monoclonal anti flag m2 antibody Millipore Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore anti flag mab
    SUMOylation does not affect the chromatin binding properties of <t>LEDGF.</t> Chromatin-binding properties of LEDGF WT and SUMOylation-deficient mutants. (a) Chromatin-binding assay. LEDGF/p75-deficient HEK 293T cells co-expressing <t>FLAG-tagged</t> LEDGF/p75 WT and
    Anti Flag Mab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 721 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag mab/product/Millipore
    Average 99 stars, based on 721 article reviews
    Price from $9.99 to $1999.99
    anti flag mab - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    84
    Millipore rrid ab 2302370 mouse monoclonal anti flag m2 antibody millipore cat
    SUMOylation does not affect the chromatin binding properties of <t>LEDGF.</t> Chromatin-binding properties of LEDGF WT and SUMOylation-deficient mutants. (a) Chromatin-binding assay. LEDGF/p75-deficient HEK 293T cells co-expressing <t>FLAG-tagged</t> LEDGF/p75 WT and
    Rrid Ab 2302370 Mouse Monoclonal Anti Flag M2 Antibody Millipore Cat, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rrid ab 2302370 mouse monoclonal anti flag m2 antibody millipore cat/product/Millipore
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rrid ab 2302370 mouse monoclonal anti flag m2 antibody millipore cat - by Bioz Stars, 2020-08
    84/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti flag
    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 <t>anti-FLAG</t> <t>M2</t> 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.
    Monoclonal Anti Flag, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1605 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag/product/Millipore
    Average 99 stars, based on 1605 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    85
    Millipore flag m2 moab
    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 <t>anti-FLAG</t> <t>M2</t> 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.
    Flag M2 Moab, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flag m2 moab/product/Millipore
    Average 85 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    flag m2 moab - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    Image Search Results


    SUMOylation does not affect the chromatin binding properties of LEDGF. Chromatin-binding properties of LEDGF WT and SUMOylation-deficient mutants. (a) Chromatin-binding assay. LEDGF/p75-deficient HEK 293T cells co-expressing FLAG-tagged LEDGF/p75 WT and

    Journal: Journal of molecular biology

    Article Title: SUMOylation of the Lens Epithelium-derived Growth Factor/p75 attenuates its transcriptional activity on the Heat Shock Protein 27 promoter

    doi: 10.1016/j.jmb.2010.03.063

    Figure Lengend Snippet: SUMOylation does not affect the chromatin binding properties of LEDGF. Chromatin-binding properties of LEDGF WT and SUMOylation-deficient mutants. (a) Chromatin-binding assay. LEDGF/p75-deficient HEK 293T cells co-expressing FLAG-tagged LEDGF/p75 WT and

    Article Snippet: FLAG-tagged LEDGF proteins were detected with anti-FLAG Mab (1/1,000, M2, Sigma), or anti-LEDGF Mab (1/250).

    Techniques: Binding Assay, Expressing

    LEDGF/p52 and LEDGF/p75 are SUMO-1 targets. (a) LEDGF/p75-deficient cells were transfected with FLAG-tagged LEDGF expression plasmids alone or together with the Dual S1/I/U plasmid encoding UBC9 and SUMO-1. Cell lysates were immunoblotted for LEDGF proteins

    Journal: Journal of molecular biology

    Article Title: SUMOylation of the Lens Epithelium-derived Growth Factor/p75 attenuates its transcriptional activity on the Heat Shock Protein 27 promoter

    doi: 10.1016/j.jmb.2010.03.063

    Figure Lengend Snippet: LEDGF/p52 and LEDGF/p75 are SUMO-1 targets. (a) LEDGF/p75-deficient cells were transfected with FLAG-tagged LEDGF expression plasmids alone or together with the Dual S1/I/U plasmid encoding UBC9 and SUMO-1. Cell lysates were immunoblotted for LEDGF proteins

    Article Snippet: FLAG-tagged LEDGF proteins were detected with anti-FLAG Mab (1/1,000, M2, Sigma), or anti-LEDGF Mab (1/250).

    Techniques: Transfection, Expressing, Plasmid Preparation

    An N-terminal p120 fragment suppresses invasiveness. A , effect of p120 N-terminal fragment overexpression on RhoA and Rac1 activities. p120-depleted MDA-MB-231 cells reexpressing murine p120 isoform 1A were infected with retroviruses expressing zeocin resistance alone ( zeo ) or together with a FLAG-tagged N-terminal p120 truncation mutant containing amino acids 1-323 ( N1 ). The relative levels of active RhoA and Rac1 were determined in stable polyclonal cell lines using pull-down assays and Western blots as described earlier. Expression of N1 did not affect endogenous levels of cadherin 11 or Rac1 activation but caused a significant increase in basal RhoA activity. B , NIH3T3 fibroblasts and MDA-MB-231 cells stably expressing zeocin resistance alone or together with the N1 p120 fragment were subjected to invasion assays to determine the effect of N1 on cell invasiveness. Results are the mean ± S.E. of three independent determinations performed in duplicate. ** , p

    Journal: The Journal of Biological Chemistry

    Article Title: A p120 Catenin Isoform Switch Affects Rho Activity, Induces Tumor Cell Invasion, and Predicts Metastatic Disease *A p120 Catenin Isoform Switch Affects Rho Activity, Induces Tumor Cell Invasion, and Predicts Metastatic Disease * S⃞

    doi: 10.1074/jbc.M801192200

    Figure Lengend Snippet: An N-terminal p120 fragment suppresses invasiveness. A , effect of p120 N-terminal fragment overexpression on RhoA and Rac1 activities. p120-depleted MDA-MB-231 cells reexpressing murine p120 isoform 1A were infected with retroviruses expressing zeocin resistance alone ( zeo ) or together with a FLAG-tagged N-terminal p120 truncation mutant containing amino acids 1-323 ( N1 ). The relative levels of active RhoA and Rac1 were determined in stable polyclonal cell lines using pull-down assays and Western blots as described earlier. Expression of N1 did not affect endogenous levels of cadherin 11 or Rac1 activation but caused a significant increase in basal RhoA activity. B , NIH3T3 fibroblasts and MDA-MB-231 cells stably expressing zeocin resistance alone or together with the N1 p120 fragment were subjected to invasion assays to determine the effect of N1 on cell invasiveness. Results are the mean ± S.E. of three independent determinations performed in duplicate. ** , p

    Article Snippet: Primary antibodies were used as follows: 0.25 μg/ml anti-p120 mAb p120 (BD Biosciences), 5 μg/ml anti-FLAG tag mAb (M2; Sigma), 1 μg/ml anti-Myc tag (9E10; Sigma), 0.5 μg/ml anti-cadherin 11 mAb (Zymed Laboratories Inc.), 0.6 μg/ml anti-RhoA mAb (26C4; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 0.75 μg/ml anti-Rac1 mAb (BD Biosciences), and 0.6 μg/ml anti-actin goat polyclonal antibody (I-19; Santa Cruz Biotechnology).

    Techniques: Over Expression, Multiple Displacement Amplification, Infection, Expressing, Mutagenesis, Western Blot, Activation Assay, Activity Assay, Stable Transfection

    Differential effects of p120 isoforms on cell invasiveness. A , p120 isoforms tested in this study include the naturally occurring isoforms 1A, 3A, and 4A. Murine p120 isoform 1A contains a coiled-coil domain and a regulatory phosphorylation domain in its N terminus, a central armadillo domain, and the alternatively spliced exon A in the C terminus. Murine p120 isoforms 3A and 4A lack the coiled-coil or the entire N-terminal domain, respectively. The ΔRho p120 mutant is identical to p120 isoform 1A with an in-frame deletion of amino acids 622-628. Finally, N1 is a FLAG-tagged N-terminal mutant of p120 lacking the central and C-terminal domains. B , MDA-MB-231 cells with knocked down expression of endogenous p120 (shRNA) were infected with retroviruses expressing neomycin resistance alone ( neo ), or together with murine p120 isoforms 1A, 3A, or 4A. After G418 selection, the invasiveness of p120-reexpressing cells was tested in vitro toward a gradient of HGF, as described under “Experimental Procedures.” After a 20-h incubation, cells on the underside of a Matrigel-coated transwell membrane were counted under a ×20 objective. Data are expressed as percentage of control and represent the means ± S.E. of three independent determinations performed in duplicate. ** , p

    Journal: The Journal of Biological Chemistry

    Article Title: A p120 Catenin Isoform Switch Affects Rho Activity, Induces Tumor Cell Invasion, and Predicts Metastatic Disease *A p120 Catenin Isoform Switch Affects Rho Activity, Induces Tumor Cell Invasion, and Predicts Metastatic Disease * S⃞

    doi: 10.1074/jbc.M801192200

    Figure Lengend Snippet: Differential effects of p120 isoforms on cell invasiveness. A , p120 isoforms tested in this study include the naturally occurring isoforms 1A, 3A, and 4A. Murine p120 isoform 1A contains a coiled-coil domain and a regulatory phosphorylation domain in its N terminus, a central armadillo domain, and the alternatively spliced exon A in the C terminus. Murine p120 isoforms 3A and 4A lack the coiled-coil or the entire N-terminal domain, respectively. The ΔRho p120 mutant is identical to p120 isoform 1A with an in-frame deletion of amino acids 622-628. Finally, N1 is a FLAG-tagged N-terminal mutant of p120 lacking the central and C-terminal domains. B , MDA-MB-231 cells with knocked down expression of endogenous p120 (shRNA) were infected with retroviruses expressing neomycin resistance alone ( neo ), or together with murine p120 isoforms 1A, 3A, or 4A. After G418 selection, the invasiveness of p120-reexpressing cells was tested in vitro toward a gradient of HGF, as described under “Experimental Procedures.” After a 20-h incubation, cells on the underside of a Matrigel-coated transwell membrane were counted under a ×20 objective. Data are expressed as percentage of control and represent the means ± S.E. of three independent determinations performed in duplicate. ** , p

    Article Snippet: Primary antibodies were used as follows: 0.25 μg/ml anti-p120 mAb p120 (BD Biosciences), 5 μg/ml anti-FLAG tag mAb (M2; Sigma), 1 μg/ml anti-Myc tag (9E10; Sigma), 0.5 μg/ml anti-cadherin 11 mAb (Zymed Laboratories Inc.), 0.6 μg/ml anti-RhoA mAb (26C4; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 0.75 μg/ml anti-Rac1 mAb (BD Biosciences), and 0.6 μg/ml anti-actin goat polyclonal antibody (I-19; Santa Cruz Biotechnology).

    Techniques: Mutagenesis, Multiple Displacement Amplification, Expressing, shRNA, Infection, Selection, In Vitro, Incubation

    Reduced NF-κB activity in c-FLIP S -Tg T cells. (A) Purified T cells from NF-κB-luciferase mice (NF-κB-luc) or NF-κB-luc/c-FLIP S double transgenic mice (c-FLIP S -Tg x NF-κB-luc) were stimulated with anti-CD3/anti-CD28 for the days indicated. *Results were statistically significant on Day 2 (p=0.021), Day 3 (p=0.008), and Day 3 (p=0.015) by paired t-test. (B) Immunoblot of whole cell lysates from purified T cells from wild-type (WT) or c-FLIP S -Tg (Tg) mice using antibodies specific for p50, RelA, c-Rel, and actin. (C) Immunoblot analysis of anti-CD3/CD28-activated T cells for phospho-IκBα versus total IκBα, and phospho-IKKα/β versus total IKKα/β. Control activation was with PMA plus ionomycin (P+I) for 60 min. (D) EMSA for NF-κB binding of nuclear lysates from purified anti-CD3/CD28-activated T cell. Phosphoimager analysis of the same assay was quantified for the p65/p50 and p50/p50 bands and the ratio displayed in the panel at the right. (E) Supershift EMSA analysis of the 1h lysates from (D) using either no antibody (−) or antibodies to p50 or p65. (F) EMSA for NF-κB and control CREB binding following 24 h or CD3/CD28 stimulation.

    Journal: European journal of immunology

    Article Title: c-FLIPS reduces activation of caspase and NF-?B pathways and decreases T cell survival

    doi: 10.1002/eji.200636956

    Figure Lengend Snippet: Reduced NF-κB activity in c-FLIP S -Tg T cells. (A) Purified T cells from NF-κB-luciferase mice (NF-κB-luc) or NF-κB-luc/c-FLIP S double transgenic mice (c-FLIP S -Tg x NF-κB-luc) were stimulated with anti-CD3/anti-CD28 for the days indicated. *Results were statistically significant on Day 2 (p=0.021), Day 3 (p=0.008), and Day 3 (p=0.015) by paired t-test. (B) Immunoblot of whole cell lysates from purified T cells from wild-type (WT) or c-FLIP S -Tg (Tg) mice using antibodies specific for p50, RelA, c-Rel, and actin. (C) Immunoblot analysis of anti-CD3/CD28-activated T cells for phospho-IκBα versus total IκBα, and phospho-IKKα/β versus total IKKα/β. Control activation was with PMA plus ionomycin (P+I) for 60 min. (D) EMSA for NF-κB binding of nuclear lysates from purified anti-CD3/CD28-activated T cell. Phosphoimager analysis of the same assay was quantified for the p65/p50 and p50/p50 bands and the ratio displayed in the panel at the right. (E) Supershift EMSA analysis of the 1h lysates from (D) using either no antibody (−) or antibodies to p50 or p65. (F) EMSA for NF-κB and control CREB binding following 24 h or CD3/CD28 stimulation.

    Article Snippet: Immunoblot using either anti-FLIP mAb (Dave-2, Apotech, Lausen, Switzerland) ( ) (or anti-FLAG mAb (M2, Sigma Chemical Co., St. Louis, MO) (data not shown) further confirmed expression of the transgene The c-FLIPS -Tg mouse strain has been backcrossed to C57BL/6 mice (The Jackson Laboratory, Bar Harbor, Maine) for 9 generations.

    Techniques: Activity Assay, Purification, Luciferase, Mouse Assay, Transgenic Assay, Activation Assay, Binding Assay

    Membrane topology of HEV ORF3 protein. ( A ) N- and C-terminal ends of HEV ORF3 protein are intracellularly exposed. S10-3 cells were transfected with pCMV_FLAG-ORF3-HA and subjected to immunfluorescence detection of HA and FLAG tags, using rabbit pAb anti-HA (Y-11) and mouse mAb anti-FLAG M2, respectively, after permeabilization with 0.5% saponin (Perm. Sap. 0.5%) or in the absence of permeabilization (No Perm.). ( B ) Similarly, S10-3 cells transfected with pCMVORF3 were subjected to immunofluorescence to detect the plasma membrane tetraspanin CD151 (mouse mAb 11G5a), the cytoplasmic protein MAVS (rabbit pAb anti-MAVS) or HEV ORF3 protein using mAb MRB198. Nuclei were stained by DAPI. ( C ) Selective membrane permeabilization. S10-3 cells were transfected with pCMVORF3 or co-transfected with pUHD15-1 and pUHD-Cp7 allowing the expression of the hepatitis C virus (HCV) core-p7 region (top panel) and cultured for 24 h. S10-3 cells were transfected with the HEV p6 infectious clone (middle panel) and cultured for 5 d. All cells were fixed and permeabilized with either 0.2% or 0.01% digitonin. Immunofluorescence detection of the cytoplasmic HCV core with mouse mAb C7-50 or the endoplasmic reticulum luminal HCV E1 glycoprotein with mouse mAb A4 served as controls for selective permeabilization of intracellular membranes. HEV ORF3 protein is detected using anti-ORF3 mAb MRB198. The lower panel shows histograms summarizing fluorescence intensities, as determined by using ImageJ software in 10 to 35 cells per condition, obtained after immunofluorescence with total (Dig. 0.2%) or selective (Dig. 0.01%) membrane permeabilization of S10-3 cells replicating the HEV p6 infectious clone. The asterisk (*) indicates statistically significant results with p

    Journal: PLoS Pathogens

    Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion

    doi: 10.1371/journal.ppat.1007471

    Figure Lengend Snippet: Membrane topology of HEV ORF3 protein. ( A ) N- and C-terminal ends of HEV ORF3 protein are intracellularly exposed. S10-3 cells were transfected with pCMV_FLAG-ORF3-HA and subjected to immunfluorescence detection of HA and FLAG tags, using rabbit pAb anti-HA (Y-11) and mouse mAb anti-FLAG M2, respectively, after permeabilization with 0.5% saponin (Perm. Sap. 0.5%) or in the absence of permeabilization (No Perm.). ( B ) Similarly, S10-3 cells transfected with pCMVORF3 were subjected to immunofluorescence to detect the plasma membrane tetraspanin CD151 (mouse mAb 11G5a), the cytoplasmic protein MAVS (rabbit pAb anti-MAVS) or HEV ORF3 protein using mAb MRB198. Nuclei were stained by DAPI. ( C ) Selective membrane permeabilization. S10-3 cells were transfected with pCMVORF3 or co-transfected with pUHD15-1 and pUHD-Cp7 allowing the expression of the hepatitis C virus (HCV) core-p7 region (top panel) and cultured for 24 h. S10-3 cells were transfected with the HEV p6 infectious clone (middle panel) and cultured for 5 d. All cells were fixed and permeabilized with either 0.2% or 0.01% digitonin. Immunofluorescence detection of the cytoplasmic HCV core with mouse mAb C7-50 or the endoplasmic reticulum luminal HCV E1 glycoprotein with mouse mAb A4 served as controls for selective permeabilization of intracellular membranes. HEV ORF3 protein is detected using anti-ORF3 mAb MRB198. The lower panel shows histograms summarizing fluorescence intensities, as determined by using ImageJ software in 10 to 35 cells per condition, obtained after immunofluorescence with total (Dig. 0.2%) or selective (Dig. 0.01%) membrane permeabilization of S10-3 cells replicating the HEV p6 infectious clone. The asterisk (*) indicates statistically significant results with p

    Article Snippet: Antibodies Monoclonal antibodies (mAb) anti-FLAG M2 and anti-β-actin were from Sigma-Aldrich (Saint-Louis, MI).

    Techniques: Transfection, Immunofluorescence, Staining, Expressing, Cell Culture, Fluorescence, Software

    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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.22.8.2575-2585.2002

    Figure Lengend 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.

    Article Snippet: The membrane filter was blocked with 5% powdered milk in TBST (Tris-buffered saline containing 0.1% [vol/vol] Tween 20) for 1 h at room temperature and then incubated with a primary antibody including a monoclonal anti-HA (12CA5; Roche Molecular Biochemicals), monoclonal anti-p73α (Ab-1; Oncogene Research Products), monoclonal anti-FLAG (M2; Sigma), polyclonal anti-ΔNp73, or monoclonal anti-p53 (DO-1; Oncogene Research Products) antibody for 1 h at room temperature.

    Techniques: 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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.22.8.2575-2585.2002

    Figure Lengend 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.

    Article Snippet: The membrane filter was blocked with 5% powdered milk in TBST (Tris-buffered saline containing 0.1% [vol/vol] Tween 20) for 1 h at room temperature and then incubated with a primary antibody including a monoclonal anti-HA (12CA5; Roche Molecular Biochemicals), monoclonal anti-p73α (Ab-1; Oncogene Research Products), monoclonal anti-FLAG (M2; Sigma), polyclonal anti-ΔNp73, or monoclonal anti-p53 (DO-1; Oncogene Research Products) antibody for 1 h at room temperature.

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

    Effect of thimerosal and IP 3 on the interaction between the IP 3 -binding and transmembrane domains of IP 3 R1 ( A ) Pull-down experiment applied for GST–1–604 with FLAG(2170–2749) in the presence of 1 mM 2-mercaptoethanol or 100 μM thimerosal. EGTA (1 mM) or 25 μM Ca 2+ was added as indicated in the Figure. The recombinant FLAG(2170–2749) was incubated with GST or GST–1–604 immobilized on glutathione–Sepharose 4B. After washing, the retained proteins were eluted. After SDS/PAGE, the proteins were analysed by immunoblotting with ANTI-FLAG® M2 monoclonal antibody. Lane ‘microsomes’ contains 0.5 μg of protein and lane ‘soluble fraction’ contains 2 μg of protein, whereas the lanes representing samples obtained after GST-pull down contain 15-fold more material than the soluble fraction. ( B ) Pull-down experiment applied for GST–1–604 with FLAG(2170–2749) in the presence of 1 mM 2-mercaptoethanol. During the incubation reaction, 20 μM IP 3 or adenophostin A was added. The experimental procedure was the same as that described in ( A ). IP 3 and adenophostin A significantly inhibited the interaction to 45±5 and 32±8% of the control respectively. Results are the means±S.D. for at least three independent experiments.

    Journal: Biochemical Journal

    Article Title: Thimerosal stimulates Ca2+ flux through inositol 1,4,5-trisphosphate receptor type 1, but not type 3, via modulation of an isoform-specific Ca2+-dependent intramolecular interaction

    doi: 10.1042/BJ20040072

    Figure Lengend Snippet: Effect of thimerosal and IP 3 on the interaction between the IP 3 -binding and transmembrane domains of IP 3 R1 ( A ) Pull-down experiment applied for GST–1–604 with FLAG(2170–2749) in the presence of 1 mM 2-mercaptoethanol or 100 μM thimerosal. EGTA (1 mM) or 25 μM Ca 2+ was added as indicated in the Figure. The recombinant FLAG(2170–2749) was incubated with GST or GST–1–604 immobilized on glutathione–Sepharose 4B. After washing, the retained proteins were eluted. After SDS/PAGE, the proteins were analysed by immunoblotting with ANTI-FLAG® M2 monoclonal antibody. Lane ‘microsomes’ contains 0.5 μg of protein and lane ‘soluble fraction’ contains 2 μg of protein, whereas the lanes representing samples obtained after GST-pull down contain 15-fold more material than the soluble fraction. ( B ) Pull-down experiment applied for GST–1–604 with FLAG(2170–2749) in the presence of 1 mM 2-mercaptoethanol. During the incubation reaction, 20 μM IP 3 or adenophostin A was added. The experimental procedure was the same as that described in ( A ). IP 3 and adenophostin A significantly inhibited the interaction to 45±5 and 32±8% of the control respectively. Results are the means±S.D. for at least three independent experiments.

    Article Snippet: For immunodetection of FLAG(2170–2749) fusion protein, ANTI-FLAG® M2 monoclonal antibody (1:1500; Sigma) was used.

    Techniques: Binding Assay, Recombinant, Incubation, SDS Page

    Binding of NP to importin α. (A) Purification of mRFP-Flag-tagged WT and mutant NP110aas from COS-7 cells and GST-tagged importin α isoforms, Rch1, Qip1 and NPI-1 from Escherichia coli . For purification of NP110aa, COS-7 cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT and mutant NP110aas, lysed and proteins were purified using ANTI-FLAG M2 Agarose beads and the 3× Flag peptide. For purification of importin α proteins, Escherichia coli expressing GST-tagged Rch1, Qip1 and NPI-1 were lysed and proteins were purified using glutathione-Sepharose 4B. Purified proteins were subjected to 10% or 15% SDS-PAGE and stained by Coomassie brilliant blue. (B) Binding of WT and mutant NP110aas to the three importin α isoforms. Both GST beads bound to Rch1, Qip1, NPI-1 and non-bound GST beads were incubated with the WT, 14-110aa, R8A or S9A NP110aa proteins. Binding of NP110aa to importin α was detected by Western blotting with anti-FLAG M2 monoclonal antibody (mAb). (C) Quantification of band intensity. Different concentrations of Rch1, Qip1 and NPI-1 proteins (0, 5, 25 and 50 µM) were subjected to 10% SDS-PAGE and Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. Band intensities were measured by Image J and the standard curves for the amount of importin α isoforms were constructed. (D) Competition binding assay of importin α to WT and S9A NP110aa-binding beads. Purified importin α isoforms, Rch1, Qip1 and NPI-1 from Escherichia coli were subjected to 10% SDS-PAGE and stained with Coomassie brilliant blue. All three importin α proteins (50 µM) were incubated with WT and S9A NP110aa-binding Flag beads at 4°C for 1 day. After washing with buffer, the binding of importin α to WT and S9A NP110aa was detected by Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. Empty Flag beads were used as the negative control. The amount of importin α proteins which bound to WT NP110aa and S9A NP110aa were calculated by analyzing the standard curves as shown in C right panel. (E) Binding of WT full length NP, WT and S9A NP110aa to the three importin α isoforms in HEK-293T cells. HEK-293T cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT full length NP, WT or S9A NP110aa, cells were lysed and proteins were purified using ANTI-FLAG M2 Agarose beads and the 3× Flag peptide. The bound importin αs to NP was detected by Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. The same amount of lysate was used for control. The amounts of importin α proteins which bound to WT NP, WT NP110aa and S9A NP110aa were calculated by analyzing the standard curves as shown in C right panel.

    Journal: PLoS ONE

    Article Title: Importin ?3/Qip1 Is Involved in Multiplication of Mutant Influenza Virus with Alanine Mutation at Amino Acid 9 Independently of Nuclear Transport Function

    doi: 10.1371/journal.pone.0055765

    Figure Lengend Snippet: Binding of NP to importin α. (A) Purification of mRFP-Flag-tagged WT and mutant NP110aas from COS-7 cells and GST-tagged importin α isoforms, Rch1, Qip1 and NPI-1 from Escherichia coli . For purification of NP110aa, COS-7 cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT and mutant NP110aas, lysed and proteins were purified using ANTI-FLAG M2 Agarose beads and the 3× Flag peptide. For purification of importin α proteins, Escherichia coli expressing GST-tagged Rch1, Qip1 and NPI-1 were lysed and proteins were purified using glutathione-Sepharose 4B. Purified proteins were subjected to 10% or 15% SDS-PAGE and stained by Coomassie brilliant blue. (B) Binding of WT and mutant NP110aas to the three importin α isoforms. Both GST beads bound to Rch1, Qip1, NPI-1 and non-bound GST beads were incubated with the WT, 14-110aa, R8A or S9A NP110aa proteins. Binding of NP110aa to importin α was detected by Western blotting with anti-FLAG M2 monoclonal antibody (mAb). (C) Quantification of band intensity. Different concentrations of Rch1, Qip1 and NPI-1 proteins (0, 5, 25 and 50 µM) were subjected to 10% SDS-PAGE and Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. Band intensities were measured by Image J and the standard curves for the amount of importin α isoforms were constructed. (D) Competition binding assay of importin α to WT and S9A NP110aa-binding beads. Purified importin α isoforms, Rch1, Qip1 and NPI-1 from Escherichia coli were subjected to 10% SDS-PAGE and stained with Coomassie brilliant blue. All three importin α proteins (50 µM) were incubated with WT and S9A NP110aa-binding Flag beads at 4°C for 1 day. After washing with buffer, the binding of importin α to WT and S9A NP110aa was detected by Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. Empty Flag beads were used as the negative control. The amount of importin α proteins which bound to WT NP110aa and S9A NP110aa were calculated by analyzing the standard curves as shown in C right panel. (E) Binding of WT full length NP, WT and S9A NP110aa to the three importin α isoforms in HEK-293T cells. HEK-293T cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT full length NP, WT or S9A NP110aa, cells were lysed and proteins were purified using ANTI-FLAG M2 Agarose beads and the 3× Flag peptide. The bound importin αs to NP was detected by Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. The same amount of lysate was used for control. The amounts of importin α proteins which bound to WT NP, WT NP110aa and S9A NP110aa were calculated by analyzing the standard curves as shown in C right panel.

    Article Snippet: The purified proteins were subjected to SDS-PAGE and Western blotting with an anti-Flag monoclonal antibody (MAb) (M2; Sigma).

    Techniques: Binding Assay, Purification, Mutagenesis, Transfection, Expressing, SDS Page, Staining, Incubation, Western Blot, Construct, Negative Control

    (A) Transient expression of IκB-SR renders SV80 cells, but not FLIP-L- or FLIP-S-expressing cells, sensitive to TRAIL in the absence of CHX. SV80, SV80 FLIP-L, and SV80 FLIP-S cells were transfected with pEGFP along with empty vector or pIκB-SR encoding a nondegradable form of IκB and were split. After 1 day of recovery they were challenged with TRAIL-Flag complexed with the anti-Flag MAb M2 (1 μg/ml) for an additional 16 h or remained untreated. Finally GFP-positive cells were analyzed for the percentage of cells with morphological features of apoptosis. −, absence of TRAIL; +, presence of TRAIL. (B) Cells were cultivated in 96-well plates (15,000 cells/well) for 24 h and were then treated 1 h with MG-132 (10 μM). Subsequently, the indicated concentrations of cross-linked TRAIL-Flag were added for an additional 16 h. Cell viability was determined using the MTT assay. wt, wild type.

    Journal: Molecular and Cellular Biology

    Article Title: NF-?B Inducers Upregulate cFLIP, a Cycloheximide-Sensitive Inhibitor of Death Receptor Signaling

    doi: 10.1128/MCB.21.12.3964-3973.2001

    Figure Lengend Snippet: (A) Transient expression of IκB-SR renders SV80 cells, but not FLIP-L- or FLIP-S-expressing cells, sensitive to TRAIL in the absence of CHX. SV80, SV80 FLIP-L, and SV80 FLIP-S cells were transfected with pEGFP along with empty vector or pIκB-SR encoding a nondegradable form of IκB and were split. After 1 day of recovery they were challenged with TRAIL-Flag complexed with the anti-Flag MAb M2 (1 μg/ml) for an additional 16 h or remained untreated. Finally GFP-positive cells were analyzed for the percentage of cells with morphological features of apoptosis. −, absence of TRAIL; +, presence of TRAIL. (B) Cells were cultivated in 96-well plates (15,000 cells/well) for 24 h and were then treated 1 h with MG-132 (10 μM). Subsequently, the indicated concentrations of cross-linked TRAIL-Flag were added for an additional 16 h. Cell viability was determined using the MTT assay. wt, wild type.

    Article Snippet: The latter had been cross-linked with anti-Flag MAb M2 (Sigma, Deisenhofen, Germany) before treatment.

    Techniques: Expressing, Transfection, Plasmid Preparation, MTT Assay

    Impact of prestimulation with IL-1 (A), TNF (B), and the agonistic CD40-specific MAb G28.5 on TNF- and TRAIL-induced cytotoxicity. SV80-CD40 cells were cultivated in 96-well plates (15,000 cells/well) for 24 h and were then treated with IL-1 (10 ng/ml), TNF (10 ng/ml), and anti-CD40 MAb G28.5 (1 μg/ml) (A to C; solid bars) or remained untreated (A to C; empty bars). After 6 h the cells were washed twice with medium and challenged with TNF (50 ng/ml) or TRAIL-Flag (100 ng/ml) complexed with the anti-Flag MAb M2 (1 μg/ml) in the presence of 25 μg of CHX/ml for an additional 8 h. Cell viability was determined using the MTT assay.

    Journal: Molecular and Cellular Biology

    Article Title: NF-?B Inducers Upregulate cFLIP, a Cycloheximide-Sensitive Inhibitor of Death Receptor Signaling

    doi: 10.1128/MCB.21.12.3964-3973.2001

    Figure Lengend Snippet: Impact of prestimulation with IL-1 (A), TNF (B), and the agonistic CD40-specific MAb G28.5 on TNF- and TRAIL-induced cytotoxicity. SV80-CD40 cells were cultivated in 96-well plates (15,000 cells/well) for 24 h and were then treated with IL-1 (10 ng/ml), TNF (10 ng/ml), and anti-CD40 MAb G28.5 (1 μg/ml) (A to C; solid bars) or remained untreated (A to C; empty bars). After 6 h the cells were washed twice with medium and challenged with TNF (50 ng/ml) or TRAIL-Flag (100 ng/ml) complexed with the anti-Flag MAb M2 (1 μg/ml) in the presence of 25 μg of CHX/ml for an additional 8 h. Cell viability was determined using the MTT assay.

    Article Snippet: The latter had been cross-linked with anti-Flag MAb M2 (Sigma, Deisenhofen, Germany) before treatment.

    Techniques: MTT Assay

    (A) FACS analysis of SV80 transfectants stably expressing FLIP-L–GFP or FLIP-S–GFP and mock-transfected SV80 cells. (B) Cells described for panel A were cultivated in 96-well plates (15,000 cells/well) for 24 h and were then treated with the indicated concentrations of TNF or TRAIL-Flag complexed with the anti-Flag MAb M2 (1 μg/ml) in the presence of 50 μg of CHX/ml for an additional 16 h. Cell viability was determined using the MTT assay. wt, wild type. (C) Cells described for panel A were challenged with TNF (10 ng/ml) and with cross-linked TRAIL-Flag in the presence of 50 μg of CHX/ml or remained untreated. Cells were lysed, and proteins were then separated by SDS-PAGE and transferred to nitrocellulose. The presence of the nonprocessed caspase 8 isoforms p53 and p55 was determined by Western blot analyses. wt, wild type; −, absence of TNF or TRAIL; +, presence of TNF or TRAIL. (D) SV80 and SV80 FLIP-S–GFP cells were incubated for the indicated times with CHX (25 μg/ml). Proteins (70 μg per lane) were then separated by SDS-PAGE and transferred to nitrocellulose, and the expression of endogenous cFLIP in SV80 cells and of cFLIP-S–GFP in the transfectants was detected on the same blot with the anti-FLIP MAb N19 and an alkaline-conjugated secondary antibody. (E) RNase protection assay analysis of various members of the TRAF and IAP protein families in SV80, SV80 FLIP-S–GFP, and SV80 FLIP-L–GFP. Cells were treated with the indicated combinations of TNF (20 ng/ml), agonistic anti-TRAIL-R2 antisera (αTR2) (1 μg/ml), z-VAD-fmk (Z) (20 μM), and CHX (C) (25 μg/ml) for 6 h. 0, untreated. Total RNAs were isolated after treatment, and 10 μg of each RNA was analyzed with the hApo-5 Multi-Probe template set to detect the indicated mRNAs. Absolute expression and normalized expression are given in arbitrary units. Relative expression levels were calculated as described in Materials and Methods. Arrows indicate the positions of the bands specific for TRAF1 and cIAP2.

    Journal: Molecular and Cellular Biology

    Article Title: NF-?B Inducers Upregulate cFLIP, a Cycloheximide-Sensitive Inhibitor of Death Receptor Signaling

    doi: 10.1128/MCB.21.12.3964-3973.2001

    Figure Lengend Snippet: (A) FACS analysis of SV80 transfectants stably expressing FLIP-L–GFP or FLIP-S–GFP and mock-transfected SV80 cells. (B) Cells described for panel A were cultivated in 96-well plates (15,000 cells/well) for 24 h and were then treated with the indicated concentrations of TNF or TRAIL-Flag complexed with the anti-Flag MAb M2 (1 μg/ml) in the presence of 50 μg of CHX/ml for an additional 16 h. Cell viability was determined using the MTT assay. wt, wild type. (C) Cells described for panel A were challenged with TNF (10 ng/ml) and with cross-linked TRAIL-Flag in the presence of 50 μg of CHX/ml or remained untreated. Cells were lysed, and proteins were then separated by SDS-PAGE and transferred to nitrocellulose. The presence of the nonprocessed caspase 8 isoforms p53 and p55 was determined by Western blot analyses. wt, wild type; −, absence of TNF or TRAIL; +, presence of TNF or TRAIL. (D) SV80 and SV80 FLIP-S–GFP cells were incubated for the indicated times with CHX (25 μg/ml). Proteins (70 μg per lane) were then separated by SDS-PAGE and transferred to nitrocellulose, and the expression of endogenous cFLIP in SV80 cells and of cFLIP-S–GFP in the transfectants was detected on the same blot with the anti-FLIP MAb N19 and an alkaline-conjugated secondary antibody. (E) RNase protection assay analysis of various members of the TRAF and IAP protein families in SV80, SV80 FLIP-S–GFP, and SV80 FLIP-L–GFP. Cells were treated with the indicated combinations of TNF (20 ng/ml), agonistic anti-TRAIL-R2 antisera (αTR2) (1 μg/ml), z-VAD-fmk (Z) (20 μM), and CHX (C) (25 μg/ml) for 6 h. 0, untreated. Total RNAs were isolated after treatment, and 10 μg of each RNA was analyzed with the hApo-5 Multi-Probe template set to detect the indicated mRNAs. Absolute expression and normalized expression are given in arbitrary units. Relative expression levels were calculated as described in Materials and Methods. Arrows indicate the positions of the bands specific for TRAF1 and cIAP2.

    Article Snippet: The latter had been cross-linked with anti-Flag MAb M2 (Sigma, Deisenhofen, Germany) before treatment.

    Techniques: FACS, Stable Transfection, Expressing, Transfection, MTT Assay, SDS Page, Western Blot, Incubation, Rnase Protection Assay, Isolation

    Purification and characterization of FLAG–Man2C1

    Journal:

    Article Title: Man2C1, an ?-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol

    doi: 10.1042/BJ20060945

    Figure Lengend Snippet: Purification and characterization of FLAG–Man2C1

    Article Snippet: Western blotting of FLAG–Man2C1 was performed using a mouse anti-FLAG monoclonal antibody (M2; Sigma) as described previously [ ] and was visualized using a LAS-3000 mini (Fujifilm).

    Techniques: Purification

    LBP mediates LPS-induced dimerization of sTLR4/MD-2 in a CD14-dependent manner. Recombinant sTLR4F/MD-2HA and sTLR4G/MD-2HA were incubated with LPS (5 μg/ml) for 30 min in the presence of FLAG-tagged sCD14, LBP, or both, followed by sTLR4G immunoprecipitation using an anti-GFP Ab-immobilized gel. Precipitated sTLR4F and sTLR4G and the added recombinant proteins (input) were detected by Western blotting using anti-FLAG M2 mAb ( sTLR4F , sCD14 , and LBP ), anti-GFP Ab ( sTLR4G ), or anti-HA mAb ( MD-2 ), respectively. Data are representative of three independent experiments. WB , Western blotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1–IKKϵ–IRF3 axis activation

    doi: 10.1074/jbc.M117.796631

    Figure Lengend Snippet: LBP mediates LPS-induced dimerization of sTLR4/MD-2 in a CD14-dependent manner. Recombinant sTLR4F/MD-2HA and sTLR4G/MD-2HA were incubated with LPS (5 μg/ml) for 30 min in the presence of FLAG-tagged sCD14, LBP, or both, followed by sTLR4G immunoprecipitation using an anti-GFP Ab-immobilized gel. Precipitated sTLR4F and sTLR4G and the added recombinant proteins (input) were detected by Western blotting using anti-FLAG M2 mAb ( sTLR4F , sCD14 , and LBP ), anti-GFP Ab ( sTLR4G ), or anti-HA mAb ( MD-2 ), respectively. Data are representative of three independent experiments. WB , Western blotting.

    Article Snippet: Other Abs were purchased from the following companies: mouse anti-FLAG M2 mAb, Sigma; rabbit anti-GFP Ab, anti-EEA1 antibody, mouse anti-HA (TANA2) mAb, MBL, Nagoya, Japan; mouse anti-human LBP mAb (biG412), Biometec GmbH, Greifswald, Germany; mouse anti-IκBα (L35A4), anti-phospho-IκBα (Ser-32/36) (5A5), rabbit anti-phospho-TBK1 (Ser-172) (D52C2), anti-phospho-IKKϵ (Ser-172) (D1B7), anti-IRF3 (D83B9), anti-phospho-IRF3 (Ser-396) (4D4G), anti-IRAK1 (D51G7), anti-TLR4 (D8L5W) mAb, anti-phospho-p44/42 MAPK (Erk1/2) (Thr-202/Tyr-204), anti-phospho-p38 MAPK (Thr-180/Tyr-182), anti-phospho-SAPK/JNK (Thr-183/Tyr-185), anti-TRAF3 Ab, and HRP-conjugated goat anti-rabbit IgG Ab, Cell Signaling Technology, Danvers, MA; mouse anti-RIP mAb (38/RIP), BD Biosciences, San Jose, CA; HRP-conjugated goat anti-mouse IgG Ab, Jackson ImmunoResearch Laboratories; mouse anti-human/mouse TLR2 (T2.5) mAb, allophycocyanin- and phycoerythrin (PE)-conjugated goat anti-mouse IgG, anti-rat IgG, PE- and HRP-conjugated streptavidin (Stv), BioLegend, San Diego, CA; and Alexa 546-conjugated F(ab′)2 goat anti-mouse IgG (H+L), Invitrogen; rabbit anti-NAK/TBK1 (EP611Y), anti-TRAF6 (EP591Y) mAb, Alexa 488-conjugated preadsorbed goat anti-rabbit IgG H & L, Abcam, Cambridge, UK.

    Techniques: Recombinant, Incubation, Immunoprecipitation, Western Blot

    Western blot analysis of fiber and pIX modifications introduced into generated Ad vectors. Electrophoretically resolved viral proteins were transferred to a PVDF membrane and probed with mAb 4D2, mAb M2, or penton-base specific rabbit sera to detect the

    Journal:

    Article Title: Targeting of Adenovirus Serotype 5 Pseudotyped with Short Fiber from Serotype 41 to c-erbB2-Positive Cells Using Bispecific Single-Chain Diabody

    doi: 10.1016/j.jmb.2009.03.016

    Figure Lengend Snippet: Western blot analysis of fiber and pIX modifications introduced into generated Ad vectors. Electrophoretically resolved viral proteins were transferred to a PVDF membrane and probed with mAb 4D2, mAb M2, or penton-base specific rabbit sera to detect the

    Article Snippet: Briefly, the pIXflagC6.5 fusion protein was detected with anti-Flag monoclonal antibody (mAb) M2 (Sigma, St. Louis, MO), while mAb 4D2 against the tail region was used to detect the recombinant 41s fiber protein.

    Techniques: Western Blot, Generated

    Importin α5/NPI-1 preferentially mediates the nuclear import of Vpr. (A) Twenty-five pmol of purified recombinant GST- and GFP-tagged Vpr (Vpr), GST- and GFP- tagged Vpr N17C74 (N17C74), GST-tagged GFP (GST-GFP) were resolved by 10% SDS-PAGE and stained with Coomassie brilliant blue (CBB). (B) Nuclear import of Vpr by importin α (Impα) isoforms. Digitonin-permeabilized HeLa cells were incubated with 1 µM of Vpr, N17C74, and GST-GFP in the absence (-) or presence of 1 µM (for Vpr and GST-GFP) or 3 µM (for N17C74) of each of the recombinant Impα isoforms, Rch1, Qip1 and NPI-1. Cells were fixed in 3.7% formaldehyde and stained with Hoechst 33342 to show the position of the nucleus (right panel). After fixation, cells were analyzed by confocal laser scanning microscopy. Bar = 10 µm. (C) Fluorescence intensity of Vpr per surface area was quantified for at least 70 nuclei in the presence of the indicated concentrations of the Impα isoforms from three independent experiments. The bar shows the standard errors of measurements. (D) In vitro nuclear import assay for GST-GFP-Vpr was performed in the absence (-) or presence of 1 µM of the Impα isoforms. After fixation, cells were analyzed by confocal microscopy. Bar = 10 µm. (E) In vitro nuclear import assay for Vpr was performed in the absence (-) or presence of 1 µM of the Impα isoforms, and 1 µM of Impα isoforms with 1 µM Impβ. N17C74, as a control, was performed with 1 µM of Rch1 and 1 µM Impβ. After fixation, cells were analyzed by confocal microscopy. Bar = 10 µm. (F) Binding assay between Vpr and the Impα isoforms. Glutathione-Sepharose beads were coupled with the GST-Impα isoforms, Rch1, Qip1 and NPI-1 or GST alone, and were incubated with Vpr protein purified from 293T cells transfected with pCAGGS mammalian vectors encoding Flag-mRFP (mRFP), or Flag-mRFP-Flag-Vpr (mRFP-Vpr). The bound fractions and 1/20 of the input of mRFP-Vpr and mRFP were analyzed by immunoblotting with an anti-Flag M2 monoclonal antibody (MAb) (right panel). Twenty-five pmol of GST or GST-Impα isoforms were resolved by 10% SDS-PAGE and stained with CBB (left panel). The positions of mRFP and mRFP-Vpr are indicated.

    Journal: PLoS ONE

    Article Title: Nuclear Exportin Receptor CAS Regulates the NPI-1-Mediated Nuclear Import of HIV-1 Vpr

    doi: 10.1371/journal.pone.0027815

    Figure Lengend Snippet: Importin α5/NPI-1 preferentially mediates the nuclear import of Vpr. (A) Twenty-five pmol of purified recombinant GST- and GFP-tagged Vpr (Vpr), GST- and GFP- tagged Vpr N17C74 (N17C74), GST-tagged GFP (GST-GFP) were resolved by 10% SDS-PAGE and stained with Coomassie brilliant blue (CBB). (B) Nuclear import of Vpr by importin α (Impα) isoforms. Digitonin-permeabilized HeLa cells were incubated with 1 µM of Vpr, N17C74, and GST-GFP in the absence (-) or presence of 1 µM (for Vpr and GST-GFP) or 3 µM (for N17C74) of each of the recombinant Impα isoforms, Rch1, Qip1 and NPI-1. Cells were fixed in 3.7% formaldehyde and stained with Hoechst 33342 to show the position of the nucleus (right panel). After fixation, cells were analyzed by confocal laser scanning microscopy. Bar = 10 µm. (C) Fluorescence intensity of Vpr per surface area was quantified for at least 70 nuclei in the presence of the indicated concentrations of the Impα isoforms from three independent experiments. The bar shows the standard errors of measurements. (D) In vitro nuclear import assay for GST-GFP-Vpr was performed in the absence (-) or presence of 1 µM of the Impα isoforms. After fixation, cells were analyzed by confocal microscopy. Bar = 10 µm. (E) In vitro nuclear import assay for Vpr was performed in the absence (-) or presence of 1 µM of the Impα isoforms, and 1 µM of Impα isoforms with 1 µM Impβ. N17C74, as a control, was performed with 1 µM of Rch1 and 1 µM Impβ. After fixation, cells were analyzed by confocal microscopy. Bar = 10 µm. (F) Binding assay between Vpr and the Impα isoforms. Glutathione-Sepharose beads were coupled with the GST-Impα isoforms, Rch1, Qip1 and NPI-1 or GST alone, and were incubated with Vpr protein purified from 293T cells transfected with pCAGGS mammalian vectors encoding Flag-mRFP (mRFP), or Flag-mRFP-Flag-Vpr (mRFP-Vpr). The bound fractions and 1/20 of the input of mRFP-Vpr and mRFP were analyzed by immunoblotting with an anti-Flag M2 monoclonal antibody (MAb) (right panel). Twenty-five pmol of GST or GST-Impα isoforms were resolved by 10% SDS-PAGE and stained with CBB (left panel). The positions of mRFP and mRFP-Vpr are indicated.

    Article Snippet: Vpr proteins purified from 293T cells transfected with pCAGGS encoding mRFP or mRFP-Vpr were incubated with GST-protein conjugated beads for 2 h at 4o C. The beads were washed four times with 500 µl washing buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.2% NP-40 and 1 mM DTT] and bound proteins were eluted by incubation with sodium lauryl sulfate (SDS) sample buffer [100 mM sodium phosphate (pH 7.2), 1% SDS, 10% glycerol, 100 mM DTT and 0.001% bromophenol blue] at 98°C for 5 min. Eluted proteins were fractionated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and detected by Western blotting with anti-Flag M2 monoclonal antibody (MAb) (Sigma-Aldrich).

    Techniques: Purification, Recombinant, SDS Page, Staining, Incubation, Confocal Laser Scanning Microscopy, Fluorescence, In Vitro, Confocal Microscopy, Binding Assay, Transfection

    The TYK2 TK domain specifically mediates alternative STAT3 tyrosine phosphorylation. ( a ) HEK293T cells were transiently co-transfected with control plasmid (C.V.) or plasmids coding for Etagged-STAT3 WT or Y705F and FLAG-tagged JAK TK domains. STAT3 was immunoprecipitated with an anti-Etag antibody and the membrane was probed using general anti-phospho-tyrosine, anti-phospho-STAT3 (pY705) and anti-Etag antibodies (panels denoted IP). Total cell lysates were collected before immunoprecipitation, similar amounts of protein extracts were blotted for each condition, as quantified using a Bradford (Bio-Rad) assay and the membrane was probed with anti-phospho-STAT3 (pY705), anti-Etag, anti-β-actin and anti-FLAG antibodies (panels denoted INPUT). n = 2, representative figures. Full-length blots are presented in Supplementary Figs S6 –S 7 . Y640 phosphorylation affects STAT3 dimer formation. ( b ) SH2 domain of STAT3 (blue) with a bound phosphopeptide (pY705) of an interacting STAT3 molecule (beige). T708 and the phosphorylated Y705 of the phosphopeptide are indicated. The hydroxyl group of Y640 and Y657 (red) are in close proximity of the binding site for this phosphopeptide. ( c ) Phosphorylation of Y640 leads to a clash Model of the SH2 domain with a phosphorylated Y640. A STAT3 phosphopeptide was placed in the SH2 domain, as in panel A. This leads to clashes between T708 and the phosphate group of Y640, indicating that Y640 phosphorylation is likely to affect the binding mode with the pY705 phosphopeptide. ( d ) Alternative phosphorylation involving STAT3 Y640. HEK293T cells were transfected with increasing amounts of plasmids coding for FLAG-tagged TYK2 TK domain and different Etagged-STAT3 wt or mutants: STAT3Y705F; STAT3 Y640F; STAT3 Y640F Y705F; STAT3 Y657F; STAT3 Y657F Y705F. STAT3 was immunoprecipitated with an anti-Etag antibody and the membrane was probed using anti-phospho-tyrosine and anti-phospho-STAT3 (pY705) antibodies (panels denoted IP). For the input, total cell lysates were collected before immunoprecipitation and similar amounts of protein extracts were blotted for each condition, as quantified using a Bradford assay and the membrane was probed with anti-phospho-STAT3 (pY705), anti-Etag, anti-β-actin and anti-FLAG antibodies (panels denoted INPUT). n = 2, representative figures. Full-length blots are presented in Supplementary Figs S8 –S 9 .

    Journal: Scientific Reports

    Article Title: TYK2-induced phosphorylation of Y640 suppresses STAT3 transcriptional activity

    doi: 10.1038/s41598-017-15912-6

    Figure Lengend Snippet: The TYK2 TK domain specifically mediates alternative STAT3 tyrosine phosphorylation. ( a ) HEK293T cells were transiently co-transfected with control plasmid (C.V.) or plasmids coding for Etagged-STAT3 WT or Y705F and FLAG-tagged JAK TK domains. STAT3 was immunoprecipitated with an anti-Etag antibody and the membrane was probed using general anti-phospho-tyrosine, anti-phospho-STAT3 (pY705) and anti-Etag antibodies (panels denoted IP). Total cell lysates were collected before immunoprecipitation, similar amounts of protein extracts were blotted for each condition, as quantified using a Bradford (Bio-Rad) assay and the membrane was probed with anti-phospho-STAT3 (pY705), anti-Etag, anti-β-actin and anti-FLAG antibodies (panels denoted INPUT). n = 2, representative figures. Full-length blots are presented in Supplementary Figs S6 –S 7 . Y640 phosphorylation affects STAT3 dimer formation. ( b ) SH2 domain of STAT3 (blue) with a bound phosphopeptide (pY705) of an interacting STAT3 molecule (beige). T708 and the phosphorylated Y705 of the phosphopeptide are indicated. The hydroxyl group of Y640 and Y657 (red) are in close proximity of the binding site for this phosphopeptide. ( c ) Phosphorylation of Y640 leads to a clash Model of the SH2 domain with a phosphorylated Y640. A STAT3 phosphopeptide was placed in the SH2 domain, as in panel A. This leads to clashes between T708 and the phosphate group of Y640, indicating that Y640 phosphorylation is likely to affect the binding mode with the pY705 phosphopeptide. ( d ) Alternative phosphorylation involving STAT3 Y640. HEK293T cells were transfected with increasing amounts of plasmids coding for FLAG-tagged TYK2 TK domain and different Etagged-STAT3 wt or mutants: STAT3Y705F; STAT3 Y640F; STAT3 Y640F Y705F; STAT3 Y657F; STAT3 Y657F Y705F. STAT3 was immunoprecipitated with an anti-Etag antibody and the membrane was probed using anti-phospho-tyrosine and anti-phospho-STAT3 (pY705) antibodies (panels denoted IP). For the input, total cell lysates were collected before immunoprecipitation and similar amounts of protein extracts were blotted for each condition, as quantified using a Bradford assay and the membrane was probed with anti-phospho-STAT3 (pY705), anti-Etag, anti-β-actin and anti-FLAG antibodies (panels denoted INPUT). n = 2, representative figures. Full-length blots are presented in Supplementary Figs S8 –S 9 .

    Article Snippet: The following antibodies were used: mouse monoclonal anti-phosphotyrosine clone 4G10 (Millipore); anti mouse monoclonal anti-Etag (Phadia); mouse monoclonal anti-STAT3 (Cell Signaling Technology); rabbit polyclonal anti-phosphorylated Y705 STAT3 (Cell Signaling Technology); mouse monoclonal anti-β-actin (Sigma Aldrich); mouse monoclonal anti-FLAG (Sigma Aldrich); either peroxidase AffiniPure goat anti-mouse IgG or peroxidase AffiniPure goat anti-rabbit IgG (Jakson, Jakson ImmunoResearch) was used as secondary antibody.

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Bradford Assay

    (a) Deuteration levels and measured protection (b) for each time point of an epitope mapping experiment for the 3X FLAG-GFP / M2 anti-FLAG complex are shown for the +5 charge state of the peptide YFQGDYKDHDGDYKDHDIDYKDDDDKMVSKGEE, which contains the entire 3X FLAG tag.

    Journal: Analytical chemistry

    Article Title: Sub-Zero Temperature Chromatography for Reduced Back-Exchange and Improved Dynamic Range in Amide Hydrogen Deuterium Exchange Mass Spectrometry

    doi: 10.1021/ac302488h

    Figure Lengend Snippet: (a) Deuteration levels and measured protection (b) for each time point of an epitope mapping experiment for the 3X FLAG-GFP / M2 anti-FLAG complex are shown for the +5 charge state of the peptide YFQGDYKDHDGDYKDHDIDYKDDDDKMVSKGEE, which contains the entire 3X FLAG tag.

    Article Snippet: Human Fibrinopeptide A, equine Cytochrome C, ethylene glycol, formic acid, trifluoroacetic acid (TFA), ethylene glycol(OD)2 , D2 -formic acid, acetonitrile (ACN), formamide, dimethylformamide (DMF), methanol, and M2 anti-FLAG monoclonal antibody were purchased from Sigma Chemical Company (St. Louis, MO).

    Techniques: FLAG-tag

    Binding of Acp3 and KatA by reverse pull-down assay. His-KatA and Acp3-FLAG were expressed in PAO1. Acp3 co-purified with His-tagged KatA after nickel-affinity chromatography, as detected by immunoblotting with anti-FLAG M2 antibody. Empty vector (pEXHTB) is a negative control. The full image of the immunoblots are shown in Supplementary Figure S1 .

    Journal: Frontiers in Microbiology

    Article Title: Acyl Carrier Protein 3 Is Involved in Oxidative Stress Response in Pseudomonas aeruginosa

    doi: 10.3389/fmicb.2018.02244

    Figure Lengend Snippet: Binding of Acp3 and KatA by reverse pull-down assay. His-KatA and Acp3-FLAG were expressed in PAO1. Acp3 co-purified with His-tagged KatA after nickel-affinity chromatography, as detected by immunoblotting with anti-FLAG M2 antibody. Empty vector (pEXHTB) is a negative control. The full image of the immunoblots are shown in Supplementary Figure S1 .

    Article Snippet: SPA-tagged ACP proteins were detected by monoclonal anti-flag M2 (1:5000) (Sigma) and goat anti-mouse IgG-HRP (1:10,000) (Santa Cruz).

    Techniques: Binding Assay, Pull Down Assay, Purification, Affinity Chromatography, Plasmid Preparation, Negative Control, Western Blot