTEN and SIPL1 in MCF7 cells. PTEN and SIPL1 were immunofluorescently stained as red and green, respectively. Typical z-stack images captured using a confocal microscope (MP Leica TCS SP5) are shown. Nuclei were counterstained with DAPI (blue). Scale bars: 10 μm. Experiments in A , B , and C were repeated 3 times.

    Techniques Used: Western Blot, Staining, Microscopy

    Characterization of the interaction between SIPL1 and PTEN. Truncation mutants of SIPL1 and PTEN used in the binding experiments were generated ( E ). ( A ) SIPL1-N and SIPL1-C mutants were coexpressed individually with PTEN in 293T cells, followed by IP with the indicated antibodies, and examined for PTEN, SIPL1-N, and SIPL1-C by Western blot. The pound symbol (#) indicates the IgG heavy chain. ( B ) SIPL1 tail (see E for details) and UBL were fused with GFP (GFP-tail and GFP-UBL), followed by examination of their interaction with PTEN by transient transfection using 293T cells. The asterisk indicates the coimmunoprecipitated GFP-UBL band. ( C ) FLAG-tagged SIPL1-ΔUBL was examined for interaction with PTEN using the IP–Western blot methodology. ( D ) PTEN-N and PTEN-C were examined for binding to FLAG-tagged SIPL1-C by the IP–Western blot system using the indicated antibodies. PTEN-N and PTEN-C bands are indicated (left and middle panels). ( E ) Mapping the PTEN-binding and SIPL1-binding motifs. ++, +, +/–, and – indicate the levels of interaction between the indicated proteins.
    Figure Legend Snippet: Characterization of the interaction between SIPL1 and PTEN. Truncation mutants of SIPL1 and PTEN used in the binding experiments were generated ( E ). ( A ) SIPL1-N and SIPL1-C mutants were coexpressed individually with PTEN in 293T cells, followed by IP with the indicated antibodies, and examined for PTEN, SIPL1-N, and SIPL1-C by Western blot. The pound symbol (#) indicates the IgG heavy chain. ( B ) SIPL1 tail (see E for details) and UBL were fused with GFP (GFP-tail and GFP-UBL), followed by examination of their interaction with PTEN by transient transfection using 293T cells. The asterisk indicates the coimmunoprecipitated GFP-UBL band. ( C ) FLAG-tagged SIPL1-ΔUBL was examined for interaction with PTEN using the IP–Western blot methodology. ( D ) PTEN-N and PTEN-C were examined for binding to FLAG-tagged SIPL1-C by the IP–Western blot system using the indicated antibodies. PTEN-N and PTEN-C bands are indicated (left and middle panels). ( E ) Mapping the PTEN-binding and SIPL1-binding motifs. ++, +, +/–, and – indicate the levels of interaction between the indicated proteins.

    Techniques Used: Binding Assay, Generated, Western Blot, Transfection

    31) Product Images from "Identification of SRPK1 and SRPK2 as the Major Cellular Protein Kinases Phosphorylating Hepatitis B Virus Core Protein"

    Article Title: Identification of SRPK1 and SRPK2 as the Major Cellular Protein Kinases Phosphorylating Hepatitis B Virus Core Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.16.8124-8137.2002

    Analysis of in vivo phosphorylation and SRPK-mediated in vitro phosphorylation of HBV core protein. (A) HuH-7 cells were transiently transfected in six-well dishes with 1.5 μg of either empty expression vector, plasmid encoding wild-type HBV core protein, or plasmid encoding mutant HBV core protein devoid of the three reported C-terminal serine phosphorylation sites (serines 155, 162, and 170) per well. On the second day after transfection, cells were labeled with 50 μCi of 33 P i per ml for 3.5 h. Lysates were then subjected to immunoprecipitation (IP) with anti-HBV core antibody. After gel electrophoresis and transfer onto a nitrocellulose membrane, HBV core protein phosphorylation was first visualized by autoradiography (upper panel), followed by immunoblotting of the same filter with anti-HBV core protein antibody (lower panel). (B) COS-7 cells were transiently transfected in six-well dishes with either control plasmids or expression constructs encoding FLAG-tagged SRPK1 or VSV-tagged SRPK2 (1.5 μg/well each). After cell lysis and immunoprecipitation (IP) with anti-FLAG or anti-VSV antibodies, in vitro kinase reactions were performed in the presence of [γ- 32 P]ATP and GST-HBV-C1 core fusion protein with (C1) or without the three previously mapped C-terminal serine phosphorylation sites (C1-AAA, serines 155, 162, and 170 mutated to alanines). After gel electrophoresis, phosphate incorporation into substrate protein was visualized by autoradiography. (C) HuH-7 cells were transiently transfected in six-well dishes with plasmids encoding either wild-type HBV core protein or its mutants lacking serine phosphorylation sites in the C-terminal part as indicated (1.5 μg/well each). After metabolic labeling with 50 μCi of 33 P i per ml for 3.5 h, cell lysates were processed as described for panel A. HBV core protein phosphorylation was visualized by autoradiography (upper panel) and quantified by a PhosphorImager, and the same filter was then probed with anti-HBV core protein antibody (lower panel).
    Figure Legend Snippet: Analysis of in vivo phosphorylation and SRPK-mediated in vitro phosphorylation of HBV core protein. (A) HuH-7 cells were transiently transfected in six-well dishes with 1.5 μg of either empty expression vector, plasmid encoding wild-type HBV core protein, or plasmid encoding mutant HBV core protein devoid of the three reported C-terminal serine phosphorylation sites (serines 155, 162, and 170) per well. On the second day after transfection, cells were labeled with 50 μCi of 33 P i per ml for 3.5 h. Lysates were then subjected to immunoprecipitation (IP) with anti-HBV core antibody. After gel electrophoresis and transfer onto a nitrocellulose membrane, HBV core protein phosphorylation was first visualized by autoradiography (upper panel), followed by immunoblotting of the same filter with anti-HBV core protein antibody (lower panel). (B) COS-7 cells were transiently transfected in six-well dishes with either control plasmids or expression constructs encoding FLAG-tagged SRPK1 or VSV-tagged SRPK2 (1.5 μg/well each). After cell lysis and immunoprecipitation (IP) with anti-FLAG or anti-VSV antibodies, in vitro kinase reactions were performed in the presence of [γ- 32 P]ATP and GST-HBV-C1 core fusion protein with (C1) or without the three previously mapped C-terminal serine phosphorylation sites (C1-AAA, serines 155, 162, and 170 mutated to alanines). After gel electrophoresis, phosphate incorporation into substrate protein was visualized by autoradiography. (C) HuH-7 cells were transiently transfected in six-well dishes with plasmids encoding either wild-type HBV core protein or its mutants lacking serine phosphorylation sites in the C-terminal part as indicated (1.5 μg/well each). After metabolic labeling with 50 μCi of 33 P i per ml for 3.5 h, cell lysates were processed as described for panel A. HBV core protein phosphorylation was visualized by autoradiography (upper panel) and quantified by a PhosphorImager, and the same filter was then probed with anti-HBV core protein antibody (lower panel).

    Techniques Used: In Vivo, In Vitro, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis, Autoradiography, Construct, Lysis

    Specific association of endogenous and ectopically expressed SRPK1 and SRPK2 with GST-HBV core protein. COS-7 cells were transiently transfected with either control vectors or plasmids encoding FLAG-tagged SRPK1 (1.5 μg/well) or VSV-tagged SRPK2 (1.0 μg/well). After 24 h, cells were lysed, and extracts were subjected to in vitro association with either GST or GST-HBV-C1 protein. Samples were then resolved in duplicate by SDS-PAGE, followed by immunoblotting with anti-SRPK1 and anti-FLAG antibodies (left panels) or anti-SRPK2 and anti-VSV antibodies (right panels).
    Figure Legend Snippet: Specific association of endogenous and ectopically expressed SRPK1 and SRPK2 with GST-HBV core protein. COS-7 cells were transiently transfected with either control vectors or plasmids encoding FLAG-tagged SRPK1 (1.5 μg/well) or VSV-tagged SRPK2 (1.0 μg/well). After 24 h, cells were lysed, and extracts were subjected to in vitro association with either GST or GST-HBV-C1 protein. Samples were then resolved in duplicate by SDS-PAGE, followed by immunoblotting with anti-SRPK1 and anti-FLAG antibodies (left panels) or anti-SRPK2 and anti-VSV antibodies (right panels).

    Techniques Used: Transfection, In Vitro, SDS Page

    32) Product Images from "Transcription of the ehx Enterohemolysin Gene Is Positively Regulated by GrlA, a Global Regulator Encoded within the Locus of Enterocyte Effacement in Enterohemorrhagic Escherichia coli "

    Article Title: Transcription of the ehx Enterohemolysin Gene Is Positively Regulated by GrlA, a Global Regulator Encoded within the Locus of Enterocyte Effacement in Enterohemorrhagic Escherichia coli

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00231-08

    Expression of EhxA-FLAG and EhxC-FLAG. The amounts of EhxA-FLAG (A) and EhxC-FLAG, EspB, and FliC (B) in whole-cell lysates were examined by Western blotting using monoclonal anti-FLAG and polyclonal anti-EspB and anti-FliC antibodies, respectively. Wild-type
    Figure Legend Snippet: Expression of EhxA-FLAG and EhxC-FLAG. The amounts of EhxA-FLAG (A) and EhxC-FLAG, EspB, and FliC (B) in whole-cell lysates were examined by Western blotting using monoclonal anti-FLAG and polyclonal anti-EspB and anti-FliC antibodies, respectively. Wild-type

    Techniques Used: Expressing, Western Blot

    33) Product Images from "DUAL AND OPPOSITE EFFECTS OF hRAD51 CHEMICAL MODULATION ON HIV-1 INTEGRATION"

    Article Title: DUAL AND OPPOSITE EFFECTS OF hRAD51 CHEMICAL MODULATION ON HIV-1 INTEGRATION

    Journal: Chemistry & biology

    doi: 10.1016/j.chembiol.2015.04.020

    Effect of hRAD51 overexpression on endogenous DNA repair and early steps of HIV-1 replication and viral DNA productions The expression of hRAD51-FLAG or BAP-FLAG in 293T cells was checked 48 hours post-transfection by western blotting using anti-FLAG antibodies ( A, lane 1: protein extract from cells expressing hRAD51-FLAG, lane 2: protein extract from cells expressing BAP-FLAG). The global hRAD51 level was determined in cells transfected with the hRAD51-FLAG (hRAD51) and BAP-FLAG (BAP) expression vectors in parallel to untransfected control cells (w/o transfection), by western blotting using anti-hRAD51 antibodies. The amounts of protein loaded were normalized to the endogenous actin protein revealed by western blotting using an anti-actin antibody ( B ). The cellular distribution of the overexpressed proteins was determined by immunolocalization using an anti-FLAG antibody ( C ). The hRAD51 activity was determined under each condition by a cisplatin resistance assay as described in Materials and Method ( D ). The cells were transduced 48 hours post-transfection with the hRAD51 or BAP expression plasmids. HIV-1 replication was evaluated from fluorescence measurement 10 days after transduction by flow cytometry. The percentage of untransfected eGFP-positive cells was normalized to 100 % ( E ). The amount of total, integrated and 2-LTR circles viral DNAs was measured by quantitative PCR as described in Materials and Methods. The proportion of the different viral DNA species produced in untransfected control cells was normalized to 100 % ( F ). Results are represented as the mean values calculated from three independent experiments. The p-values are shown as *p
    Figure Legend Snippet: Effect of hRAD51 overexpression on endogenous DNA repair and early steps of HIV-1 replication and viral DNA productions The expression of hRAD51-FLAG or BAP-FLAG in 293T cells was checked 48 hours post-transfection by western blotting using anti-FLAG antibodies ( A, lane 1: protein extract from cells expressing hRAD51-FLAG, lane 2: protein extract from cells expressing BAP-FLAG). The global hRAD51 level was determined in cells transfected with the hRAD51-FLAG (hRAD51) and BAP-FLAG (BAP) expression vectors in parallel to untransfected control cells (w/o transfection), by western blotting using anti-hRAD51 antibodies. The amounts of protein loaded were normalized to the endogenous actin protein revealed by western blotting using an anti-actin antibody ( B ). The cellular distribution of the overexpressed proteins was determined by immunolocalization using an anti-FLAG antibody ( C ). The hRAD51 activity was determined under each condition by a cisplatin resistance assay as described in Materials and Method ( D ). The cells were transduced 48 hours post-transfection with the hRAD51 or BAP expression plasmids. HIV-1 replication was evaluated from fluorescence measurement 10 days after transduction by flow cytometry. The percentage of untransfected eGFP-positive cells was normalized to 100 % ( E ). The amount of total, integrated and 2-LTR circles viral DNAs was measured by quantitative PCR as described in Materials and Methods. The proportion of the different viral DNA species produced in untransfected control cells was normalized to 100 % ( F ). Results are represented as the mean values calculated from three independent experiments. The p-values are shown as *p

    Techniques Used: Over Expression, Expressing, Transfection, Western Blot, Activity Assay, Fluorescence, Transduction, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Produced

    34) Product Images from "RFXB and its splice variant RFXBSV mediate the antagonism between IFN? and TGF? on COL1A2 transcription in vascular smooth muscle cells"

    Article Title: RFXB and its splice variant RFXBSV mediate the antagonism between IFN? and TGF? on COL1A2 transcription in vascular smooth muscle cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp398

    RFXB and RFXBSV compete for the binding of HDAC2, thus limiting its recruitment to the collagen transcription start site. ( A ) 293FT cells were transfected with V5-tagged RFXB or V5-tagged RFXBSV along with either FLAG-tagged RFX5 or FLAG-tagged HDAC2. Immunoprecipitations were performed as described under ‘Materials and Methods’. Ten percent of the starting material was also loaded as input. ( B ) 293FT cells were transfected with FLAG-tagged RFXB, FLAG-tagged RFXBSV or an empty vector (top panel). Alternatively, HASMCs were infected with pLenti/V5-RFXB, pLenti/V5-RFXBSV or an empty vector (bottom panel). DNA affinity pull-down assays were performed as described. Binding of RFX5 and HDAC2 to the collagen probe in the presence of RFXB and RFXBSV was normalized to the control group and expressed as relative binding affinity. Shown in the graph are data collected from three independent experiments and expressed as average ± SD. ( C, D ) 293FT cells were transfected with FLAG-tagged RFXB and/or FLAG-tagged RFXBSV (C). Alternatively, HASMCs were infected with pLenti/V5-RFXB and/or pLenti/V5-RFXBSV (D). Chromatin immunoprecipitation assays were performed with anti-RFX5, anti-HDAC2, anti-FLAG, anti-V5 or pre-immune IgG as indicated. Precipitated DNA was amplified by real-time PCR with primers spanning the COL1A2 transcription start site (* P
    Figure Legend Snippet: RFXB and RFXBSV compete for the binding of HDAC2, thus limiting its recruitment to the collagen transcription start site. ( A ) 293FT cells were transfected with V5-tagged RFXB or V5-tagged RFXBSV along with either FLAG-tagged RFX5 or FLAG-tagged HDAC2. Immunoprecipitations were performed as described under ‘Materials and Methods’. Ten percent of the starting material was also loaded as input. ( B ) 293FT cells were transfected with FLAG-tagged RFXB, FLAG-tagged RFXBSV or an empty vector (top panel). Alternatively, HASMCs were infected with pLenti/V5-RFXB, pLenti/V5-RFXBSV or an empty vector (bottom panel). DNA affinity pull-down assays were performed as described. Binding of RFX5 and HDAC2 to the collagen probe in the presence of RFXB and RFXBSV was normalized to the control group and expressed as relative binding affinity. Shown in the graph are data collected from three independent experiments and expressed as average ± SD. ( C, D ) 293FT cells were transfected with FLAG-tagged RFXB and/or FLAG-tagged RFXBSV (C). Alternatively, HASMCs were infected with pLenti/V5-RFXB and/or pLenti/V5-RFXBSV (D). Chromatin immunoprecipitation assays were performed with anti-RFX5, anti-HDAC2, anti-FLAG, anti-V5 or pre-immune IgG as indicated. Precipitated DNA was amplified by real-time PCR with primers spanning the COL1A2 transcription start site (* P

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Infection, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    RFXB silencing favors the TGF-β antagonism of IFN-γ induced collagen repression. ( A ) HEK293 cells were transfected with FLAG-tagged RFXB or RFXBSV plasmids in the presence of shB, shB/SV or an empty vector (EV). Silencing efficiency was measured by Western blot. ( B, C, D ) HASMCs were infected with viral stocks carrying shB, shB/SV or an EV followed by treatment with IFN-γ and/or TGF-β as indicated. Message (B) and protein (C) levels of collagen type I were assessed by real-time PCR and Western blot, respectively as described in Figure 1 . ChIP assays (D) with anti-AcH3, -HDAC2 and –RFX5 antibodies were performed as described in Figure 5 . ( E ) A collagen type I promoter construct (pH 20) was co-transfected with expression constructs for HDAC2 into HASMCs along with shB, shB/SV or an EV as indicated, followed by treatment with IFN-γ and/or TGF-β. Luciferase activities were normalized by both protein concentration and GFP fluorescence, and presented as average ± SD (* P
    Figure Legend Snippet: RFXB silencing favors the TGF-β antagonism of IFN-γ induced collagen repression. ( A ) HEK293 cells were transfected with FLAG-tagged RFXB or RFXBSV plasmids in the presence of shB, shB/SV or an empty vector (EV). Silencing efficiency was measured by Western blot. ( B, C, D ) HASMCs were infected with viral stocks carrying shB, shB/SV or an EV followed by treatment with IFN-γ and/or TGF-β as indicated. Message (B) and protein (C) levels of collagen type I were assessed by real-time PCR and Western blot, respectively as described in Figure 1 . ChIP assays (D) with anti-AcH3, -HDAC2 and –RFX5 antibodies were performed as described in Figure 5 . ( E ) A collagen type I promoter construct (pH 20) was co-transfected with expression constructs for HDAC2 into HASMCs along with shB, shB/SV or an EV as indicated, followed by treatment with IFN-γ and/or TGF-β. Luciferase activities were normalized by both protein concentration and GFP fluorescence, and presented as average ± SD (* P

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Infection, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Construct, Expressing, Luciferase, Protein Concentration, Fluorescence

    35) Product Images from "Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar"

    Article Title: Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00040-15

    Western blot detection of archaellin FlaB2 and pilin EpdE-FLAG from in-frame deletion mutants. (A) Blots were developed with M. maripaludis anti-FlaB2. FlaB2 modified by known glycan lengths is as indicated for the wild type (Mm900), glycosyltransferase
    Figure Legend Snippet: Western blot detection of archaellin FlaB2 and pilin EpdE-FLAG from in-frame deletion mutants. (A) Blots were developed with M. maripaludis anti-FlaB2. FlaB2 modified by known glycan lengths is as indicated for the wild type (Mm900), glycosyltransferase

    Techniques Used: Western Blot, Modification

    36) Product Images from "Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi"

    Article Title: Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2007.05969.x

    Expression and cellular localization of the BBB26-FLAG and BBB27-FLAG proteins. A. Proteins lysates from E. coli (Ec) or B. burgdorferi B31-A34 (Bb) harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG were separated by SDS-PAGE and analysed by immunoblot with anti-FLAG antibodies. The mobilities of size standards (molecular weights in kDa) are indicated to the left of the figure. B. Protein lysates from B. burgdorferi clone A34 harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG were harvested and separated into soluble and membrane fractions by ultracentrifugation. Protein fractions from equivalent numbers of spirochetes were subjected to SDS-PAGE and analysed by immunoblot with FLAG (BBB26 and BBB27), OppAIV (inner membrane), OspB (outer membrane) and SodA (cytoplasmic) antisera. Representative results for the localization of the OppAIV, OspB and SodA proteins from B. burgdorferi clone A34 harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG are shown. L, total cell lysate; S, soluble protein fraction; M, membrane protein fraction. C. Equal numbers of whole or 0.1% SDS-treated (+0.1% SDS) cells of B. burgdorferi clone A34 harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG were incubated with different concentrations (μg ml −1 ) of proteinase K (PK). Lysates of PK-treated bacteria were separated by SDS-PAGE and analysed by immunoblot with FLAG (BBB26 and BBB27), flagellin (FlaB, periplasmic marker), OppAIV (inner membrane marker) and OspB (surface exposed, outer membrane marker) antisera. Representative results for the proteinase K sensitivity of the FlaB, OppAIV and OspB proteins from B. burgdorferi clone A34 harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG are shown.
    Figure Legend Snippet: Expression and cellular localization of the BBB26-FLAG and BBB27-FLAG proteins. A. Proteins lysates from E. coli (Ec) or B. burgdorferi B31-A34 (Bb) harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG were separated by SDS-PAGE and analysed by immunoblot with anti-FLAG antibodies. The mobilities of size standards (molecular weights in kDa) are indicated to the left of the figure. B. Protein lysates from B. burgdorferi clone A34 harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG were harvested and separated into soluble and membrane fractions by ultracentrifugation. Protein fractions from equivalent numbers of spirochetes were subjected to SDS-PAGE and analysed by immunoblot with FLAG (BBB26 and BBB27), OppAIV (inner membrane), OspB (outer membrane) and SodA (cytoplasmic) antisera. Representative results for the localization of the OppAIV, OspB and SodA proteins from B. burgdorferi clone A34 harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG are shown. L, total cell lysate; S, soluble protein fraction; M, membrane protein fraction. C. Equal numbers of whole or 0.1% SDS-treated (+0.1% SDS) cells of B. burgdorferi clone A34 harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG were incubated with different concentrations (μg ml −1 ) of proteinase K (PK). Lysates of PK-treated bacteria were separated by SDS-PAGE and analysed by immunoblot with FLAG (BBB26 and BBB27), flagellin (FlaB, periplasmic marker), OppAIV (inner membrane marker) and OspB (surface exposed, outer membrane marker) antisera. Representative results for the proteinase K sensitivity of the FlaB, OppAIV and OspB proteins from B. burgdorferi clone A34 harbouring either pBSV2ex bbb26 -FLAG or pBSV2ex bbb27 -FLAG are shown.

    Techniques Used: Expressing, SDS Page, Incubation, Marker

    37) Product Images from "Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation *Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation * S⃞"

    Article Title: Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation *Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M710608200

    Kinase domain of Plk1 is essential for the interaction with p73. A , schematic drawing of wild-type Plk1 and its deletion mutants. KD , kinase domain; PB , polo-box domain. B , Coomassie Brilliant Blue staining of GST-p73α-(1–130) used for this study. C, in vitro pulldown assay. Equal amount of GST-p73α-(1–130) was incubated with radiolabeled FLAG-Plk1 deletion mutants ( left panel ). After incubation, GST-p73α-(1–130) was precipitated by glutathione-Sepharose beads, and bound materials were separated by SDS-PAGE followed by autoradiography.
    Figure Legend Snippet: Kinase domain of Plk1 is essential for the interaction with p73. A , schematic drawing of wild-type Plk1 and its deletion mutants. KD , kinase domain; PB , polo-box domain. B , Coomassie Brilliant Blue staining of GST-p73α-(1–130) used for this study. C, in vitro pulldown assay. Equal amount of GST-p73α-(1–130) was incubated with radiolabeled FLAG-Plk1 deletion mutants ( left panel ). After incubation, GST-p73α-(1–130) was precipitated by glutathione-Sepharose beads, and bound materials were separated by SDS-PAGE followed by autoradiography.

    Techniques Used: Staining, In Vitro, Incubation, SDS Page, Autoradiography

    Kinase activity of Plk1 is required for the inhibition of p73. A , Plk1(K82M) retains an ability to interact with p73 in cells. COS7 cells were transiently co-transfected with the expression plasmids for HA-p73α and FLAG-Plk1(K82M). Forty-eight hours after transfection, whole cell lysates were prepared and subjected to IP with NMS or with anti-FLAG antibody. The immunoprecipitates were analyzed by IB with anti-p73 ( 1st panel ) or with anti-FLAG ( 2nd panel ) antibody. Input lysates were processed for IB with the indicated antibodies. Right panels show the results of the reciprocal experiments. B–D , luciferase reporter assay. H1299 cells were transiently co-transfected with the constant amount of HA-p73α expression plasmid (25 ng), 100 ng of luciferase reporter construct carrying p53/p73-responsive element derived from p21 WAF1 ( B ), Bax ( C ), or MDM2 ( D ) promoter and 10 ng of pRL-TK together with or without the increasing amounts of the expression plasmid for FLAG-Plk1(K82M) (50 and 100 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities determined. Firefly luminescence signal was normalized based on the Renilla luminescence signal. Results were shown as fold induction of the firefly luciferase activity compared with control cells transfected with the empty plasmid alone.
    Figure Legend Snippet: Kinase activity of Plk1 is required for the inhibition of p73. A , Plk1(K82M) retains an ability to interact with p73 in cells. COS7 cells were transiently co-transfected with the expression plasmids for HA-p73α and FLAG-Plk1(K82M). Forty-eight hours after transfection, whole cell lysates were prepared and subjected to IP with NMS or with anti-FLAG antibody. The immunoprecipitates were analyzed by IB with anti-p73 ( 1st panel ) or with anti-FLAG ( 2nd panel ) antibody. Input lysates were processed for IB with the indicated antibodies. Right panels show the results of the reciprocal experiments. B–D , luciferase reporter assay. H1299 cells were transiently co-transfected with the constant amount of HA-p73α expression plasmid (25 ng), 100 ng of luciferase reporter construct carrying p53/p73-responsive element derived from p21 WAF1 ( B ), Bax ( C ), or MDM2 ( D ) promoter and 10 ng of pRL-TK together with or without the increasing amounts of the expression plasmid for FLAG-Plk1(K82M) (50 and 100 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities determined. Firefly luminescence signal was normalized based on the Renilla luminescence signal. Results were shown as fold induction of the firefly luciferase activity compared with control cells transfected with the empty plasmid alone.

    Techniques Used: Activity Assay, Inhibition, Transfection, Expressing, Luciferase, Reporter Assay, Plasmid Preparation, Construct, Derivative Assay

    NH 2 -terminal small domain of p73 is required for the interaction with Plk1. A , domain structure of wild-type p73α and schematic representation of GST-tagged p73α deletion mutants. TA , transactivation domain; DB , DNA-binding domain; OD , oligomerization domain; SAM , sterileα-motif domain. Numbers indicate amino acid positions. B , GST and GST-p73α fusion proteins were purified by glutathione-Sepharose beads and separated by 10% SDS-PAGE followed by Coomassie Brilliant Blue staining. C, in vitro pulldown assay. Equal amount of radiolabeled FLAG-Plk1 was incubated with GST or with the indicated GST-p73α fusion proteins. After incubation, GST or GST-p73α fusion proteins were recovered by glutathione-Sepharose beads, and bound materials were resolved by 10% SDS-PAGE followed by autoradiography.
    Figure Legend Snippet: NH 2 -terminal small domain of p73 is required for the interaction with Plk1. A , domain structure of wild-type p73α and schematic representation of GST-tagged p73α deletion mutants. TA , transactivation domain; DB , DNA-binding domain; OD , oligomerization domain; SAM , sterileα-motif domain. Numbers indicate amino acid positions. B , GST and GST-p73α fusion proteins were purified by glutathione-Sepharose beads and separated by 10% SDS-PAGE followed by Coomassie Brilliant Blue staining. C, in vitro pulldown assay. Equal amount of radiolabeled FLAG-Plk1 was incubated with GST or with the indicated GST-p73α fusion proteins. After incubation, GST or GST-p73α fusion proteins were recovered by glutathione-Sepharose beads, and bound materials were resolved by 10% SDS-PAGE followed by autoradiography.

    Techniques Used: Binding Assay, Purification, SDS Page, Staining, In Vitro, Incubation, Autoradiography

    Plk1 inhibits the pro-apoptotic activity of p73. A , apoptotic assay. H1299 cells were seeded at a density of 2 × 10 5 cells/6-well tissue culture plate and allowed to attach overnight. Next day, cells were co-transfected with the constant amount of GFP (100 ng) and HA-p73α (900 ng) expression plasmids together with or without the increasing amounts of FLAG-Plk1 expression plasmid (500 and 1000 ng). Total amount of plasmid DNA was kept constant (2 μg) with pcDNA3. Forty-eight hours after transfection, cells were fixe, and cell nuclei were stained with DAPI. The percentages of GFP-positive cells with apoptotic nuclei were plotted. B , FACS analysis. H1299 cells were co-transfected with the constant amount of the expression plasmid encoding HA-p73α (250 ng) together with or without the increasing amounts of FLAG-Plk1 expression plasmid (100 or 200 ng). Forty-eight hours after transfection, attached and floating cells were collected, stained with PI, and their cell cycle distributions analyzed by flow cytometry.
    Figure Legend Snippet: Plk1 inhibits the pro-apoptotic activity of p73. A , apoptotic assay. H1299 cells were seeded at a density of 2 × 10 5 cells/6-well tissue culture plate and allowed to attach overnight. Next day, cells were co-transfected with the constant amount of GFP (100 ng) and HA-p73α (900 ng) expression plasmids together with or without the increasing amounts of FLAG-Plk1 expression plasmid (500 and 1000 ng). Total amount of plasmid DNA was kept constant (2 μg) with pcDNA3. Forty-eight hours after transfection, cells were fixe, and cell nuclei were stained with DAPI. The percentages of GFP-positive cells with apoptotic nuclei were plotted. B , FACS analysis. H1299 cells were co-transfected with the constant amount of the expression plasmid encoding HA-p73α (250 ng) together with or without the increasing amounts of FLAG-Plk1 expression plasmid (100 or 200 ng). Forty-eight hours after transfection, attached and floating cells were collected, stained with PI, and their cell cycle distributions analyzed by flow cytometry.

    Techniques Used: Activity Assay, Transfection, Expressing, Plasmid Preparation, Staining, FACS, Flow Cytometry, Cytometry

    Plk1 represses the p73-mediated transcriptional activation. A–C , H1299 cells (5 × 10 4 cells) were co-transfected with the constant amount of HA-p73α expression plasmid (25 ng), 100 ng of p53/p73-responsive luciferase reporter construct bearing p21 WAF1 ( A ), BAX ( B ), or MDM2 ( C ) promoter and 10 ng of Renilla luciferase reporter plasmid (pRL-TK) in the presence or absence of the increasing amounts of FLAG-Plk1 expression plasmid (50, 100, and 200 ng). To standardize the amounts of plasmid DNA per transfection, pcDNA3 was added to yield a total of 510 ng of plasmid. Forty-eight hours after transfection, cells were lysed, and their luciferase activities were measured. Data were normalized and presented as mean values ± S.D. of three independent experiments. D , RT-PCR analysis. H1299 cells were co-transfected with the constant amount of HA-p73α together with or without the increasing amounts of FLAG-Plk1 expression plasmid. Forty-eight hours after transfection, total RNA was prepared and analyzed for the expression levels of p21 WAF1 , BAX, and MDM2 by RT-PCR. Amplification of GAPDH serves as an internal control.
    Figure Legend Snippet: Plk1 represses the p73-mediated transcriptional activation. A–C , H1299 cells (5 × 10 4 cells) were co-transfected with the constant amount of HA-p73α expression plasmid (25 ng), 100 ng of p53/p73-responsive luciferase reporter construct bearing p21 WAF1 ( A ), BAX ( B ), or MDM2 ( C ) promoter and 10 ng of Renilla luciferase reporter plasmid (pRL-TK) in the presence or absence of the increasing amounts of FLAG-Plk1 expression plasmid (50, 100, and 200 ng). To standardize the amounts of plasmid DNA per transfection, pcDNA3 was added to yield a total of 510 ng of plasmid. Forty-eight hours after transfection, cells were lysed, and their luciferase activities were measured. Data were normalized and presented as mean values ± S.D. of three independent experiments. D , RT-PCR analysis. H1299 cells were co-transfected with the constant amount of HA-p73α together with or without the increasing amounts of FLAG-Plk1 expression plasmid. Forty-eight hours after transfection, total RNA was prepared and analyzed for the expression levels of p21 WAF1 , BAX, and MDM2 by RT-PCR. Amplification of GAPDH serves as an internal control.

    Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Construct, Reverse Transcription Polymerase Chain Reaction, Amplification

    38) Product Images from "Oncogenic LMO3 Collaborates with HEN2 to Enhance Neuroblastoma Cell Growth through Transactivation of Mash1"

    Article Title: Oncogenic LMO3 Collaborates with HEN2 to Enhance Neuroblastoma Cell Growth through Transactivation of Mash1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019297

    LMO3/HEN2-mediated transcriptional induction of Mash1 . (A) RT-PCR. SH-SY5Y cells were infected with empty adenovirus or with the indicated combinations of recombinant adenovirus encoding HA-LMO3 or FLAG-HEN2. At the indicated time points after infection, total RNA was analyzed for expression levels of LMO3 , HEN2 and Mash1 by RT-PCR. GAPDH was used as an internal control. (B) Schematic drawing of human Mash1 promoter. Nucleotide positions were indicated relative to transcriptional initiation site (+1). The putative HES1-binding sites and E-box were depicted by filled and open boxes, respectively. This genomic fragment was subcloned into appropriate restriction sites of pGL3-Basic Vector to give pluc-hMash1. (C) siRNA-mediated knockdown of LMO3 reduces the promoter activity of Mash1 . SH-SY5Y cells were co-transfected with constant amount of pluc-Mash1 (100 ng) and pRL-CMV (0.2 ng) in the presence or absence of increasing amounts of expression plasmid for siRNA against human LMO3 (100 or 400 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were measured. (D) LMO3 transactivates Mash1 promoter. Mouse neuroblastoma Neuro2a cells (1×10 5 cells/24-well plate) were co-transfected with constant amount of pluc-hMash1 (100 ng) and pRL-CMV (0.2 ng) together with or without expression plasmid for HA-LMO3 (150 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were measured. (E) HEN2 inhibits Mash1 promoter activity. Luciferase activities were measured in Neuro2a cells with or without FLAG-HEN2 (100 ng). (F) LMO3 interferes with negative effect of HEN2 on Mash1 transcription in Neuro2a cells. Luciferase activities were measured in Neuro2a cells transfected with HA-LMO3 (150 ng), FLAG-HEN2 (100 ng) or both of them.
    Figure Legend Snippet: LMO3/HEN2-mediated transcriptional induction of Mash1 . (A) RT-PCR. SH-SY5Y cells were infected with empty adenovirus or with the indicated combinations of recombinant adenovirus encoding HA-LMO3 or FLAG-HEN2. At the indicated time points after infection, total RNA was analyzed for expression levels of LMO3 , HEN2 and Mash1 by RT-PCR. GAPDH was used as an internal control. (B) Schematic drawing of human Mash1 promoter. Nucleotide positions were indicated relative to transcriptional initiation site (+1). The putative HES1-binding sites and E-box were depicted by filled and open boxes, respectively. This genomic fragment was subcloned into appropriate restriction sites of pGL3-Basic Vector to give pluc-hMash1. (C) siRNA-mediated knockdown of LMO3 reduces the promoter activity of Mash1 . SH-SY5Y cells were co-transfected with constant amount of pluc-Mash1 (100 ng) and pRL-CMV (0.2 ng) in the presence or absence of increasing amounts of expression plasmid for siRNA against human LMO3 (100 or 400 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were measured. (D) LMO3 transactivates Mash1 promoter. Mouse neuroblastoma Neuro2a cells (1×10 5 cells/24-well plate) were co-transfected with constant amount of pluc-hMash1 (100 ng) and pRL-CMV (0.2 ng) together with or without expression plasmid for HA-LMO3 (150 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were measured. (E) HEN2 inhibits Mash1 promoter activity. Luciferase activities were measured in Neuro2a cells with or without FLAG-HEN2 (100 ng). (F) LMO3 interferes with negative effect of HEN2 on Mash1 transcription in Neuro2a cells. Luciferase activities were measured in Neuro2a cells transfected with HA-LMO3 (150 ng), FLAG-HEN2 (100 ng) or both of them.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Recombinant, Expressing, Binding Assay, Plasmid Preparation, Activity Assay, Transfection, Luciferase

    Interaction between HEN2 and HES1 in cells. (A) Neuro2a cells were co-transfected with the indicated combinations of expression plasmids. Forty-eight hours after transfection, cells were lysed and immunoprecipitated with anti-FLAG (left panel) or with anti-Myc tag antibody (right panel) and the immunoprecipitates were analyzed by immunoblotting with anti-HES1 or with anti-FLAG antibody, respectively. Aliquots of cell lysates were subjected to immunoblotting with anti-HES1, anti-FLAG or with anti-Myc tag antibody. (B) In vitro pull-down assay. Radio-labeled FLAG-HEN2 was incubated with cell lysates prepared from Neuro2a cells transfected with Myc-HES1 expression plasmid. The reaction mixture was immunoprecipitated with normal rabbit serum (NRS) or with polyclonal anti-Myc tag antibody and separated by SDS-PAGE followed by autoradiography. 1/5 inputs were also shown.
    Figure Legend Snippet: Interaction between HEN2 and HES1 in cells. (A) Neuro2a cells were co-transfected with the indicated combinations of expression plasmids. Forty-eight hours after transfection, cells were lysed and immunoprecipitated with anti-FLAG (left panel) or with anti-Myc tag antibody (right panel) and the immunoprecipitates were analyzed by immunoblotting with anti-HES1 or with anti-FLAG antibody, respectively. Aliquots of cell lysates were subjected to immunoblotting with anti-HES1, anti-FLAG or with anti-Myc tag antibody. (B) In vitro pull-down assay. Radio-labeled FLAG-HEN2 was incubated with cell lysates prepared from Neuro2a cells transfected with Myc-HES1 expression plasmid. The reaction mixture was immunoprecipitated with normal rabbit serum (NRS) or with polyclonal anti-Myc tag antibody and separated by SDS-PAGE followed by autoradiography. 1/5 inputs were also shown.

    Techniques Used: Transfection, Expressing, Immunoprecipitation, In Vitro, Pull Down Assay, Labeling, Incubation, Plasmid Preparation, SDS Page, Autoradiography

    LMO3/HEN2 attenuates HES1-dependent down-regulation of Mash1 . (A) Luciferase reporter assay. Neuro2a cells were co-transfected with constant amount of pluc-hMash1 (100 ng), pRL-CMV (0.2 ng) and expression plasmid for HES1 (50 ng) or HEN2 (50 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were examined. (B) Luciferase reporter assay. Neuro2a cells were co-transfected with constant amount of pluc-hMash1 (100 ng), pRL-CMV (0.2 ng) and expression plasmid for HES1 (5 ng) in the presence or absence of expression plasmid for HA-LMO3 (150 ng) together with or without increasing amounts of FLAG-HEN2 expression plasmid (100, 200 or 300 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were examined. (C) Schematic representation of mouse Mash1 promoter. The canonical HES1-binding sites and E-box were indicated by filled and open boxes, respectively. The positions of primer sets used for chromatin immunoprecipitation (ChIP) assays were also indicated. (D) ChIP assay. Cross-linked chromatin prepared from Neuro2a cells transfected with the indicated combinations of expression plasmids was sonicated and immunoprecipitated with normal rabbit serum (NRS), polyclonal anti-Myc tag or with polyclonal anti-FLAG antibody. The genomic DNA was purified from the immunoprecipitates and amplified by PCR.
    Figure Legend Snippet: LMO3/HEN2 attenuates HES1-dependent down-regulation of Mash1 . (A) Luciferase reporter assay. Neuro2a cells were co-transfected with constant amount of pluc-hMash1 (100 ng), pRL-CMV (0.2 ng) and expression plasmid for HES1 (50 ng) or HEN2 (50 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were examined. (B) Luciferase reporter assay. Neuro2a cells were co-transfected with constant amount of pluc-hMash1 (100 ng), pRL-CMV (0.2 ng) and expression plasmid for HES1 (5 ng) in the presence or absence of expression plasmid for HA-LMO3 (150 ng) together with or without increasing amounts of FLAG-HEN2 expression plasmid (100, 200 or 300 ng). Forty-eight hours after transfection, cells were lysed and their luciferase activities were examined. (C) Schematic representation of mouse Mash1 promoter. The canonical HES1-binding sites and E-box were indicated by filled and open boxes, respectively. The positions of primer sets used for chromatin immunoprecipitation (ChIP) assays were also indicated. (D) ChIP assay. Cross-linked chromatin prepared from Neuro2a cells transfected with the indicated combinations of expression plasmids was sonicated and immunoprecipitated with normal rabbit serum (NRS), polyclonal anti-Myc tag or with polyclonal anti-FLAG antibody. The genomic DNA was purified from the immunoprecipitates and amplified by PCR.

    Techniques Used: Luciferase, Reporter Assay, Transfection, Expressing, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Purification, Amplification, Polymerase Chain Reaction

    LMO3 attenuates binding of HES1 to Mash1 promoter and promotes that of HEN2. (A) Complex formation between LMO3 and HES1 in cells. Neuro2a cells were transiently transfected with the indicated combinations of the expression plasmids. Forty-eight hours after transfection, cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-Myc tag antibody (top panel). Expressions of FLAG-LMO3 and Myc-HES1 are also shown (lower panels). (B) ChIP assay. Cross-linked chromatin prepared from Neuro2a cells transfected with the indicated combinations of expression plasmids was sonicated and immunoprecipitated with normal rabbit serum (NRS), polyclonal anti-Myc tag or with polyclonal anti-FLAG antibody. The genomic DNA was purified from the immunoprecipitates and amplified by PCR.
    Figure Legend Snippet: LMO3 attenuates binding of HES1 to Mash1 promoter and promotes that of HEN2. (A) Complex formation between LMO3 and HES1 in cells. Neuro2a cells were transiently transfected with the indicated combinations of the expression plasmids. Forty-eight hours after transfection, cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-Myc tag antibody (top panel). Expressions of FLAG-LMO3 and Myc-HES1 are also shown (lower panels). (B) ChIP assay. Cross-linked chromatin prepared from Neuro2a cells transfected with the indicated combinations of expression plasmids was sonicated and immunoprecipitated with normal rabbit serum (NRS), polyclonal anti-Myc tag or with polyclonal anti-FLAG antibody. The genomic DNA was purified from the immunoprecipitates and amplified by PCR.

    Techniques Used: Binding Assay, Transfection, Expressing, Immunoprecipitation, Chromatin Immunoprecipitation, Sonication, Purification, Amplification, Polymerase Chain Reaction

    39) Product Images from "Recurrent viral infections associated with a homozygous CORO1A mutation that disrupts oligomerization and cytoskeletal association"

    Article Title: Recurrent viral infections associated with a homozygous CORO1A mutation that disrupts oligomerization and cytoskeletal association

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2015.08.020

    Oligomerization and cytoskeletal distribution of the CORO1A S401fs mutant ( A ) Co-immunoprecipitation of WT and Mut CORO1A in lysates from HEK293T cells co-transfected with combinations of Myc- and FLAG-tagged WT and Mut-CORO1A and FLAG-tagged PYK2 as a negative control. Myc- and FLAG-IPs and total cell lysates were immunoblotted with anti-Myc antibody. ( B ) Immunoblot analysis of CORO1A in cytosolic and cytoskeletal fractions from EBV-B cells of normal control (NL) or patient (P1). GAPDH and PARP were immunoblotted as markers for cytosolic and cytoskeleton-associated proteins, respectively. ( C ) Quantification of CORO1A distribution in the cytosolic and cytoskeletal fractions. Columns and bars represent mean and standard error of two independent experiments, *p
    Figure Legend Snippet: Oligomerization and cytoskeletal distribution of the CORO1A S401fs mutant ( A ) Co-immunoprecipitation of WT and Mut CORO1A in lysates from HEK293T cells co-transfected with combinations of Myc- and FLAG-tagged WT and Mut-CORO1A and FLAG-tagged PYK2 as a negative control. Myc- and FLAG-IPs and total cell lysates were immunoblotted with anti-Myc antibody. ( B ) Immunoblot analysis of CORO1A in cytosolic and cytoskeletal fractions from EBV-B cells of normal control (NL) or patient (P1). GAPDH and PARP were immunoblotted as markers for cytosolic and cytoskeleton-associated proteins, respectively. ( C ) Quantification of CORO1A distribution in the cytosolic and cytoskeletal fractions. Columns and bars represent mean and standard error of two independent experiments, *p

    Techniques Used: Mutagenesis, Immunoprecipitation, Transfection, Negative Control

    40) Product Images from "Lithobates catesbeianus (American Bullfrog) oocytes: a novel heterologous expression system for aquaporins"

    Article Title: Lithobates catesbeianus (American Bullfrog) oocytes: a novel heterologous expression system for aquaporins

    Journal: Biology Open

    doi: 10.1242/bio.031880

    Osmotic water permeability ( P f ) values of oocytes expressing hAQP1, hAQP1 C189S , hAQP1 FLAG , rAQP3, hAQP7, hAQP8 and rAQP9 versus their day-matched H 2 O-injected control oocytes, before and after the treatment with pCMBS (1 mM for 30 min). The P f values of AQP-expressing oocytes (black bars) are significantly greater than those of the P f of day-matched H 2 O-injected controls (gray bars) ( P
    Figure Legend Snippet: Osmotic water permeability ( P f ) values of oocytes expressing hAQP1, hAQP1 C189S , hAQP1 FLAG , rAQP3, hAQP7, hAQP8 and rAQP9 versus their day-matched H 2 O-injected control oocytes, before and after the treatment with pCMBS (1 mM for 30 min). The P f values of AQP-expressing oocytes (black bars) are significantly greater than those of the P f of day-matched H 2 O-injected controls (gray bars) ( P

    Techniques Used: Permeability, Expressing, Injection

    Surface expression of hAQP1, hAQP1 FLAG , hAQP1 C189S , rAQP3, hAQP7, hAQP8 and rAQP9 versus H 2 O-injected control oocytes. The surface expression of hAQP1, hAQP1 C189S mutant, rAQP3, hAQP7, hAQP8 and hAQP9 monomers (∼28 kDa) is shown by immunoreactive bands detected at a molecular weight (MW) between 25 and 37 kDa, using polyclonal antibodies (anti-AQP1, anti-AQP3, anti-AQP7, anti-AQP8 and anti-AQP9, respectively). The surface expression of hAQP1 FLAG monomer is shown by a band at a MW between 25 and 37 kDa, using a monoclonal antibody anti-FLAG. All the western blots show the absence of this immunoreactive bands in the H 2 O-injected control oocytes. Oocytes from 8–18 different frogs (i.e. batches of oocytes) were analyzed for surface expression, depending on the AQP construct used.
    Figure Legend Snippet: Surface expression of hAQP1, hAQP1 FLAG , hAQP1 C189S , rAQP3, hAQP7, hAQP8 and rAQP9 versus H 2 O-injected control oocytes. The surface expression of hAQP1, hAQP1 C189S mutant, rAQP3, hAQP7, hAQP8 and hAQP9 monomers (∼28 kDa) is shown by immunoreactive bands detected at a molecular weight (MW) between 25 and 37 kDa, using polyclonal antibodies (anti-AQP1, anti-AQP3, anti-AQP7, anti-AQP8 and anti-AQP9, respectively). The surface expression of hAQP1 FLAG monomer is shown by a band at a MW between 25 and 37 kDa, using a monoclonal antibody anti-FLAG. All the western blots show the absence of this immunoreactive bands in the H 2 O-injected control oocytes. Oocytes from 8–18 different frogs (i.e. batches of oocytes) were analyzed for surface expression, depending on the AQP construct used.

    Techniques Used: Expressing, Injection, Mutagenesis, Molecular Weight, Western Blot, Construct

    Channel-dependent osmotic water permeability ( P f *) of oocytes expressing hAQP1, hAQP1 FLAG hAQP1 C189S , rAQP3, hAQP7, hAQP8 and rAQP9 before and after treatment with pCMBS (1 mM for 30 min). Subtracting the P f value for day-matched H 2 O-injected control oocytes from the P f of each AQP-expressing oocyte, before and after pCMBS treatment yields the channel-dependent osmotic water permeability ( P f *). Treatment with pCMBS reduces the P f * of hAQP1 ( P =0.01, t -test), hAQP1 FLAG ( P =0.03, t -test), hAQP8 ( P =0.03, t -test) and rAQP9 ( P =0.02, t -test), but has no effect on P f * of hAQP C189S mutant ( P =0.85, t -test), AQP3 ( P =0.10, t -test), and AQP7 ( P =0.54, t -test). A one-way ANOVA, followed by an SNK post hoc analysis to compare P f * before and after treatment with pCMBS was also performed. Overall P values: P =0.27 before pCBMS treatment and P =0.0001 after pCMBS treatment).
    Figure Legend Snippet: Channel-dependent osmotic water permeability ( P f *) of oocytes expressing hAQP1, hAQP1 FLAG hAQP1 C189S , rAQP3, hAQP7, hAQP8 and rAQP9 before and after treatment with pCMBS (1 mM for 30 min). Subtracting the P f value for day-matched H 2 O-injected control oocytes from the P f of each AQP-expressing oocyte, before and after pCMBS treatment yields the channel-dependent osmotic water permeability ( P f *). Treatment with pCMBS reduces the P f * of hAQP1 ( P =0.01, t -test), hAQP1 FLAG ( P =0.03, t -test), hAQP8 ( P =0.03, t -test) and rAQP9 ( P =0.02, t -test), but has no effect on P f * of hAQP C189S mutant ( P =0.85, t -test), AQP3 ( P =0.10, t -test), and AQP7 ( P =0.54, t -test). A one-way ANOVA, followed by an SNK post hoc analysis to compare P f * before and after treatment with pCMBS was also performed. Overall P values: P =0.27 before pCBMS treatment and P =0.0001 after pCMBS treatment).

    Techniques Used: Permeability, Expressing, Injection, Mutagenesis

    Related Articles

    Transfection:

    Article Title: Mutation in BAG3 Causes Severe Dominant Childhood Muscular Dystrophy
    Article Snippet: .. The transfected cells were visualized with monoclonal anti-FLAG antibody (Sigma-Aldrich, St Louis, MO) and FITC conjugated second antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). .. The preparations were examined with a Zeiss Axiovert epifluorescence microscope using apotome optics, Axiovision 4.4 software, and a 63x objective (numerical aperture 1.4).

    Luciferase:

    Article Title: A Nucleotide Exchange Factor Promotes Endoplasmic Reticulum-to-Cytosol Membrane Penetration of the Nonenveloped Virus Simian Virus 40
    Article Snippet: .. Polyclonal Hsp90 and SV40 large T-antigen antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), polyclonal BiP and monoclonal PDI antibodies from Abcam (Cambridge, MA), a monoclonal BiP antibody from BD (Franklin, NJ), monoclonal Grp170 and polyclonal Sil1 antibodies from GeneTex (Los Angeles, CA), monoclonal FLAG antibody and luciferase from Sigma-Aldrich (St. Louis, MO), monoclonal green fluorescent protein (GFP) antibody from Protein Tech Group (Chicago, IL), and monoclonal BAP31 antibody from Thermo Fisher Scientific (Waltham, MA). ..

    Magnetic Beads:

    Article Title: Multiple Host Factors Interact with the Hypervariable Domain of Chikungunya Virus nsP3 and Determine Viral Replication in Cell-Specific Mode
    Article Snippet: .. Protein complexes were isolated from the postnuclear fraction of the NP-40-lysed cells using magnetic beads loaded with the Flag-specific monoclonal antibody (MAb) (Sigma) as described elsewhere ( ). .. Proteins were separated on 4 to 12% NuPAGE gels (Invitrogen), stained with Coomassie blue, and identified by mass spectrometry as previously described ( ).

    Isolation:

    Article Title: Multiple Host Factors Interact with the Hypervariable Domain of Chikungunya Virus nsP3 and Determine Viral Replication in Cell-Specific Mode
    Article Snippet: .. Protein complexes were isolated from the postnuclear fraction of the NP-40-lysed cells using magnetic beads loaded with the Flag-specific monoclonal antibody (MAb) (Sigma) as described elsewhere ( ). .. Proteins were separated on 4 to 12% NuPAGE gels (Invitrogen), stained with Coomassie blue, and identified by mass spectrometry as previously described ( ).

    Western Blot:

    Article Title: A Role for Huntington Disease Protein in Dendritic RNA Granules *
    Article Snippet: .. The following antibodies were used: α-Htt (Millipore 2166 and 5492 antibodies gave same results, immunofluorescence (IF)-dilution: 1:250, Western blot (WB)-dilution 1:1,000), rabbit polyclonal α-Staufen1 (Millipore AB5781, IF-dilution 1:250, WB-dilution 1:500), rabbit polyclonal α-Staufen (gift of Susana de Lucas (National Centre for Biotechnology, Madrid, Spain), 1:400), chicken polyclonal α-MAP2 (Covance PCK-554P, IF-dilution 1:10,000), α-FLAG (Sigma, M2 monoclonal, IF-dilution 1:100), rabbit polyclonal α-DDX6 (Bethyl A300-460A, IF-dilution 1:500), rabbit polyclonal α-Dcp1a (gift of J. Lykke-Andersen (University of Colorado, Boulder), IF-dilution 1:200), α-rpS6 (Cell Signaling 2217, IF-dilution 1:500), rabbit polyclonal α-GFP (gift of E. Ziff (NYU School of Medicine, New York, NY), IF-dilution 1:1,000), rabbit polyclonal α-eIF4E (Cell Signaling 9742, WB-dilution 1:1,000), anti-β-tubulin (MMS-410P, WB-dilution 1:1,000), α-Ago2 (Cell Signaling 2897, WB-dilution 1:1,000), α-Ago2 (gift of Ramin Shiekhattar (The Wistar Institute, PA), IF-dilution 1:1,000), α-HA (12CA5, WB-dilution 1:1,000), and GAPDH (Millipore MAB374, WB-dilution 1:300). .. Rat hippocampal neurons were cultured (DIV 14) as described above.

    Immunofluorescence:

    Article Title: A Role for Huntington Disease Protein in Dendritic RNA Granules *
    Article Snippet: .. The following antibodies were used: α-Htt (Millipore 2166 and 5492 antibodies gave same results, immunofluorescence (IF)-dilution: 1:250, Western blot (WB)-dilution 1:1,000), rabbit polyclonal α-Staufen1 (Millipore AB5781, IF-dilution 1:250, WB-dilution 1:500), rabbit polyclonal α-Staufen (gift of Susana de Lucas (National Centre for Biotechnology, Madrid, Spain), 1:400), chicken polyclonal α-MAP2 (Covance PCK-554P, IF-dilution 1:10,000), α-FLAG (Sigma, M2 monoclonal, IF-dilution 1:100), rabbit polyclonal α-DDX6 (Bethyl A300-460A, IF-dilution 1:500), rabbit polyclonal α-Dcp1a (gift of J. Lykke-Andersen (University of Colorado, Boulder), IF-dilution 1:200), α-rpS6 (Cell Signaling 2217, IF-dilution 1:500), rabbit polyclonal α-GFP (gift of E. Ziff (NYU School of Medicine, New York, NY), IF-dilution 1:1,000), rabbit polyclonal α-eIF4E (Cell Signaling 9742, WB-dilution 1:1,000), anti-β-tubulin (MMS-410P, WB-dilution 1:1,000), α-Ago2 (Cell Signaling 2897, WB-dilution 1:1,000), α-Ago2 (gift of Ramin Shiekhattar (The Wistar Institute, PA), IF-dilution 1:1,000), α-HA (12CA5, WB-dilution 1:1,000), and GAPDH (Millipore MAB374, WB-dilution 1:300). .. Rat hippocampal neurons were cultured (DIV 14) as described above.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore rabbit anti gfp
    Knockdown of KIF21A inhibits the axonal transport of <t>NCKX2.</t> A , B , shRNA-mediated depletion of KIF21A. A , KIF21A-targeting shRNA (shKIF21A) was expressed by pLentiLox3.7 plasmids (pLL) encoding EGFP or mRFP <t>(pLL-GFP-shKIF21A</t> or pLL-RFP-shKIF21A). FLAG-KIF21A was cotransfected with shKIF21A or empty pLL vectors into HEK293 cells. shKIF21A completely depleted FLAG-KIF21A, but the empty pLL did not. B , Endogenous KIF21A was remarkably depleted in cultured hippocampal neurons infected with lentivirus encoding shKIF21A but not in those with lentivirus encoding nontargeting shRNA (NT control). Time-dependent knockdown of endogenous KIF21A is shown in the right bar graph. The noninfected control is shown in the leftmost bar and the NT control is in the rightmost bar. In A and B , β-actin was detected as a loading control. C , D , The axonal transport of endogenous NCKX2 (green) in the shKIF21A-transfected ( D ) or untransfected control ( C ) neurons. KIF21A-depleted neuron was identified by red fluorescence of mRFP coexpressed with shRNA (insets on DIC images). Dendrites were identified by MAP2 immunofluorescence (red). Neurites that are MAP2 negative but clearly seen in the DIC images (indicated by arrowheads) were regarded as axons. E , Analysis of ADR of endogenous NCKX2. Dendritic ROIs (left; red dotted polygons) were drawn on the endogenous NCKX2 immunofluorescence image (same as in C ). After nullifying pixels that overlap the binary mask of MAP2-positive neurites (middle) from the endogenous NCKX2 image, the axonal ROI was set on the NCKX2 image (right; red dotted polygon). F , The mean ADR (black bars) and DSR (gray bars) of endogenous NCKX2 estimated from the untransfected control ( n = 8) or KIF21A-depleted neurons ( n = 6). ADR of NCKX2 in the KIF21A-depleted group is significantly lower than that in the control group. ** p
    Rabbit Anti Gfp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti gfp/product/Millipore
    Average 99 stars, based on 83 article reviews
    Price from $9.99 to $1999.99
    rabbit anti gfp - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    85
    Millipore mouse monoclonal anti hsp90α
    Subcellular distribution of hMSH5 fusion proteins. ( A ) The intracellular distribution of the hMSH5 protein fused to an N-terminal GFP-tag (GFP-C1-hMSH5), a C-terminal GFP-tag (GFP-N1-hMSH5) or an N-terminal FLAG-tag (FLAG-hMSH5) was examined in HeLa cells by laser confocal fluorescence (GFP-tagged proteins) or immunofluorescence (FLAG-hMSH5) microscopy. The images shown are representative of all the transfected cells. Upper panels show the distribution of the indicated fusion protein. Cells were counterstained with DAPI to visualize nuclei (lower panels). ( B ) HeLa cells expressing the indicated proteins were collected and fractionated into cytosolic (C) and nuclear extracts (N). Equal cell amounts from cytosolic and nuclear extracts were immunoblotted with anti-GFP or anti-FLAG antibodies, as indicated. The efficiency of fractionation was verified by staining for <t>HSP90α</t> as a cytoplasmic marker, and PARP-1 as a nuclear marker. ( C ) HeLa cells transiently expressing the indicated fusion proteins were incubated without (–LMB) or with leptomycin B (+LMB) for 6 h. The localization of hMSH5 fusion proteins was determined by fluorescence and immunofluorescence microscopy. Left panels show representative micrographs. The right panel presents a quantitative analysis of the localization data. For each cell, the fluorescence intensities in the entire cell area and in the nuclear area were evaluated, as detailed in ‘Material and Methods’ section and the nuclear fluorescence was calculated as a percentage of total (nuclear plus cytoplasmic) cell fluorescence (N/N+C). As many as 30–35 cells were analyzed per experiment, for each protein and each condition (±LMB). The graph represents the mean N/N+C values with standard deviations (error bars) across at least three independent experiments.
    Mouse Monoclonal Anti Hsp90α, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti hsp90α/product/Millipore
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti hsp90α - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti flag m2 antibody
    V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an <t>anti-c-Myc</t> antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with <t>anti-FLAG</t> antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2 antibody/product/Millipore
    Average 99 stars, based on 796 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag m2 antibody - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Knockdown of KIF21A inhibits the axonal transport of NCKX2. A , B , shRNA-mediated depletion of KIF21A. A , KIF21A-targeting shRNA (shKIF21A) was expressed by pLentiLox3.7 plasmids (pLL) encoding EGFP or mRFP (pLL-GFP-shKIF21A or pLL-RFP-shKIF21A). FLAG-KIF21A was cotransfected with shKIF21A or empty pLL vectors into HEK293 cells. shKIF21A completely depleted FLAG-KIF21A, but the empty pLL did not. B , Endogenous KIF21A was remarkably depleted in cultured hippocampal neurons infected with lentivirus encoding shKIF21A but not in those with lentivirus encoding nontargeting shRNA (NT control). Time-dependent knockdown of endogenous KIF21A is shown in the right bar graph. The noninfected control is shown in the leftmost bar and the NT control is in the rightmost bar. In A and B , β-actin was detected as a loading control. C , D , The axonal transport of endogenous NCKX2 (green) in the shKIF21A-transfected ( D ) or untransfected control ( C ) neurons. KIF21A-depleted neuron was identified by red fluorescence of mRFP coexpressed with shRNA (insets on DIC images). Dendrites were identified by MAP2 immunofluorescence (red). Neurites that are MAP2 negative but clearly seen in the DIC images (indicated by arrowheads) were regarded as axons. E , Analysis of ADR of endogenous NCKX2. Dendritic ROIs (left; red dotted polygons) were drawn on the endogenous NCKX2 immunofluorescence image (same as in C ). After nullifying pixels that overlap the binary mask of MAP2-positive neurites (middle) from the endogenous NCKX2 image, the axonal ROI was set on the NCKX2 image (right; red dotted polygon). F , The mean ADR (black bars) and DSR (gray bars) of endogenous NCKX2 estimated from the untransfected control ( n = 8) or KIF21A-depleted neurons ( n = 6). ADR of NCKX2 in the KIF21A-depleted group is significantly lower than that in the control group. ** p

    Journal: The Journal of Neuroscience

    Article Title: KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

    doi: 10.1523/JNEUROSCI.6331-11.2012

    Figure Lengend Snippet: Knockdown of KIF21A inhibits the axonal transport of NCKX2. A , B , shRNA-mediated depletion of KIF21A. A , KIF21A-targeting shRNA (shKIF21A) was expressed by pLentiLox3.7 plasmids (pLL) encoding EGFP or mRFP (pLL-GFP-shKIF21A or pLL-RFP-shKIF21A). FLAG-KIF21A was cotransfected with shKIF21A or empty pLL vectors into HEK293 cells. shKIF21A completely depleted FLAG-KIF21A, but the empty pLL did not. B , Endogenous KIF21A was remarkably depleted in cultured hippocampal neurons infected with lentivirus encoding shKIF21A but not in those with lentivirus encoding nontargeting shRNA (NT control). Time-dependent knockdown of endogenous KIF21A is shown in the right bar graph. The noninfected control is shown in the leftmost bar and the NT control is in the rightmost bar. In A and B , β-actin was detected as a loading control. C , D , The axonal transport of endogenous NCKX2 (green) in the shKIF21A-transfected ( D ) or untransfected control ( C ) neurons. KIF21A-depleted neuron was identified by red fluorescence of mRFP coexpressed with shRNA (insets on DIC images). Dendrites were identified by MAP2 immunofluorescence (red). Neurites that are MAP2 negative but clearly seen in the DIC images (indicated by arrowheads) were regarded as axons. E , Analysis of ADR of endogenous NCKX2. Dendritic ROIs (left; red dotted polygons) were drawn on the endogenous NCKX2 immunofluorescence image (same as in C ). After nullifying pixels that overlap the binary mask of MAP2-positive neurites (middle) from the endogenous NCKX2 image, the axonal ROI was set on the NCKX2 image (right; red dotted polygon). F , The mean ADR (black bars) and DSR (gray bars) of endogenous NCKX2 estimated from the untransfected control ( n = 8) or KIF21A-depleted neurons ( n = 6). ADR of NCKX2 in the KIF21A-depleted group is significantly lower than that in the control group. ** p

    Article Snippet: For surface immunostaining of NCKX2, live cells were incubated with rabbit anti-NCKX2ext (1:100) or rabbit anti-GFP (1:100; Millipore Bioscience Research Reagents) in serum-free culture medium for 15 min at 36°C or at 4°C, rinsed with culture medium, fixed with ice-cold 4% PFA or 3.8% formaldehyde in PBS for 20 min, and washed with PBS.

    Techniques: shRNA, Cell Culture, Infection, Transfection, Fluorescence, Immunofluorescence

    Axonal transport of NCKX2 is inhibited by dnKIF21A. A , Test for functional competence of NCKX2-GFP. Aa , Overexpressed NCKX2-GFP was localized both in the cytoplasm and the plasma membrane in the HEK293 cell. A line profile of GFP fluorescence along the white line is shown in the inset of Aa . Scale bar, 10 μm. Ab , A whole-cell current at the holding potential of 0 mV was recorded from a HEK293 cell expressing NCKX2-GFP using high-Na + and high-BAPTA internal pipette solution. Reverse-mode NCKX current could be induced by bath application of Ca 2+ plus K + , but not by Ca 2+ plus Cs + or Ca 2+ only. B , Overexpression of NCKX2-GFP alone (left) or coexpression of NCKX2-GFP and dnKIF21A (right) in the hippocampal neurons. GFP fluorescence was inverted into gray color to improve contrast. Scale bar, 100 μm. The longest neurites were indicated by arrowheads. C , D , Coexpression of NCKX2-GFP together with KIF21A ( C ) or dnKIF21A ( D ) in hippocampal neurons. The longest neurite was traced from DsRed fluorescence using NeuronJ, and then fluorescence intensity of NCKX2-GFP was measured along the traced line (rightmost graphs). The traced line is overlaid as a red line on each DsRed image (gray). Scale bar, 100 μm. E , Mean NCKX2-GFP fluorescence profiles of the longest neurites. Ten points smoothing was performed with all data. In the range of 50–250 μm, intensity values for NCKX2-GFP from neurons expressing dnKIF21A (red; n = 11) were significantly lower than those from three different control groups: (1) neurons expressing NCKX2-GFP alone (green; n = 19) at all 43 points, (2) neurons expressing KIF21A (black; n = 10) at 39 points, and (3) neurons expressing dnKIF21B (blue; n = 20) at 36 points (mean ± SEM; p

    Journal: The Journal of Neuroscience

    Article Title: KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

    doi: 10.1523/JNEUROSCI.6331-11.2012

    Figure Lengend Snippet: Axonal transport of NCKX2 is inhibited by dnKIF21A. A , Test for functional competence of NCKX2-GFP. Aa , Overexpressed NCKX2-GFP was localized both in the cytoplasm and the plasma membrane in the HEK293 cell. A line profile of GFP fluorescence along the white line is shown in the inset of Aa . Scale bar, 10 μm. Ab , A whole-cell current at the holding potential of 0 mV was recorded from a HEK293 cell expressing NCKX2-GFP using high-Na + and high-BAPTA internal pipette solution. Reverse-mode NCKX current could be induced by bath application of Ca 2+ plus K + , but not by Ca 2+ plus Cs + or Ca 2+ only. B , Overexpression of NCKX2-GFP alone (left) or coexpression of NCKX2-GFP and dnKIF21A (right) in the hippocampal neurons. GFP fluorescence was inverted into gray color to improve contrast. Scale bar, 100 μm. The longest neurites were indicated by arrowheads. C , D , Coexpression of NCKX2-GFP together with KIF21A ( C ) or dnKIF21A ( D ) in hippocampal neurons. The longest neurite was traced from DsRed fluorescence using NeuronJ, and then fluorescence intensity of NCKX2-GFP was measured along the traced line (rightmost graphs). The traced line is overlaid as a red line on each DsRed image (gray). Scale bar, 100 μm. E , Mean NCKX2-GFP fluorescence profiles of the longest neurites. Ten points smoothing was performed with all data. In the range of 50–250 μm, intensity values for NCKX2-GFP from neurons expressing dnKIF21A (red; n = 11) were significantly lower than those from three different control groups: (1) neurons expressing NCKX2-GFP alone (green; n = 19) at all 43 points, (2) neurons expressing KIF21A (black; n = 10) at 39 points, and (3) neurons expressing dnKIF21B (blue; n = 20) at 36 points (mean ± SEM; p

    Article Snippet: For surface immunostaining of NCKX2, live cells were incubated with rabbit anti-NCKX2ext (1:100) or rabbit anti-GFP (1:100; Millipore Bioscience Research Reagents) in serum-free culture medium for 15 min at 36°C or at 4°C, rinsed with culture medium, fixed with ice-cold 4% PFA or 3.8% formaldehyde in PBS for 20 min, and washed with PBS.

    Techniques: Functional Assay, Fluorescence, Expressing, Transferring, Over Expression

    Colocalization of NCKX2 and KIF21A. A , Reliability test of NCKX2 ext antibody. Aa , The HEK293 cells expressing NCKX2-GFP (green) were specifically labeled with anti-NCKX2 ext antibody (red). Untransfected cells were visualized by DAPI staining (blue). Scale bar, 20 μm. Ab , Because the anti-NCKX2 ext antibody was raised against a synthetic peptide within the N-terminal extracellular region (residues 90–102), HEK293 cells expressing NCKX2-FLAG, in which the FLAG tag ( 90 DYKDDDDK 97 ) replaces the N-terminal 8 amino acids of the epitope, were not immunoreactive to the anti-NCKX2 ext (green). Expression of NCKX2-FLAG was detected by anti-FLAG (red), and untransfected cells were visualized by DAPI staining (blue). Scale bar, 20 μm. B , Preabsorption test of anti-NCKX2 ext antibody. DIV18 hippocampal neuron lysate was immunoblotted using anti-NCKX2 ext antibody without (left lane) or with (right lane) preincubation of the antigen peptide. β-Actin was detected as a loading control (bottom panel). C , Endogenous NCKX2 (green) and transfected FLAG-KIF21A (red) were colocalized not only in the somatodendritic region but also in the axon terminals of the cultured hippocampal neuron. Scale bar, 50 μm. D , Higher magnification images of the axonal bouton (dotted box in C ), the soma of which was marked by an arrow in C . The axon was identified by NF-H immunofluorescence (blue). Scale bar, 20 μm. E–G , Quantitative analysis of colocalization. Ea , Higher magnification image of the FLAG-positive axon (boxed region in C ). Other FLAG-negative neurites were eliminated using the binary mask made from NF-H fluorescence image. Eb , The PDM value was calculated at each pixel and shown as the pixel value at its location. For clarity, the contrast of this PDM image was adjusted where the brightness is saturated at 0.2. Scale bar, 50 μm. F , A scattered plot of green and red pixel intensities against their PDM value at each paired pixel. G , The line profiles of NCKX2 (green) and FLAG-KIF21A (red) fluorescence intensities along two ROI lines (red lines in the left panel). The locations of arrowheads in the right two panels correspond to puncta indicated by arrowheads in Eb . *PDM > 0.1; **PDM > 0.2.

    Journal: The Journal of Neuroscience

    Article Title: KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

    doi: 10.1523/JNEUROSCI.6331-11.2012

    Figure Lengend Snippet: Colocalization of NCKX2 and KIF21A. A , Reliability test of NCKX2 ext antibody. Aa , The HEK293 cells expressing NCKX2-GFP (green) were specifically labeled with anti-NCKX2 ext antibody (red). Untransfected cells were visualized by DAPI staining (blue). Scale bar, 20 μm. Ab , Because the anti-NCKX2 ext antibody was raised against a synthetic peptide within the N-terminal extracellular region (residues 90–102), HEK293 cells expressing NCKX2-FLAG, in which the FLAG tag ( 90 DYKDDDDK 97 ) replaces the N-terminal 8 amino acids of the epitope, were not immunoreactive to the anti-NCKX2 ext (green). Expression of NCKX2-FLAG was detected by anti-FLAG (red), and untransfected cells were visualized by DAPI staining (blue). Scale bar, 20 μm. B , Preabsorption test of anti-NCKX2 ext antibody. DIV18 hippocampal neuron lysate was immunoblotted using anti-NCKX2 ext antibody without (left lane) or with (right lane) preincubation of the antigen peptide. β-Actin was detected as a loading control (bottom panel). C , Endogenous NCKX2 (green) and transfected FLAG-KIF21A (red) were colocalized not only in the somatodendritic region but also in the axon terminals of the cultured hippocampal neuron. Scale bar, 50 μm. D , Higher magnification images of the axonal bouton (dotted box in C ), the soma of which was marked by an arrow in C . The axon was identified by NF-H immunofluorescence (blue). Scale bar, 20 μm. E–G , Quantitative analysis of colocalization. Ea , Higher magnification image of the FLAG-positive axon (boxed region in C ). Other FLAG-negative neurites were eliminated using the binary mask made from NF-H fluorescence image. Eb , The PDM value was calculated at each pixel and shown as the pixel value at its location. For clarity, the contrast of this PDM image was adjusted where the brightness is saturated at 0.2. Scale bar, 50 μm. F , A scattered plot of green and red pixel intensities against their PDM value at each paired pixel. G , The line profiles of NCKX2 (green) and FLAG-KIF21A (red) fluorescence intensities along two ROI lines (red lines in the left panel). The locations of arrowheads in the right two panels correspond to puncta indicated by arrowheads in Eb . *PDM > 0.1; **PDM > 0.2.

    Article Snippet: For surface immunostaining of NCKX2, live cells were incubated with rabbit anti-NCKX2ext (1:100) or rabbit anti-GFP (1:100; Millipore Bioscience Research Reagents) in serum-free culture medium for 15 min at 36°C or at 4°C, rinsed with culture medium, fixed with ice-cold 4% PFA or 3.8% formaldehyde in PBS for 20 min, and washed with PBS.

    Techniques: Expressing, Labeling, Staining, FLAG-tag, Transfection, Cell Culture, Immunofluorescence, Fluorescence

    Immunocytochemical localization of endogenous ( A ) or exogenous ( B ) NCKX2 in cultured hippocampal neurons. Aa , DIV23 hippocampal neurons were stained for endogenous NCKX2 (green) and dendritic marker MAP2 (red). Ab–Ae , Surface NCKX2 (s-NCKX2) was immunolabeled by incubating live cells with anti-NCKX2 ext (green) at 36°C, and then cells were fixed, permeabilized, and immunostained with antibodies against axonal marker Tau-1 (red; Ab , Ac ) or presynaptic marker synaptophysin (red; Ad ) or dendritic maker MAP2 (red; Ae ). Higher magnification image of the dashed box in Ab is shown in Ac . The sites where endogenous s-NCKX2 was colocalized with Tau-1 ( Ac ) or synaptophysin ( Ad ) are marked with arrows. Af , Endogenous s-NCKX2 (green) was immunolabeled by the same manner as in Ae except incubating live cells with anti-NCKX2 ext at 4°C before fixation. Scale bars: Aa , 10 μm; Ab , Ae , Af , 50 μm; Ac , Ad , 5 μm. Ba , A DIV7 hippocampal neuron transfected with NCKX2-GFP and DsRed. Scale bar, 50 μm. Bb , A DIV13 hippocampal neuron transfected with NCKX2-GFP (green). Surface NCKX2-GFP was visualized by live-cell immunolabeling with antibody against GFP (s-NCKX2-GFP; red), and then stained for MAP2 (blue). Scale bar, 10 μm. The open and solid arrowheads indicate dendrites and axons, respectively. The asterisks show location of somata.

    Journal: The Journal of Neuroscience

    Article Title: KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

    doi: 10.1523/JNEUROSCI.6331-11.2012

    Figure Lengend Snippet: Immunocytochemical localization of endogenous ( A ) or exogenous ( B ) NCKX2 in cultured hippocampal neurons. Aa , DIV23 hippocampal neurons were stained for endogenous NCKX2 (green) and dendritic marker MAP2 (red). Ab–Ae , Surface NCKX2 (s-NCKX2) was immunolabeled by incubating live cells with anti-NCKX2 ext (green) at 36°C, and then cells were fixed, permeabilized, and immunostained with antibodies against axonal marker Tau-1 (red; Ab , Ac ) or presynaptic marker synaptophysin (red; Ad ) or dendritic maker MAP2 (red; Ae ). Higher magnification image of the dashed box in Ab is shown in Ac . The sites where endogenous s-NCKX2 was colocalized with Tau-1 ( Ac ) or synaptophysin ( Ad ) are marked with arrows. Af , Endogenous s-NCKX2 (green) was immunolabeled by the same manner as in Ae except incubating live cells with anti-NCKX2 ext at 4°C before fixation. Scale bars: Aa , 10 μm; Ab , Ae , Af , 50 μm; Ac , Ad , 5 μm. Ba , A DIV7 hippocampal neuron transfected with NCKX2-GFP and DsRed. Scale bar, 50 μm. Bb , A DIV13 hippocampal neuron transfected with NCKX2-GFP (green). Surface NCKX2-GFP was visualized by live-cell immunolabeling with antibody against GFP (s-NCKX2-GFP; red), and then stained for MAP2 (blue). Scale bar, 10 μm. The open and solid arrowheads indicate dendrites and axons, respectively. The asterisks show location of somata.

    Article Snippet: For surface immunostaining of NCKX2, live cells were incubated with rabbit anti-NCKX2ext (1:100) or rabbit anti-GFP (1:100; Millipore Bioscience Research Reagents) in serum-free culture medium for 15 min at 36°C or at 4°C, rinsed with culture medium, fixed with ice-cold 4% PFA or 3.8% formaldehyde in PBS for 20 min, and washed with PBS.

    Techniques: Cell Culture, Staining, Marker, Immunolabeling, Transfection

    Subcellular distribution of hMSH5 fusion proteins. ( A ) The intracellular distribution of the hMSH5 protein fused to an N-terminal GFP-tag (GFP-C1-hMSH5), a C-terminal GFP-tag (GFP-N1-hMSH5) or an N-terminal FLAG-tag (FLAG-hMSH5) was examined in HeLa cells by laser confocal fluorescence (GFP-tagged proteins) or immunofluorescence (FLAG-hMSH5) microscopy. The images shown are representative of all the transfected cells. Upper panels show the distribution of the indicated fusion protein. Cells were counterstained with DAPI to visualize nuclei (lower panels). ( B ) HeLa cells expressing the indicated proteins were collected and fractionated into cytosolic (C) and nuclear extracts (N). Equal cell amounts from cytosolic and nuclear extracts were immunoblotted with anti-GFP or anti-FLAG antibodies, as indicated. The efficiency of fractionation was verified by staining for HSP90α as a cytoplasmic marker, and PARP-1 as a nuclear marker. ( C ) HeLa cells transiently expressing the indicated fusion proteins were incubated without (–LMB) or with leptomycin B (+LMB) for 6 h. The localization of hMSH5 fusion proteins was determined by fluorescence and immunofluorescence microscopy. Left panels show representative micrographs. The right panel presents a quantitative analysis of the localization data. For each cell, the fluorescence intensities in the entire cell area and in the nuclear area were evaluated, as detailed in ‘Material and Methods’ section and the nuclear fluorescence was calculated as a percentage of total (nuclear plus cytoplasmic) cell fluorescence (N/N+C). As many as 30–35 cells were analyzed per experiment, for each protein and each condition (±LMB). The graph represents the mean N/N+C values with standard deviations (error bars) across at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: hMSH5 is a nucleocytoplasmic shuttling protein whose stability depends on its subcellular localization

    doi: 10.1093/nar/gkq098

    Figure Lengend Snippet: Subcellular distribution of hMSH5 fusion proteins. ( A ) The intracellular distribution of the hMSH5 protein fused to an N-terminal GFP-tag (GFP-C1-hMSH5), a C-terminal GFP-tag (GFP-N1-hMSH5) or an N-terminal FLAG-tag (FLAG-hMSH5) was examined in HeLa cells by laser confocal fluorescence (GFP-tagged proteins) or immunofluorescence (FLAG-hMSH5) microscopy. The images shown are representative of all the transfected cells. Upper panels show the distribution of the indicated fusion protein. Cells were counterstained with DAPI to visualize nuclei (lower panels). ( B ) HeLa cells expressing the indicated proteins were collected and fractionated into cytosolic (C) and nuclear extracts (N). Equal cell amounts from cytosolic and nuclear extracts were immunoblotted with anti-GFP or anti-FLAG antibodies, as indicated. The efficiency of fractionation was verified by staining for HSP90α as a cytoplasmic marker, and PARP-1 as a nuclear marker. ( C ) HeLa cells transiently expressing the indicated fusion proteins were incubated without (–LMB) or with leptomycin B (+LMB) for 6 h. The localization of hMSH5 fusion proteins was determined by fluorescence and immunofluorescence microscopy. Left panels show representative micrographs. The right panel presents a quantitative analysis of the localization data. For each cell, the fluorescence intensities in the entire cell area and in the nuclear area were evaluated, as detailed in ‘Material and Methods’ section and the nuclear fluorescence was calculated as a percentage of total (nuclear plus cytoplasmic) cell fluorescence (N/N+C). As many as 30–35 cells were analyzed per experiment, for each protein and each condition (±LMB). The graph represents the mean N/N+C values with standard deviations (error bars) across at least three independent experiments.

    Article Snippet: Antibodies Mouse monoclonal anti-GFP (B-2) and goat polyclonal anti-Lamin B (C-20) were from Santa Cruz Biotechnology; mouse monoclonal anti-Flag (M2) and mouse monoclonal anti-ß-tubulin were purchased from Sigma–Aldrich, mouse monoclonal anti-Hsp90α and mouse monoclonal anti-Poly(ADP-Ribose) Polymerase-1 (PARP-1) were from Calbiochem.

    Techniques: FLAG-tag, Fluorescence, Immunofluorescence, Microscopy, Transfection, Expressing, Fractionation, Staining, Marker, Incubation

    V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an anti-c-Myc antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with anti-FLAG antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an anti-c-Myc antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with anti-FLAG antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Recombinant, Immunoprecipitation, SDS Page, Western Blot, Negative Control

    Vacuum infiltration and purification of V H H9-sIgA. N. benthamiana leaves were vacuum infiltrated with a mixture of V H H9-Fc/SC/JC and p19. Tissue was collected at 12 dpi. Cell extracts were prepared under native conditions and separated with SDS-PAGE under non-reducing conditions. (A) Secretory IgA was purified with peptide M Agarose. Western blots were detected with anti-c-Myc antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1), tetrameric (No. 2, ∼176 kDa), trimeric (No. 3, ∼132 kDa), dimeric (No. 4, ∼88 kDa), and monomeric (No. 5, ∼44 kDa) V H H9-Fc. (B) Secretory IgA was purified with anti-FLAG agarose. Western blots were detected with anti-Flag antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1, ∼66 kDa), SC/trimeric V H H9-Fc/JC (No. 2, ∼206 kDa), SC/dimeric V H H9-Fc (No. 3, ∼160), SC/monomeric V H H9-Fc (No. 4, ∼110 kDa), and monomeric SC (No. 5). 10 μl of cell extract was loaded as a snapshot of the antibody produced in vivo .

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Vacuum infiltration and purification of V H H9-sIgA. N. benthamiana leaves were vacuum infiltrated with a mixture of V H H9-Fc/SC/JC and p19. Tissue was collected at 12 dpi. Cell extracts were prepared under native conditions and separated with SDS-PAGE under non-reducing conditions. (A) Secretory IgA was purified with peptide M Agarose. Western blots were detected with anti-c-Myc antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1), tetrameric (No. 2, ∼176 kDa), trimeric (No. 3, ∼132 kDa), dimeric (No. 4, ∼88 kDa), and monomeric (No. 5, ∼44 kDa) V H H9-Fc. (B) Secretory IgA was purified with anti-FLAG agarose. Western blots were detected with anti-Flag antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1, ∼66 kDa), SC/trimeric V H H9-Fc/JC (No. 2, ∼206 kDa), SC/dimeric V H H9-Fc (No. 3, ∼160), SC/monomeric V H H9-Fc (No. 4, ∼110 kDa), and monomeric SC (No. 5). 10 μl of cell extract was loaded as a snapshot of the antibody produced in vivo .

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Purification, SDS Page, Western Blot, Produced, In Vivo

    Binding of plant-produced V H H9-sIgA to EHEC O157:H7 intimin. (A) SPR binding of V H H9-sIgA purified using peptide M. Either plant-produced V H H9-sIgA (top) or E. coli -produced V H H9 monomer (bottom) was immobilized on CM5 Series S sensor chips via amine coupling and MBP-Int277 was flowed over the resulting surfaces at concentrations ranging from 0.3 to 5 nM. The experiment was conducted in duplicate. Black lines show data and red lines show fits. (B) ELISA binding of plant-produced V H H9-sIgA purified using either peptide M (left) or anti-FLAG antibody (right) and detected using either anti-bovine IgA antibody (top) or anti-FLAG antibody (bottom). Results are representative of two independent experiments.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Binding of plant-produced V H H9-sIgA to EHEC O157:H7 intimin. (A) SPR binding of V H H9-sIgA purified using peptide M. Either plant-produced V H H9-sIgA (top) or E. coli -produced V H H9 monomer (bottom) was immobilized on CM5 Series S sensor chips via amine coupling and MBP-Int277 was flowed over the resulting surfaces at concentrations ranging from 0.3 to 5 nM. The experiment was conducted in duplicate. Black lines show data and red lines show fits. (B) ELISA binding of plant-produced V H H9-sIgA purified using either peptide M (left) or anti-FLAG antibody (right) and detected using either anti-bovine IgA antibody (top) or anti-FLAG antibody (bottom). Results are representative of two independent experiments.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Binding Assay, Produced, SPR Assay, Purification, Enzyme-linked Immunosorbent Assay

    Design and production of individual subunits required for chimeric secretory IgA assembly. (A) Schematic of all produced subunits fully assembled into a chimeric antibody intended for secretory IgA functionality. It notably differs from the structure of native secretory IgA by the replacement of the Fab region with a camelid-derived variable heavy chain fragment (V H H). (B) Schematic representation of constructs used for Agrobacterium -mediated transient expression in N. benthamiana leaves. CaMV 35S, cauliflower mosaic virus 35S promoter; CPMV 5′UTR, 5′-untranslated region of Cowpea mosaic virus; PR1b, tobacco pathogenesis-related protein 1b signal peptide; V H Hx-Fc, fusion of a camelid-derived V H H to a bovine Fc where x is either 1, 3, 9, or 10, corresponding to the isolated V H Hs; SC, bovine secretory component; JC, bovine JC; c-Myc, FLAG, HA, detection tags; KDEL, endoplasmic reticulum retrieval tetra-peptide; CPMV 3′UTR, 3′-untranslated region of Cowpea mosaic virus; nos, nopaline synthase terminator sequence; the cassettes were cloned into pEAQ-DEST-1 plant expression vectors. Schematic not drawn to scale. Bold outlines indicate translated regions. (C) Monovalent affinities and kinetics of the interaction between V H Hs and MBP-Int277 by SPR (pH 7.4, 25°C). (D) Predicted protein size and number of glycosylation sites for each subunit. (E–G) Western blots of crude extract from leaves of N. benthamiana harvested at 6 dpi expressing V H H1, 3, 9, and 10-Fc along with p19, a suppressor of gene silencing (E) , SC (F) , and JC (G) . 10 μg of TSP was loaded in each lane.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Design and production of individual subunits required for chimeric secretory IgA assembly. (A) Schematic of all produced subunits fully assembled into a chimeric antibody intended for secretory IgA functionality. It notably differs from the structure of native secretory IgA by the replacement of the Fab region with a camelid-derived variable heavy chain fragment (V H H). (B) Schematic representation of constructs used for Agrobacterium -mediated transient expression in N. benthamiana leaves. CaMV 35S, cauliflower mosaic virus 35S promoter; CPMV 5′UTR, 5′-untranslated region of Cowpea mosaic virus; PR1b, tobacco pathogenesis-related protein 1b signal peptide; V H Hx-Fc, fusion of a camelid-derived V H H to a bovine Fc where x is either 1, 3, 9, or 10, corresponding to the isolated V H Hs; SC, bovine secretory component; JC, bovine JC; c-Myc, FLAG, HA, detection tags; KDEL, endoplasmic reticulum retrieval tetra-peptide; CPMV 3′UTR, 3′-untranslated region of Cowpea mosaic virus; nos, nopaline synthase terminator sequence; the cassettes were cloned into pEAQ-DEST-1 plant expression vectors. Schematic not drawn to scale. Bold outlines indicate translated regions. (C) Monovalent affinities and kinetics of the interaction between V H Hs and MBP-Int277 by SPR (pH 7.4, 25°C). (D) Predicted protein size and number of glycosylation sites for each subunit. (E–G) Western blots of crude extract from leaves of N. benthamiana harvested at 6 dpi expressing V H H1, 3, 9, and 10-Fc along with p19, a suppressor of gene silencing (E) , SC (F) , and JC (G) . 10 μg of TSP was loaded in each lane.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Produced, Derivative Assay, Construct, Expressing, Isolation, Sequencing, Clone Assay, SPR Assay, Western Blot