monoclonal anti flag m2 antibody Millipore Search Results


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  • 99
    Millipore monoclonal anti flag m2 antibody
    Cysteine trapping of the complex between ACKR3 and CXCL12. ( a ) CRS2 interactions detected using SDS–PAGE of purified ACKR3:CXCL12 complexes in the absence of reducing agent. Upper bands correspond to the crosslinked complex and lower bands to free receptor. Crosslinking of thermostabilized apocytochrome b562 (bRIL)-R197C-ACKR3 with Cys mutants in the N terminus of CXCL12. No crosslinking is seen for control samples N45C-CXCL12 or WT-CXCL12. ( b ) Crosslinking of Y7C-CXCL12 with ECL2 mutants of ACKR3. R197C-ACKR3 efficiently crosslinks with the chemokine, while no crosslinking is detected for F199C-ACKR3 or WT-ACKR3. ( c ) Schematic summary of disulfide trapping experiments involving ECL2 of ACKR3 and the N terminus of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Crosslinked sites are connected with solid lines, where the line thickness is proportional to crosslinking efficiency. Dashed lines indicate unsuccessful cross-links. ( d ) Cysteine trapping of CRS1 interactions detected by western blot of ACKR3:CXCL12 complexes under non-reducing conditions. <t>ACKR3-FLAG</t> and CXCL12-HA were detected with primary <t>anti-FLAG</t> and anti-HA antibodies and fluorescent secondary antibodies; 16, 17 and 20 refer to the residue in CXCL12 mutated to Cys. ( e ) Schematic representation of CRS1 disulfide trapping experiments. Mutation of C21 or C26 to Ser gives a free Cys side chain that can be crosslinked to residue 20 of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Full, uncropped versions of the gels and blots are shown in Supplementary Fig. 7 .
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 40465 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti flag m2 peroxidase hrp antibody
    The γ subunits associate with Ca v 1.2 in the presence of α2/δ-1 subunit. A–D ) The α1c, β1b, and γ4 ( A ), γ6 ( B ), γ7 ( C ), and γ8 ( D ) subunits were expressed in HEK293 cells in the absence (−) or presence (+) of the α2/δ1 subunit. Lysates were prepared, and γ subunits were immunoprecipitated (IP) with <t>anti-FLAG</t> antibody. Negative controls were preimmune serum and cells without α2/δ-1 coexpression. Top panels: immunoblot with anti-α2 antibody. Bottom panels: immunoblot with <t>HRP-conjugated,</t> anti-FLAG antibody. Cont, control. E ) Same lysates as in ( B ). The α1c subunit was immunoprecipitated with an anti-α1c antibody. Top panel: immunoblot with HRP-conjugated, anti-FLAG antibody. Bottom panel: immunoblot with anti-α1c antibody. Results are representative of ≥3 similar experiments for each condition. F ) Coimmunoprecipitation of α1c and γ6 from mouse heart. The α1c immunoprecipitates of Triton X-100 extracts (1.5 mg) of mouse heart homogenates were size-fractionated on SDS-PAGE, transferred to nitrocellulose, and probed with anti-γ6 antibody. HEK cells were transfected with FLAG-γ6 and α1c. The α1c immunoprecipitates were probed with anti-γ6 antibody. The α1c subunit is truncated in heart (bottom arrow). Results are representative of 3 similar experiments.
    Monoclonal Anti Flag M2 Peroxidase Hrp Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2 peroxidase hrp antibody/product/Millipore
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    99
    Millipore anti flag
    <t>Clusterin</t> binds APP with intact and wild-type JH intracellularly. ( A ) Coimmunoprecipitation (CoIP) of endogenous APP and clusterin from brain homogenate. Anti-GAPDH was used as control IP antibody. ( B ) <t>Clusterin-FLAG</t> and APP WT without tag were either coexpressed or separately expressed in HEK293 cells. Cells with both clusterin and APP were directly lysed (coexpression), and cells with only clusterin or APP overexpression were lysed and the lysates were combined (pooled lysates). After IP with FLAG-agarose, the precipitated proteins were blotted with C20 antibody for APP and FLAG antibody for clusterin. ( C ) Schematic diagram showing the position of the JH in different CTFs. ( D ) C99, C83, and C80 were coexpressed with clusterin-FLAG in HEK293, and the lysates were subjected to anti-FLAG CoIP. C20 antibody was used to detect CTFs in the precipitates. ( E ) Clusterin-FLAG was coexpressed with wild-type C99 (C99 WT ), F615P containing C99 (C99 F615P ), and Flemish mutation containing C99 (C99 Fle ) in HEK293 cells, and cell lysates were immunoprecipitated using anti-FLAG antibody. Precipitates were Western blotted using C20 for C99 variants and anti-FLAG for clusterin. ( F ) CoIP of clusterin-FLAG with APP variants in HEK293 cells. IP was by anti-FLAG antibody, and APP detection was by C20 antibody.
    Anti Flag, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 23400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 712 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore flag peptide
    Analysis of <t>AKT3</t> activity in vitro. ( A ) The primary structure of AKT3 showing the relative positions of the pleckstrin homology (PH) domain for lipid binding the catalytic kinase domain and C-terminal (C-ter) region. Mutations identified to date are shown along with the numbers of patients with these mutations in brackets. ( B ) Catalytic kinase domain and C-terminal localizing patient-derived AKT3 mutations are associated with elevated kinase activity. Ectopically expressed wild-type (WT) AKT, a kinase dead variant K177M, the E17K activating pleckstrin homology domain mutant and various patient mutants were assessed for kinase activity using a GSK3β peptide as a substrate in an ex vivo kinase assay. The upper panel shows immune detection of phosphorylated GSK3β peptide following western blotting with anti-phospho-GSK3β (Ser9/Ser21) antibody. The patient mutants all exhibit elevated phospho-activity compared to wild-type. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal (a.u. = arbitrary units). Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. ( C ) Pleckstrin homology domain localizing patient mutations are associated with elevated kinase activity and altered phospholipid-binding profile. Left panels show western blot analysis of phospho-GSK3β (Ser9/Ser21) of ectopically expressed wild-type, K177M kinase dead and three pleckstrin homology domain patient mutants; E17K, N53K and F54Y. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal. Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. The bottom panels depict PIP-membranes seeded with various lipids and phospholipids for dot blot binding analysis. Ectopically expressed <t>FLAG-tagged</t> wild-type and AKT3 pleckstrin homology domain mutants were incubated with the PIP Strips and bound protein detected by western blotting using anti-FLAG. All three pleckstrin homology domain mutants exhibit altered and elevated binding to specific phospholipids compared to wild-type. DMEG = dysplastic megalencephaly; HMEG = hemimegalencephaly; LPA = lysophophatidic acid; LPC = lysophosphocholine; MEG = megalencephaly; P = phosphate; PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PMG = polymicrogryria; PS = phosphatidylserine; PtdIns = phosphatidylinositol; S1P = sphingosine-1-phosphate.
    Flag Peptide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16747 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cysteine trapping of the complex between ACKR3 and CXCL12. ( a ) CRS2 interactions detected using SDS–PAGE of purified ACKR3:CXCL12 complexes in the absence of reducing agent. Upper bands correspond to the crosslinked complex and lower bands to free receptor. Crosslinking of thermostabilized apocytochrome b562 (bRIL)-R197C-ACKR3 with Cys mutants in the N terminus of CXCL12. No crosslinking is seen for control samples N45C-CXCL12 or WT-CXCL12. ( b ) Crosslinking of Y7C-CXCL12 with ECL2 mutants of ACKR3. R197C-ACKR3 efficiently crosslinks with the chemokine, while no crosslinking is detected for F199C-ACKR3 or WT-ACKR3. ( c ) Schematic summary of disulfide trapping experiments involving ECL2 of ACKR3 and the N terminus of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Crosslinked sites are connected with solid lines, where the line thickness is proportional to crosslinking efficiency. Dashed lines indicate unsuccessful cross-links. ( d ) Cysteine trapping of CRS1 interactions detected by western blot of ACKR3:CXCL12 complexes under non-reducing conditions. ACKR3-FLAG and CXCL12-HA were detected with primary anti-FLAG and anti-HA antibodies and fluorescent secondary antibodies; 16, 17 and 20 refer to the residue in CXCL12 mutated to Cys. ( e ) Schematic representation of CRS1 disulfide trapping experiments. Mutation of C21 or C26 to Ser gives a free Cys side chain that can be crosslinked to residue 20 of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Full, uncropped versions of the gels and blots are shown in Supplementary Fig. 7 .

    Journal: Nature Communications

    Article Title: Structural basis of ligand interaction with atypical chemokine receptor 3

    doi: 10.1038/ncomms14135

    Figure Lengend Snippet: Cysteine trapping of the complex between ACKR3 and CXCL12. ( a ) CRS2 interactions detected using SDS–PAGE of purified ACKR3:CXCL12 complexes in the absence of reducing agent. Upper bands correspond to the crosslinked complex and lower bands to free receptor. Crosslinking of thermostabilized apocytochrome b562 (bRIL)-R197C-ACKR3 with Cys mutants in the N terminus of CXCL12. No crosslinking is seen for control samples N45C-CXCL12 or WT-CXCL12. ( b ) Crosslinking of Y7C-CXCL12 with ECL2 mutants of ACKR3. R197C-ACKR3 efficiently crosslinks with the chemokine, while no crosslinking is detected for F199C-ACKR3 or WT-ACKR3. ( c ) Schematic summary of disulfide trapping experiments involving ECL2 of ACKR3 and the N terminus of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Crosslinked sites are connected with solid lines, where the line thickness is proportional to crosslinking efficiency. Dashed lines indicate unsuccessful cross-links. ( d ) Cysteine trapping of CRS1 interactions detected by western blot of ACKR3:CXCL12 complexes under non-reducing conditions. ACKR3-FLAG and CXCL12-HA were detected with primary anti-FLAG and anti-HA antibodies and fluorescent secondary antibodies; 16, 17 and 20 refer to the residue in CXCL12 mutated to Cys. ( e ) Schematic representation of CRS1 disulfide trapping experiments. Mutation of C21 or C26 to Ser gives a free Cys side chain that can be crosslinked to residue 20 of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Full, uncropped versions of the gels and blots are shown in Supplementary Fig. 7 .

    Article Snippet: Mouse anti-Flag M2 primary antibody (1:5,000 dilution, F3165; Sigma Aldrich) and IRDye 680-conjugated donkey anti-mouse IgG (1:20,000 dilution; LI-COR Biosciences) secondary antibody were used to detect the FLAG-tagged receptor.

    Techniques: SDS Page, Purification, Footprinting, Western Blot, Mutagenesis

    SsSSVP1 is a Sclerotinia- and Botryotinia- specific, cysteine-rich, small, secreted protein. ( A ) A predicted structure diagram of SsSSVP1 which comprises 163 aa. A putative N-terminal SP (aa 1 to 17) and the position of the eight cysteine residues of SsSSVP1 are present (C 38 , C 44 , C 54 , C 79 , C 81 , C 92 , C 95 and C 117 ). ( B ) Multiple alignments indicate the homologs of SsSSVP1 are only present in Sclerotinia - and Botryotinia in the organisms sequenced so far and the eight cysteine residues are conserved in these homologs. Red rectangle labels the sites of the eight cysteine residues in multiple alignments. Protein sequences from top to bottom are derived from B . cinerea T4, B . cinerea B05.10, B . cinerea BcDW1, Sclerotinia borealis F-4157 and S . sclerotiorum Ep-1PNA367 respectively. The protein sequences of SsSSVP1 in S . sclerotiorum Ep-1PNA367 and 1980 are the same. ( C ) Western blot analysis with total proteins isolated from the liquid CM culture of the wild-type strain and SsSSVP1-FLAG engineered strains. SDS-polyacrylamide gel electrophoresis shows the equal loading amount of proteins used for the west blot analysis. Horseradish peroxidase conjugated secondary antibody detected an approximate 17 kDa band in SsSSVP1-FLAG engineered strains, but not in the wild-type strain.

    Journal: PLoS Pathogens

    Article Title: A Small Secreted Virulence-Related Protein Is Essential for the Necrotrophic Interactions of Sclerotinia sclerotiorum with Its Host Plants

    doi: 10.1371/journal.ppat.1005435

    Figure Lengend Snippet: SsSSVP1 is a Sclerotinia- and Botryotinia- specific, cysteine-rich, small, secreted protein. ( A ) A predicted structure diagram of SsSSVP1 which comprises 163 aa. A putative N-terminal SP (aa 1 to 17) and the position of the eight cysteine residues of SsSSVP1 are present (C 38 , C 44 , C 54 , C 79 , C 81 , C 92 , C 95 and C 117 ). ( B ) Multiple alignments indicate the homologs of SsSSVP1 are only present in Sclerotinia - and Botryotinia in the organisms sequenced so far and the eight cysteine residues are conserved in these homologs. Red rectangle labels the sites of the eight cysteine residues in multiple alignments. Protein sequences from top to bottom are derived from B . cinerea T4, B . cinerea B05.10, B . cinerea BcDW1, Sclerotinia borealis F-4157 and S . sclerotiorum Ep-1PNA367 respectively. The protein sequences of SsSSVP1 in S . sclerotiorum Ep-1PNA367 and 1980 are the same. ( C ) Western blot analysis with total proteins isolated from the liquid CM culture of the wild-type strain and SsSSVP1-FLAG engineered strains. SDS-polyacrylamide gel electrophoresis shows the equal loading amount of proteins used for the west blot analysis. Horseradish peroxidase conjugated secondary antibody detected an approximate 17 kDa band in SsSSVP1-FLAG engineered strains, but not in the wild-type strain.

    Article Snippet: About 5 μl ANTI-FLAG M2 monoclonal antibody (Sigma, Saint Louis, Missouri, USA) was added to 1 ml protein extracts and then was incubated at room temperature for 2 hours.

    Techniques: Derivative Assay, Western Blot, Isolation, Polyacrylamide Gel Electrophoresis

    Interaction of WDRPUH with HSP70, CCT1-δ, CCT1-α, or BRCA2. HEK293 cells were transiently transfected with p3xFLAG-WDRPUH or p3xFLAG empty vector (Mock). Cell lysates were immunoprecipitated with anti-FLAG M2 antibody (mouse) conjugated with agarose beads or specific antibodies to interacting protein. (A and B) Coimmunoprecipitation of FLAG-WDRPUH and endogenous HSP70. Whole cell extracts from the cells exogenously expressing FLAG-WDRPUH were used as positive controls. (C) Coimmunoprecipitation of FLAG-WDRPUH and endogenous CCT1-δ. (D) Coimmunoprecipitation of FLAG-WDRPUH and endogenous CCT1-α. (E and F) Coimmunoprecipitation of FLAG-WDRPUH and endogenous BRCA2.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: WDRPUH, A Novel WD-Repeat-Containing Protein, Is Highly Expressed in Human Hepatocellular Carcinoma and Involved in Cell Proliferation 1WDRPUH, A Novel WD-Repeat-Containing Protein, Is Highly Expressed in Human Hepatocellular Carcinoma and Involved in Cell Proliferation 1 *

    doi:

    Figure Lengend Snippet: Interaction of WDRPUH with HSP70, CCT1-δ, CCT1-α, or BRCA2. HEK293 cells were transiently transfected with p3xFLAG-WDRPUH or p3xFLAG empty vector (Mock). Cell lysates were immunoprecipitated with anti-FLAG M2 antibody (mouse) conjugated with agarose beads or specific antibodies to interacting protein. (A and B) Coimmunoprecipitation of FLAG-WDRPUH and endogenous HSP70. Whole cell extracts from the cells exogenously expressing FLAG-WDRPUH were used as positive controls. (C) Coimmunoprecipitation of FLAG-WDRPUH and endogenous CCT1-δ. (D) Coimmunoprecipitation of FLAG-WDRPUH and endogenous CCT1-α. (E and F) Coimmunoprecipitation of FLAG-WDRPUH and endogenous BRCA2.

    Article Snippet: Proteins were separated by 10% SDS-PAGE and immunoblotted with mouse anti-FLAG M2 (Sigma) antibody, anti-HSP70 antibody (Stressgen, Victoria, Canada), anti-chaperonin-containing TCP-1 (CCT1) delta subunity (Santa Cruz Biotechnology, Santa Cruz, CA), anti-CTT1-alpha subunity (Stressgen), or anti-BRCA2 (Stressgen).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing

    Eya1 interacts with the Sipl1-related protein Rbck1. (A) Rbck1 protein domain structure. RING, really interesting new gene; IBR, in-between RING; C/H, cysteine/histidine rich. (B) Eya1 and Rbck1 bind directly to each other. A GST pulldown assay was performed by incubating in vitro -synthesized HA-Eya1 with GST-Rbck1 or GST alone as a control. HA-Eya1 was detected by immunoblotting using anti-HA 6E2 antibody (top). The input levels of GST fusion proteins were determined by SDS-PAGE and Coomassie blue staining (bottom). The asterisk indicates bacterial protein copurifying with GST. (C) Eya1 interacts with Rbck1 in mammalian cells. For coimmunoprecipitation analysis, Cos-7 cells were transfected with expression constructs for HA-Eya1 and Flag-Rbck1. After lysis of the cells, HA-Eya1 was precipitated using anti-HA 12CA5 antibody. Whole-cell extracts and immunocomplexes were analyzed by SDS-PAGE and immunoblotting. HA-Eya1 was detected with anti-HA 6E2 antibody, and Flag-Sipl1 was detected with anti-Flag M2 antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿

    doi: 10.1128/MCB.01645-09

    Figure Lengend Snippet: Eya1 interacts with the Sipl1-related protein Rbck1. (A) Rbck1 protein domain structure. RING, really interesting new gene; IBR, in-between RING; C/H, cysteine/histidine rich. (B) Eya1 and Rbck1 bind directly to each other. A GST pulldown assay was performed by incubating in vitro -synthesized HA-Eya1 with GST-Rbck1 or GST alone as a control. HA-Eya1 was detected by immunoblotting using anti-HA 6E2 antibody (top). The input levels of GST fusion proteins were determined by SDS-PAGE and Coomassie blue staining (bottom). The asterisk indicates bacterial protein copurifying with GST. (C) Eya1 interacts with Rbck1 in mammalian cells. For coimmunoprecipitation analysis, Cos-7 cells were transfected with expression constructs for HA-Eya1 and Flag-Rbck1. After lysis of the cells, HA-Eya1 was precipitated using anti-HA 12CA5 antibody. Whole-cell extracts and immunocomplexes were analyzed by SDS-PAGE and immunoblotting. HA-Eya1 was detected with anti-HA 6E2 antibody, and Flag-Sipl1 was detected with anti-Flag M2 antibody.

    Article Snippet: Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

    Techniques: GST Pulldown Assay, In Vitro, Synthesized, SDS Page, Staining, Transfection, Expressing, Construct, Lysis

    Sipl1 is a novel interaction partner of Eya1. (A) Sipl1 protein domain structure. Ubl, ubiquitin-like; ZnF, zinc finger. (B) Eya1 and Sipl1 interact directly with each other. GST pulldown assay with in vitro -synthesized HA-Eya1 and recombinant GST-Sipl1 or GST as a control. HA-Eya1 was visualized by immunoblotting (IB) with anti-HA 6E2 antibody (top) and GST fusion proteins by SDS-PAGE and Coomassie blue staining (bottom). The asterisk indicates bacterial protein copurifying with GST. (C) Sipl1 and Eya1 interact in mammalian cells. Cos-7 cells were transfected with expression constructs for HA-Eya1 and Flag-Sipl1. At 48 h posttransfection, cells were lysed, and HA-Eya1 was precipitated with anti-HA 12CA5 antibody. Cell lysates before immunoprecipitation (IP) and precipitated complexes were analyzed by immunoblotting using anti-HA 6E2 antibody for the detection of HA-Eya1 and anti-Flag M2 antibody for the detection of Flag-Sipl1. (D) Sipl1 interacts with Eya1 and Eya2 but not with Eya3 and Eya4. Cotransformation of S. cerevisiae KFY1 with constructs encoding the C-terminal fragments of Eya1-4 and pGADT7-Sipl1 or empty vector. In each case, the interaction strength was determined by β-Gal liquid assay from three pooled colonies in triplicate. (E) Eya1 binds to the conserved Ubl domain of Sipl1. S. cerevisiae KFY1 was transformed with a pGBT9 vector encoding the C terminus of Eya1 and pGADT7 encoding the indicated Sipl1 deletion fragments. For each sample, a β-Gal liquid assay was performed with 3 pooled colonies and measured in triplicate. The data represent the means and standard deviations from the results of one representative experiment. Expression of the yeast constructs was confirmed by protein extraction and immunoblot analysis (insets).

    Journal: Molecular and Cellular Biology

    Article Title: Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development ▿

    doi: 10.1128/MCB.01645-09

    Figure Lengend Snippet: Sipl1 is a novel interaction partner of Eya1. (A) Sipl1 protein domain structure. Ubl, ubiquitin-like; ZnF, zinc finger. (B) Eya1 and Sipl1 interact directly with each other. GST pulldown assay with in vitro -synthesized HA-Eya1 and recombinant GST-Sipl1 or GST as a control. HA-Eya1 was visualized by immunoblotting (IB) with anti-HA 6E2 antibody (top) and GST fusion proteins by SDS-PAGE and Coomassie blue staining (bottom). The asterisk indicates bacterial protein copurifying with GST. (C) Sipl1 and Eya1 interact in mammalian cells. Cos-7 cells were transfected with expression constructs for HA-Eya1 and Flag-Sipl1. At 48 h posttransfection, cells were lysed, and HA-Eya1 was precipitated with anti-HA 12CA5 antibody. Cell lysates before immunoprecipitation (IP) and precipitated complexes were analyzed by immunoblotting using anti-HA 6E2 antibody for the detection of HA-Eya1 and anti-Flag M2 antibody for the detection of Flag-Sipl1. (D) Sipl1 interacts with Eya1 and Eya2 but not with Eya3 and Eya4. Cotransformation of S. cerevisiae KFY1 with constructs encoding the C-terminal fragments of Eya1-4 and pGADT7-Sipl1 or empty vector. In each case, the interaction strength was determined by β-Gal liquid assay from three pooled colonies in triplicate. (E) Eya1 binds to the conserved Ubl domain of Sipl1. S. cerevisiae KFY1 was transformed with a pGBT9 vector encoding the C terminus of Eya1 and pGADT7 encoding the indicated Sipl1 deletion fragments. For each sample, a β-Gal liquid assay was performed with 3 pooled colonies and measured in triplicate. The data represent the means and standard deviations from the results of one representative experiment. Expression of the yeast constructs was confirmed by protein extraction and immunoblot analysis (insets).

    Article Snippet: Mouse monoclonal anti-Flag M2 (Sigma), anti-HA 6E2 and anti-c-Myc 9B11 (Cell Signaling), and anti-β-actin (ab8224; Abcam) antibodies were purchased from the indicated manufacturers.

    Techniques: GST Pulldown Assay, In Vitro, Synthesized, Recombinant, SDS Page, Staining, Transfection, Expressing, Construct, Immunoprecipitation, Plasmid Preparation, Transformation Assay, Protein Extraction

    Phosphorylation of NS1 T49 leads to reduced vRNA, RIG‐I and TRIM25 binding as well as structural destabilization of the RBD. A. A549 cells were transfected with pcDNA3 plasmids containing wt NS1 or NS1 with indicated mutations or were mock transfected. Cell lysates were subjected to immunoprecipitations 24 h p.i. using mouse anti‐NS1 antibody. Washed beads were incubated with vRNA and RNA bound to immunocomplexes was extracted. The relative amount of viral NS1 mRNA was determined by qRT‐PCR as described previously (Habjan et al ., 2008 ). Data represents mean ± SD of two independently repeated experiments. B. Comparative binding scores of RNA to RBD T49E, T49A and T49 as calculated using random forest model from the RNA–protein interaction prediction (RPISeq) tool (Muppirala et al ., 2011 ). The RNA sequence was obtained from Protein Data Bank (PDB) ID 2ZKO (Cheng et al ., 2009 ). C. Structural simulation on 2ZKO structure employing DUET (Pires et al ., 2014b ) was predicted as destabilizing (−0.17 Kcal/mol) resulting in an unfavourable RNA binding ability. The figure shows the stereo‐chemical effect of E49 compared with T49. The RNA helix is shown in yellow, while the two monomers of the NS1 dimer are shown in green and blue. NS1 residues T49 and E49 are shown in red and purple respectively. Molecular graphic simulation was performed using Bioblender (Andrei et al ., 2012 ). D. For analysis of NS1‐RIG‐I interaction, HEK293 cells transiently expressing FLAG‐tagged RIG‐I and the indicated NS1 proteins were subjected to crosslinking with DSP after 48 h, followed by quenching with glycine. Control cells were mock transfected. FLAG‐tagged RIG‐I was immunoprecipitated with anti‐FLAG M2 antibody. Detection of FLAG‐tagged RIG‐I and co‐precipitated NS1 protein was performed by Western blotting. Detection of FLAG‐tagged RIG‐I and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels. E. For analysis of NS1‐TRIM25 interaction HEK293 cells were infected with PR8/NS1‐T49, PR8/NS1‐T49A or PR8/NS1‐T49E (MOI of 1, 5 or 10 respectively) or were mock infected (control).Eighteen h p.i. cells were lysed and lysates subjected to immunoprecipitation with mouse anti‐TRIM25 antibody. TRIM25 and the co‐precipitated NS1 were detected by Western blotting using mouse anti‐TRIM25 antibody or mouse anti‐NS1 antibody. Detection of TRIM25 and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels.

    Journal: Cellular Microbiology

    Article Title: Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity) Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity

    doi: 10.1111/cmi.12559

    Figure Lengend Snippet: Phosphorylation of NS1 T49 leads to reduced vRNA, RIG‐I and TRIM25 binding as well as structural destabilization of the RBD. A. A549 cells were transfected with pcDNA3 plasmids containing wt NS1 or NS1 with indicated mutations or were mock transfected. Cell lysates were subjected to immunoprecipitations 24 h p.i. using mouse anti‐NS1 antibody. Washed beads were incubated with vRNA and RNA bound to immunocomplexes was extracted. The relative amount of viral NS1 mRNA was determined by qRT‐PCR as described previously (Habjan et al ., 2008 ). Data represents mean ± SD of two independently repeated experiments. B. Comparative binding scores of RNA to RBD T49E, T49A and T49 as calculated using random forest model from the RNA–protein interaction prediction (RPISeq) tool (Muppirala et al ., 2011 ). The RNA sequence was obtained from Protein Data Bank (PDB) ID 2ZKO (Cheng et al ., 2009 ). C. Structural simulation on 2ZKO structure employing DUET (Pires et al ., 2014b ) was predicted as destabilizing (−0.17 Kcal/mol) resulting in an unfavourable RNA binding ability. The figure shows the stereo‐chemical effect of E49 compared with T49. The RNA helix is shown in yellow, while the two monomers of the NS1 dimer are shown in green and blue. NS1 residues T49 and E49 are shown in red and purple respectively. Molecular graphic simulation was performed using Bioblender (Andrei et al ., 2012 ). D. For analysis of NS1‐RIG‐I interaction, HEK293 cells transiently expressing FLAG‐tagged RIG‐I and the indicated NS1 proteins were subjected to crosslinking with DSP after 48 h, followed by quenching with glycine. Control cells were mock transfected. FLAG‐tagged RIG‐I was immunoprecipitated with anti‐FLAG M2 antibody. Detection of FLAG‐tagged RIG‐I and co‐precipitated NS1 protein was performed by Western blotting. Detection of FLAG‐tagged RIG‐I and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels. E. For analysis of NS1‐TRIM25 interaction HEK293 cells were infected with PR8/NS1‐T49, PR8/NS1‐T49A or PR8/NS1‐T49E (MOI of 1, 5 or 10 respectively) or were mock infected (control).Eighteen h p.i. cells were lysed and lysates subjected to immunoprecipitation with mouse anti‐TRIM25 antibody. TRIM25 and the co‐precipitated NS1 were detected by Western blotting using mouse anti‐TRIM25 antibody or mouse anti‐NS1 antibody. Detection of TRIM25 and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels.

    Article Snippet: Finally, the cells were lysed with RIPA buffer and RIG‐I was immunoprecipitated with mouse anti‐FLAG M2 antibody (Sigma‐Aldrich) coupled to protein G agarose beads (Roche) overnight at 4°C.

    Techniques: Binding Assay, Transfection, Incubation, Quantitative RT-PCR, Sequencing, RNA Binding Assay, Expressing, Immunoprecipitation, Western Blot, Infection

    (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

    T cell multiepitopic B (TMEP-B) design, construction of pcDNA-TMEP-B plasmid, and TMEP-B expression analysis by Western blot. ( A ) Scheme of TMEP-B protein. ( B ) Map of the plasmid pcDNA-TMEP-B. ( C ) Expression of TMEP-B construct by Western blot. The 293T cells were mock-infected or infected with 5 pfu/cell of Western Reserve (WR) or vaccinia virus (VACV) that expresses the T7 RNA polymerase (VT7) viruses, and transfected 1 h later with 5 μg of pcDNA-TMEP-B or pMax-GFP. At 6 h post-infection, cells were harvested and lysed in Laemmli buffer with mercapoethanol and cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody to evaluate TMEP-B expression.

    Journal: Viruses

    Article Title: Potent HIV-1-Specific CD8 T Cell Responses Induced in Mice after Priming with a Multiepitopic DNA-TMEP and Boosting with the HIV Vaccine MVA-B

    doi: 10.3390/v10080424

    Figure Lengend Snippet: T cell multiepitopic B (TMEP-B) design, construction of pcDNA-TMEP-B plasmid, and TMEP-B expression analysis by Western blot. ( A ) Scheme of TMEP-B protein. ( B ) Map of the plasmid pcDNA-TMEP-B. ( C ) Expression of TMEP-B construct by Western blot. The 293T cells were mock-infected or infected with 5 pfu/cell of Western Reserve (WR) or vaccinia virus (VACV) that expresses the T7 RNA polymerase (VT7) viruses, and transfected 1 h later with 5 μg of pcDNA-TMEP-B or pMax-GFP. At 6 h post-infection, cells were harvested and lysed in Laemmli buffer with mercapoethanol and cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody to evaluate TMEP-B expression.

    Article Snippet: At 6 h post-infection, cells were harvested, washed with PBS and lysed in Laemmli buffer with β-mercaptoethanol; cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody (1:1000; Sigma-Aldrich) to evaluate TMEP-B expression.

    Techniques: Plasmid Preparation, Expressing, Western Blot, Construct, Infection, Transfection, SDS Page

    Polyubiquitination is required for IFNAR1 degradation but is dispensable for the phosphorylation of IFNAR1 and its interaction with βTrcp as well as for assembly of the SCF βTrcp E3 ubiquitin ligase. (A) Treatment of cells with IFN stimulates the degradation of endogenous IFNAR1, which was measured as IFNAR1 levels in lysates from cells treated by cycloheximide (a cycloheximide chase) detected by immunoprecipitation/immunoblotting using anti-IFNAR1 antibodies. Control precipitation (NS) was performed using an isotype monoclonal antibody. Positions of fully glycosylated mature IFNAR1 ( Ragimbeau et al., 2003 ) and heavy chain of Igs are indicated by the arrows. (B) Degradation of endogenous IFNAR1 in the presence of IFN-α was analyzed by cycloheximide chase in as in A. (C) Degradation of exogenous IFNAR1 in cells coexpressed with either wild-type ubiquitin or K0 mutant analyzed by cycloheximide chase in the presence of IFN-α using anti-Flag antibody. Results are graphed as a percentage of IFNAR1 remaining at the indicated time points of a chase. (D) Ubiquitination of endogenous IFNAR1 in the IFN-α–treated cells expressing either wild-type or K0 ubiquitin mutant was assessed as in Fig. 1 A . (E) Ser535 phosphorylation of endogenous IFNAR1 in cells expressing either wild-type or K0 ubiquitin mutant and treated or not treated with IFN-α was assessed by immunoprecipitation followed by immunoblotting using the indicated antibodies. (F and G) Effects of K0 expression on the recruitment of myc-tagged βTrcp to either Flag-tagged IFNAR1 (F) or Flag-tagged Cullin1 (G) were analyzed by coimmunoprecipitation followed by immunoblotting using the indicated antibodies. WCE, whole cell extract.

    Journal: The Journal of Cell Biology

    Article Title: Site-specific ubiquitination exposes a linear motif to promote interferon-? receptor endocytosis

    doi: 10.1083/jcb.200706034

    Figure Lengend Snippet: Polyubiquitination is required for IFNAR1 degradation but is dispensable for the phosphorylation of IFNAR1 and its interaction with βTrcp as well as for assembly of the SCF βTrcp E3 ubiquitin ligase. (A) Treatment of cells with IFN stimulates the degradation of endogenous IFNAR1, which was measured as IFNAR1 levels in lysates from cells treated by cycloheximide (a cycloheximide chase) detected by immunoprecipitation/immunoblotting using anti-IFNAR1 antibodies. Control precipitation (NS) was performed using an isotype monoclonal antibody. Positions of fully glycosylated mature IFNAR1 ( Ragimbeau et al., 2003 ) and heavy chain of Igs are indicated by the arrows. (B) Degradation of endogenous IFNAR1 in the presence of IFN-α was analyzed by cycloheximide chase in as in A. (C) Degradation of exogenous IFNAR1 in cells coexpressed with either wild-type ubiquitin or K0 mutant analyzed by cycloheximide chase in the presence of IFN-α using anti-Flag antibody. Results are graphed as a percentage of IFNAR1 remaining at the indicated time points of a chase. (D) Ubiquitination of endogenous IFNAR1 in the IFN-α–treated cells expressing either wild-type or K0 ubiquitin mutant was assessed as in Fig. 1 A . (E) Ser535 phosphorylation of endogenous IFNAR1 in cells expressing either wild-type or K0 ubiquitin mutant and treated or not treated with IFN-α was assessed by immunoprecipitation followed by immunoblotting using the indicated antibodies. (F and G) Effects of K0 expression on the recruitment of myc-tagged βTrcp to either Flag-tagged IFNAR1 (F) or Flag-tagged Cullin1 (G) were analyzed by coimmunoprecipitation followed by immunoblotting using the indicated antibodies. WCE, whole cell extract.

    Article Snippet: Cells were washed, blocked, and incubated with anti-IFNAR1 antibody (AA3 for endogenous IFNAR1) or anti-FLAG antibody (monoclonal Flag M2 for exogenous IFNAR1 and its mutants; Sigma-Aldrich) for 1 h, washed, and incubated with HRP-conjugated goat anti–mouse secondary antibody (Invitrogen) for 1 h. After extensive washing, cells were incubated with AmplexRed Ultra Reagent (10-acetyl-3,7-dihydroxyphenoxazine; Invitrogen).

    Techniques: Immunoprecipitation, Mutagenesis, Expressing

    Role of ubiquitination in IFNAR1 internalization. (A) Effect of IFN-α (black squares) on the internalization of endogenous IFNAR1 measured by a fluorescence assay. All other experiments were performed in the presence of IFN-α. (B and C) Effect of βTrcp2 knockdown (shBTR) on IFNAR1 internalization observed by fluorescence assay (B; white squares) or biotinylation assay (C). (C) The internalized, biotinylated IFNAR1 analyzed by immunoblotting using anti-Flag antibody appears at the time points indicated in minutes on the top. 100% indicates the sample that represents the total amount of biotinylated IFNAR1. Quantification of these panels was calculated as (signal at time point X – signal at time point 0)/signal at 100%. (D and E) Comparison of the internalization of wild-type IFNAR1 (black squares) and its ubiquitination-deficient mutants IFNAR1 SA and IFNAR1 SA-RPT (D; white squares and diamonds, respectively) and IFNAR1 KR (E; triangles) that was assessed by biotinylation assays. NB, nonbiotinylated control. (F) Effect of expression of the indicated ubiquitin mutants on the internalization of IFNAR1 assessed by fluorescent assay. Data are depicted as the percentage of IFNAR1 internalization detected in cells expressing wild-type ubiquitin (100%) at 15 min. *, P

    Journal: The Journal of Cell Biology

    Article Title: Site-specific ubiquitination exposes a linear motif to promote interferon-? receptor endocytosis

    doi: 10.1083/jcb.200706034

    Figure Lengend Snippet: Role of ubiquitination in IFNAR1 internalization. (A) Effect of IFN-α (black squares) on the internalization of endogenous IFNAR1 measured by a fluorescence assay. All other experiments were performed in the presence of IFN-α. (B and C) Effect of βTrcp2 knockdown (shBTR) on IFNAR1 internalization observed by fluorescence assay (B; white squares) or biotinylation assay (C). (C) The internalized, biotinylated IFNAR1 analyzed by immunoblotting using anti-Flag antibody appears at the time points indicated in minutes on the top. 100% indicates the sample that represents the total amount of biotinylated IFNAR1. Quantification of these panels was calculated as (signal at time point X – signal at time point 0)/signal at 100%. (D and E) Comparison of the internalization of wild-type IFNAR1 (black squares) and its ubiquitination-deficient mutants IFNAR1 SA and IFNAR1 SA-RPT (D; white squares and diamonds, respectively) and IFNAR1 KR (E; triangles) that was assessed by biotinylation assays. NB, nonbiotinylated control. (F) Effect of expression of the indicated ubiquitin mutants on the internalization of IFNAR1 assessed by fluorescent assay. Data are depicted as the percentage of IFNAR1 internalization detected in cells expressing wild-type ubiquitin (100%) at 15 min. *, P

    Article Snippet: Cells were washed, blocked, and incubated with anti-IFNAR1 antibody (AA3 for endogenous IFNAR1) or anti-FLAG antibody (monoclonal Flag M2 for exogenous IFNAR1 and its mutants; Sigma-Aldrich) for 1 h, washed, and incubated with HRP-conjugated goat anti–mouse secondary antibody (Invitrogen) for 1 h. After extensive washing, cells were incubated with AmplexRed Ultra Reagent (10-acetyl-3,7-dihydroxyphenoxazine; Invitrogen).

    Techniques: Fluorescence, Cell Surface Biotinylation Assay, Expressing

    Role of IFNAR1 polyubiquitination in mediating the efficient postinternalization sorting of IFNAR1. (A) Localization of internalized Flag-tagged IFNAR1 wt and IFNAR1 KR . IFNAR1 proteins were labeled for 1 h in the presence of IFN-α, anti-Flag antibody, and TRITC-conjugated secondary Fab and were washed and chased (allowed to internalize) for an additional 30 min. Lysosomes and recycling endosomes were labeled with FITC-dextran and FITC-transferrin (Tfr), respectively. Dextran completely colocalized with Lamp1 (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200706034/DC1 ). Single optical sections of representative cells obtained by laser confocal fluorescence microscopy are depicted. A similar localization of IFNAR1 KR was also observed when chase time was increased to 90 min. Bars, 10 μm. (B) Vesicular pH (pH v ) of Flag-tagged IFNAR1-containing endosomes/lysosomes in transiently transfected HEK293T cells. Endosomal pH was measured by FRIA upon labeling and 30-min chase, and the frequency distribution of the pH v among the indicated number of analyzed vesicles ( n ) was plotted. After calibration, the ratios are expressed in pH values. A similar distribution for IFNAR1 KR was also observed at 90-min chase. The IFNAR1 KR-RPT mutant contains an additional ubiquitination repeat module and is ubiquitination competent (see Fig. 6 ). (C) Postendocytotic sorting of Flag-tagged IFNAR1 wt in cells that coexpress ubiquitin constructs (as indicated, upon labeling and chase for 30 min) was monitored by pH v measurements as described in B. Data are expressed as the frequency of pH v and mean ± SEM ( n = 3).

    Journal: The Journal of Cell Biology

    Article Title: Site-specific ubiquitination exposes a linear motif to promote interferon-? receptor endocytosis

    doi: 10.1083/jcb.200706034

    Figure Lengend Snippet: Role of IFNAR1 polyubiquitination in mediating the efficient postinternalization sorting of IFNAR1. (A) Localization of internalized Flag-tagged IFNAR1 wt and IFNAR1 KR . IFNAR1 proteins were labeled for 1 h in the presence of IFN-α, anti-Flag antibody, and TRITC-conjugated secondary Fab and were washed and chased (allowed to internalize) for an additional 30 min. Lysosomes and recycling endosomes were labeled with FITC-dextran and FITC-transferrin (Tfr), respectively. Dextran completely colocalized with Lamp1 (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200706034/DC1 ). Single optical sections of representative cells obtained by laser confocal fluorescence microscopy are depicted. A similar localization of IFNAR1 KR was also observed when chase time was increased to 90 min. Bars, 10 μm. (B) Vesicular pH (pH v ) of Flag-tagged IFNAR1-containing endosomes/lysosomes in transiently transfected HEK293T cells. Endosomal pH was measured by FRIA upon labeling and 30-min chase, and the frequency distribution of the pH v among the indicated number of analyzed vesicles ( n ) was plotted. After calibration, the ratios are expressed in pH values. A similar distribution for IFNAR1 KR was also observed at 90-min chase. The IFNAR1 KR-RPT mutant contains an additional ubiquitination repeat module and is ubiquitination competent (see Fig. 6 ). (C) Postendocytotic sorting of Flag-tagged IFNAR1 wt in cells that coexpress ubiquitin constructs (as indicated, upon labeling and chase for 30 min) was monitored by pH v measurements as described in B. Data are expressed as the frequency of pH v and mean ± SEM ( n = 3).

    Article Snippet: Cells were washed, blocked, and incubated with anti-IFNAR1 antibody (AA3 for endogenous IFNAR1) or anti-FLAG antibody (monoclonal Flag M2 for exogenous IFNAR1 and its mutants; Sigma-Aldrich) for 1 h, washed, and incubated with HRP-conjugated goat anti–mouse secondary antibody (Invitrogen) for 1 h. After extensive washing, cells were incubated with AmplexRed Ultra Reagent (10-acetyl-3,7-dihydroxyphenoxazine; Invitrogen).

    Techniques: Labeling, Fluorescence, Microscopy, Transfection, Mutagenesis, Construct

    Site-specific ubiquitination is required for efficient internalization of IFNAR1. (A) Diagram of the intracellular domains of IFNAR1 proteins, including generated IFNAR1 mutants. Positions of S535 and S539 residues within the phosphodegron, of ubiquitin-acceptor cluster K501/525/526, and of Y466 within the linear endocytic motif are shown. (B) Ser535 phosphorylation of Flag-tagged IFNAR1 proteins expressed in IFN-α–treated 293T cells was analyzed by immunoblotting as in Fig. 1 F . Vec, empty vector. (C) Interaction of HA-tagged βTrcp with coexpressed Flag-tagged IFNAR1 proteins in IFN-α–treated 293T cells was analyzed by immunoprecipitation/immunoblotting using the indicated antibodies. (D) Ubiquitination of IFNAR1 proteins in IFN-α–treated 293T cells was analyzed as in Fig. 3 C . Ratios between ubiquitin and IFNAR1 signals were calculated as the percentage of such for wild-type IFNAR1. (E and F) Internalization of Flag-tagged IFNAR1 proteins was measured similar to the experiment shown in Fig. 5 A using anti-Flag antibodies. Error bars represent SEM.

    Journal: The Journal of Cell Biology

    Article Title: Site-specific ubiquitination exposes a linear motif to promote interferon-? receptor endocytosis

    doi: 10.1083/jcb.200706034

    Figure Lengend Snippet: Site-specific ubiquitination is required for efficient internalization of IFNAR1. (A) Diagram of the intracellular domains of IFNAR1 proteins, including generated IFNAR1 mutants. Positions of S535 and S539 residues within the phosphodegron, of ubiquitin-acceptor cluster K501/525/526, and of Y466 within the linear endocytic motif are shown. (B) Ser535 phosphorylation of Flag-tagged IFNAR1 proteins expressed in IFN-α–treated 293T cells was analyzed by immunoblotting as in Fig. 1 F . Vec, empty vector. (C) Interaction of HA-tagged βTrcp with coexpressed Flag-tagged IFNAR1 proteins in IFN-α–treated 293T cells was analyzed by immunoprecipitation/immunoblotting using the indicated antibodies. (D) Ubiquitination of IFNAR1 proteins in IFN-α–treated 293T cells was analyzed as in Fig. 3 C . Ratios between ubiquitin and IFNAR1 signals were calculated as the percentage of such for wild-type IFNAR1. (E and F) Internalization of Flag-tagged IFNAR1 proteins was measured similar to the experiment shown in Fig. 5 A using anti-Flag antibodies. Error bars represent SEM.

    Article Snippet: Cells were washed, blocked, and incubated with anti-IFNAR1 antibody (AA3 for endogenous IFNAR1) or anti-FLAG antibody (monoclonal Flag M2 for exogenous IFNAR1 and its mutants; Sigma-Aldrich) for 1 h, washed, and incubated with HRP-conjugated goat anti–mouse secondary antibody (Invitrogen) for 1 h. After extensive washing, cells were incubated with AmplexRed Ultra Reagent (10-acetyl-3,7-dihydroxyphenoxazine; Invitrogen).

    Techniques: Generated, Plasmid Preparation, Immunoprecipitation

    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

    A : identification of 2 CTRP9 isoforms in plasma. gCTRP9, globular domain isoform; fCTRP9, recombinant full-length isoform. a : mouse plasma CTRP9 determined by anti-gCTRP9. b : fCTRP9 and gCTRP9 expressed in E. coli . Typical blots from ≥5 independent experiments. B : overexpressed CTRP9 in mammalian cell produce 2 protein isoforms in medium. a : COOH-terminal FLAG-tagged fCTRP9 was expressed in 3T3-L1 cells, CTRP9 in cell lysates ( lane 1 ) or conditioned medium ( lane 2 ) was detected by antibody against FLAG. b : Flag-Myc duo-tagged fCTRP9 was expressed in HEK 293T cells, and CTRP9 in medium was detected by antibody against either Flag ( lane 1 ) or Myc ( lane 2 ). Typical blots from ≥5 independent experiments. C : purified fCTRP9 protein is proteolytic modified by heart tissue lyses. a : cardiac endogenous CTRP9 was detected with antibody against gCTRP9. b : HEK 293T cells expressed fCTRP9-FLAG-Tag (purified from cell lysate) was incubated with homogenized tissue buffer ( lane 1 ) or cardiac tissue extracts in the absence ( lane 2 ) or presence ( lane 3 ) of protease inhibitor cocktail. CTRP9 is detected with antibody against FLAG. Typical blots from ≥5 independent experiments. D : Coomassie blue and Western blot analysis of purified fCTRP9 protein. Left : Coomassie brilliant blue staining (on M2 affinity gel) of fCTRP9-transfacted HEK 293T cell lysates (CL) or purified fCTRP9 from transfected cells (fCTRP9). Right : Western blot analysis (anti-CTRP9) of fCTRP9-transfacted HEK 293T cell lysates (CL) or purified fCTRP9 from the transfected cells (fCTRP9). E : Western blot determining presence of gCTRP9 fragment (anti-Myc) in cultured medium with varying FBS %concentration. M, marker; S, nontransfected HEK 293 cells, cultured in 10% FBS-MEM; C, pCMV24-3XFLAG-fCTRP9-Myc transfected HEK 293T cells, cultured in 0% FBS-MEM medium; 1%–10%: pCMV24-3XFLAG-fCTRP9-Myc transfected HEK 293T cells, cultured in indicated %FBS-MEM medium for 48 h.

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: C1q-TNF-related protein-9, a novel cardioprotetcive cardiokine, requires proteolytic cleavage to generate a biologically active globular domain isoform

    doi: 10.1152/ajpendo.00450.2014

    Figure Lengend Snippet: A : identification of 2 CTRP9 isoforms in plasma. gCTRP9, globular domain isoform; fCTRP9, recombinant full-length isoform. a : mouse plasma CTRP9 determined by anti-gCTRP9. b : fCTRP9 and gCTRP9 expressed in E. coli . Typical blots from ≥5 independent experiments. B : overexpressed CTRP9 in mammalian cell produce 2 protein isoforms in medium. a : COOH-terminal FLAG-tagged fCTRP9 was expressed in 3T3-L1 cells, CTRP9 in cell lysates ( lane 1 ) or conditioned medium ( lane 2 ) was detected by antibody against FLAG. b : Flag-Myc duo-tagged fCTRP9 was expressed in HEK 293T cells, and CTRP9 in medium was detected by antibody against either Flag ( lane 1 ) or Myc ( lane 2 ). Typical blots from ≥5 independent experiments. C : purified fCTRP9 protein is proteolytic modified by heart tissue lyses. a : cardiac endogenous CTRP9 was detected with antibody against gCTRP9. b : HEK 293T cells expressed fCTRP9-FLAG-Tag (purified from cell lysate) was incubated with homogenized tissue buffer ( lane 1 ) or cardiac tissue extracts in the absence ( lane 2 ) or presence ( lane 3 ) of protease inhibitor cocktail. CTRP9 is detected with antibody against FLAG. Typical blots from ≥5 independent experiments. D : Coomassie blue and Western blot analysis of purified fCTRP9 protein. Left : Coomassie brilliant blue staining (on M2 affinity gel) of fCTRP9-transfacted HEK 293T cell lysates (CL) or purified fCTRP9 from transfected cells (fCTRP9). Right : Western blot analysis (anti-CTRP9) of fCTRP9-transfacted HEK 293T cell lysates (CL) or purified fCTRP9 from the transfected cells (fCTRP9). E : Western blot determining presence of gCTRP9 fragment (anti-Myc) in cultured medium with varying FBS %concentration. M, marker; S, nontransfected HEK 293 cells, cultured in 10% FBS-MEM; C, pCMV24-3XFLAG-fCTRP9-Myc transfected HEK 293T cells, cultured in 0% FBS-MEM medium; 1%–10%: pCMV24-3XFLAG-fCTRP9-Myc transfected HEK 293T cells, cultured in indicated %FBS-MEM medium for 48 h.

    Article Snippet: Monoclonal antibody for ANTI-FLAG M2 was purchased from Sigma (St. Louis, MO).

    Techniques: Recombinant, Purification, Modification, FLAG-tag, Incubation, Protease Inhibitor, Western Blot, Staining, Transfection, Cell Culture, Concentration Assay, Marker

    Fusion activity of Chi proteins in heterotypic and homotypic complexes as assessed in vitro. (A) Mitochondria were isolated from wild-type cells (Mfn1, Mfn2) or clonal populations of Mfn1-null cells transduced with empty vector (Mfn2) or expressing either Mfn1-FLAG (Mfn1*Mfn2), Mfn2-FLAG (Mfn2*Mfn2), Chi3-FLAG (Chi3*Mfn2), or Chi5-FLAG (Chi5*Mfn2), where the asterisk indicates the nonendogenous protein. The indicated mitochondrial combinations were subject to in vitro fusion conditions at 37°C for 60 min and data are expressed as a relative amount of wild-type controls, performed in parallel. Black bars indicate homotypic reactions, where both mitochondrial fusion partners possess the same mitofusin proteins. Gray bars indicate heterotypic fusion reactions, where one of the mitochondrial fusion partners is wild type. Error bars indicate mean + SD from at least four independent experiments and the statistical significance indicated on the graphs was deter­mined by paired t test analysis (one tail); *, P

    Journal: Molecular Biology of the Cell

    Article Title: Identification of a mitofusin specificity region that confers unique activities to Mfn1 and Mfn2

    doi: 10.1091/mbc.E19-05-0291

    Figure Lengend Snippet: Fusion activity of Chi proteins in heterotypic and homotypic complexes as assessed in vitro. (A) Mitochondria were isolated from wild-type cells (Mfn1, Mfn2) or clonal populations of Mfn1-null cells transduced with empty vector (Mfn2) or expressing either Mfn1-FLAG (Mfn1*Mfn2), Mfn2-FLAG (Mfn2*Mfn2), Chi3-FLAG (Chi3*Mfn2), or Chi5-FLAG (Chi5*Mfn2), where the asterisk indicates the nonendogenous protein. The indicated mitochondrial combinations were subject to in vitro fusion conditions at 37°C for 60 min and data are expressed as a relative amount of wild-type controls, performed in parallel. Black bars indicate homotypic reactions, where both mitochondrial fusion partners possess the same mitofusin proteins. Gray bars indicate heterotypic fusion reactions, where one of the mitochondrial fusion partners is wild type. Error bars indicate mean + SD from at least four independent experiments and the statistical significance indicated on the graphs was deter­mined by paired t test analysis (one tail); *, P

    Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-FLAG (Sigma; 1:1000); mouse monoclonal anti-Mfn2 (Sigma clone 4H8; 1:1000); mouse monoclonal anti-tubulin (Thermo Fisher Scientific clone DM1A; 1:5000); rabbit polyclonal anti-Mfn1 (gift from Jodi Nunnari, University of California, Davis; 1:500); and VDAC (Thermo Fisher Scientific, polyclonal PA1-954A; 1:1000).

    Techniques: Activity Assay, In Vitro, Isolation, Transduction, Plasmid Preparation, Expressing

    Mitofusin-specific nucleotide-dependent assembly. (A) Mitochondria were isolated from clonal populations of Mfn1-null cells expressing Mfn1-FLAG, Mfn2-FLAG, Chi3-FLAG, or Chi5-FLAG. Mitochondria were either untreated (−) or incubated with the specified nucleotide conditions and subsequently subjected to detergent solubilization and analysis by BN–PAGE and immunoblotting with anti-FLAG. The positions of the molecular weight markers are shown on the left. The predicted dimer is indicated with a line (–); the ∼320 kDa species is indicated with a double arrow; the ∼450 kDa species is indicated with an arrow; a nonspecific band is highlighted with an asterisk (*). The percentage of total protein in each oligomeric state for each condition is represented in the bar graph as mean + SD of three independent experiments. (B) Mitochondria were isolated from clonal populations of Mfn1/2-null cells expressing Mfn1-FLAG, Mfn2-FLAG, Chi3-FLAG, or Chi5-FLAG. Mitochondria were either untreated (−) or incubated with the specified nucleotide conditions and subsequently subjected to detergent solubilization and analysis by BN–PAGE and immunoblotting with anti-FLAG. The positions of the molecular weight markers are shown on the left. The predicted dimer is indicated with a line (–); the ∼320 kDa species is indicated with a double arrow; the ∼450 kDa species is indicated with an arrow; a nonspecific band is highlighted with an asterisk (*). The percentage of total protein in each oligomeric state for each condition is represented in the bar graph as mean + SD of three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Identification of a mitofusin specificity region that confers unique activities to Mfn1 and Mfn2

    doi: 10.1091/mbc.E19-05-0291

    Figure Lengend Snippet: Mitofusin-specific nucleotide-dependent assembly. (A) Mitochondria were isolated from clonal populations of Mfn1-null cells expressing Mfn1-FLAG, Mfn2-FLAG, Chi3-FLAG, or Chi5-FLAG. Mitochondria were either untreated (−) or incubated with the specified nucleotide conditions and subsequently subjected to detergent solubilization and analysis by BN–PAGE and immunoblotting with anti-FLAG. The positions of the molecular weight markers are shown on the left. The predicted dimer is indicated with a line (–); the ∼320 kDa species is indicated with a double arrow; the ∼450 kDa species is indicated with an arrow; a nonspecific band is highlighted with an asterisk (*). The percentage of total protein in each oligomeric state for each condition is represented in the bar graph as mean + SD of three independent experiments. (B) Mitochondria were isolated from clonal populations of Mfn1/2-null cells expressing Mfn1-FLAG, Mfn2-FLAG, Chi3-FLAG, or Chi5-FLAG. Mitochondria were either untreated (−) or incubated with the specified nucleotide conditions and subsequently subjected to detergent solubilization and analysis by BN–PAGE and immunoblotting with anti-FLAG. The positions of the molecular weight markers are shown on the left. The predicted dimer is indicated with a line (–); the ∼320 kDa species is indicated with a double arrow; the ∼450 kDa species is indicated with an arrow; a nonspecific band is highlighted with an asterisk (*). The percentage of total protein in each oligomeric state for each condition is represented in the bar graph as mean + SD of three independent experiments.

    Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-FLAG (Sigma; 1:1000); mouse monoclonal anti-Mfn2 (Sigma clone 4H8; 1:1000); mouse monoclonal anti-tubulin (Thermo Fisher Scientific clone DM1A; 1:5000); rabbit polyclonal anti-Mfn1 (gift from Jodi Nunnari, University of California, Davis; 1:500); and VDAC (Thermo Fisher Scientific, polyclonal PA1-954A; 1:1000).

    Techniques: Isolation, Expressing, Incubation, Polyacrylamide Gel Electrophoresis, Molecular Weight

    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

    Exogenous expression of PC1 reduces Ca 2+ oscillation frequency in HEK293 cells . (a) Representative Ca 2+ oscillation patterns from individual HEK293 pSsiPKD1 (red) and HEK293 pSUPER (blue) cells before (upper traces) and after (lower traces) transient transfection with the mouse PC1 expressing plasmid (mPC1). Cells were grown on coverslips, transfected with the plasmid DNA as described in the Materials and Methods section, loaded with Fura-2-AM after 48 h and stimulated with 1% FBS, as described in Fig. 2 . Inset: the expression of the mouse PC1 was confirmed by Western blotting and immunofluorescence analysis, as shown in HEK293 pSUPER (pS) and HEK293 pSsiPKD1 (pSsiPKD1) transfected cells, the latter showing that mPC1 is not silenced by human PKD1 siRNA. Cells were lysed and total extracts were analysed by immunoblotting with the anti-FLAG M2 mouse monoclonal antibody recognizing the FLAG-tagged mouse PC1, as described in the Materials and Methods section. The antibody identified a band of about 400 kDa in only mPKD1 cDNA-transfected HEK293 pSUPER and HEK293 pSsiPKD1 cells. The ~200 kDa band was deemed to be aspecific as present in all samples. For immunofluorescence analysis, cells were fixed and treated with M2 antibody, as described in the Materials and Methods section. Upper and lower panels: contrast phase and fluorescence images. Staining of plasma membranes was indicated by arrows, mainly at cell–cell interactions. (b) Average percent reduction in Ca 2+ oscillation frequency in a 15-min period obtained after transient transfection with the mouse PC1 expressing plasmid of HEK293 pSsiPKD1 clone b 3 (red bars; n = 122 cells, five experiments) and HEK293 pSUPER cells (blue bars, n = 60, three experiments). (c) Reduction in cell proliferation in HEK293 cells stably expressing the full length mouse PC1. pCDNA3 stably transfected control cells (white bar) and cells stably transfected with the full length mouse PKD1 cDNA plasmid (grey bar) were grown for 3 days in presence of 1% FBS. Data are expressed as the average values (± SD; * P

    Journal: Cell Proliferation

    Article Title: Novel role for polycystin-1 in modulating cell proliferation through calcium oscillations in kidney cells

    doi: 10.1111/j.1365-2184.2008.00529.x

    Figure Lengend Snippet: Exogenous expression of PC1 reduces Ca 2+ oscillation frequency in HEK293 cells . (a) Representative Ca 2+ oscillation patterns from individual HEK293 pSsiPKD1 (red) and HEK293 pSUPER (blue) cells before (upper traces) and after (lower traces) transient transfection with the mouse PC1 expressing plasmid (mPC1). Cells were grown on coverslips, transfected with the plasmid DNA as described in the Materials and Methods section, loaded with Fura-2-AM after 48 h and stimulated with 1% FBS, as described in Fig. 2 . Inset: the expression of the mouse PC1 was confirmed by Western blotting and immunofluorescence analysis, as shown in HEK293 pSUPER (pS) and HEK293 pSsiPKD1 (pSsiPKD1) transfected cells, the latter showing that mPC1 is not silenced by human PKD1 siRNA. Cells were lysed and total extracts were analysed by immunoblotting with the anti-FLAG M2 mouse monoclonal antibody recognizing the FLAG-tagged mouse PC1, as described in the Materials and Methods section. The antibody identified a band of about 400 kDa in only mPKD1 cDNA-transfected HEK293 pSUPER and HEK293 pSsiPKD1 cells. The ~200 kDa band was deemed to be aspecific as present in all samples. For immunofluorescence analysis, cells were fixed and treated with M2 antibody, as described in the Materials and Methods section. Upper and lower panels: contrast phase and fluorescence images. Staining of plasma membranes was indicated by arrows, mainly at cell–cell interactions. (b) Average percent reduction in Ca 2+ oscillation frequency in a 15-min period obtained after transient transfection with the mouse PC1 expressing plasmid of HEK293 pSsiPKD1 clone b 3 (red bars; n = 122 cells, five experiments) and HEK293 pSUPER cells (blue bars, n = 60, three experiments). (c) Reduction in cell proliferation in HEK293 cells stably expressing the full length mouse PC1. pCDNA3 stably transfected control cells (white bar) and cells stably transfected with the full length mouse PKD1 cDNA plasmid (grey bar) were grown for 3 days in presence of 1% FBS. Data are expressed as the average values (± SD; * P

    Article Snippet: Reagents Dulbeccos's modified Eagle's medium/F12 and minimum essential medium media, G418 antibiotic, bovine serum albumin (BSA), 1-(beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF96365), cyclosporin A, gadolinium and anti-FLAG M2 mouse monoclonal antibody were obtained from Sigma-Aldrich (Milano, Italy), foetal bovine serum (FBS) was obtained from Eurobio (Celbio, Milan, Italy) and selective cell-permeable inhibitors of protein kinase C (PKC)-α and PKC-β1 (Ro-320432 and hispidin, respectively), were purchased from Calbiochem (La Jolla, CA, USA) and protease inhibitors were from Roche Diagnostics (Monza, Italy).

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Fluorescence, Staining, Stable Transfection

    TMEM2 is expressed as a type II transmembrane protein. A, domain structure of TMEM2 and CEMIP. The location of the FLAG epitope tag added to recombinant proteins is also indicated. B, live immunostaining of MG-63 cells transiently transfected with TMEM2

    Journal: The Journal of Biological Chemistry

    Article Title: A mammalian homolog of the zebrafish transmembrane protein 2 (TMEM2) is the long-sought-after cell-surface hyaluronidase

    doi: 10.1074/jbc.M116.770149

    Figure Lengend Snippet: TMEM2 is expressed as a type II transmembrane protein. A, domain structure of TMEM2 and CEMIP. The location of the FLAG epitope tag added to recombinant proteins is also indicated. B, live immunostaining of MG-63 cells transiently transfected with TMEM2

    Article Snippet: TMEM2 was detected by immunoblotting with mouse monoclonal anti-FLAG antibody (M2, Sigma).

    Techniques: FLAG-tag, Recombinant, Immunostaining, Transfection

    Determination of the sensitivity of the parent antibody 1E8 (a) and the recombinant Fab antibody 1E8-4b (b) to the amyloid peptides Aβ[1–40], Aβ[1–42] and Aβ[1–43] by ELISA. The peptides were serially diluted with doubling dilutions starting from 50 ng/ml while the concentrations of 1E8 and 1E8-4b were kept constant at 5 μg/ml. 1E8 was detected with antimouse-HRP conjugated antibody while 1E8-4b was probed with M2 anti-Flag antibody and then detected with antimouse-HRP conjugated antibody. Detection was via an ‘ o ’-phenylenediamine development mixture. The isotype control used was M2 anti-Flag antibody which is a mouse IgG 1 .

    Journal: Clinical and Experimental Immunology

    Article Title: Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related A? peptide

    doi: 10.1046/j.1365-2249.2002.01905.x

    Figure Lengend Snippet: Determination of the sensitivity of the parent antibody 1E8 (a) and the recombinant Fab antibody 1E8-4b (b) to the amyloid peptides Aβ[1–40], Aβ[1–42] and Aβ[1–43] by ELISA. The peptides were serially diluted with doubling dilutions starting from 50 ng/ml while the concentrations of 1E8 and 1E8-4b were kept constant at 5 μg/ml. 1E8 was detected with antimouse-HRP conjugated antibody while 1E8-4b was probed with M2 anti-Flag antibody and then detected with antimouse-HRP conjugated antibody. Detection was via an ‘ o ’-phenylenediamine development mixture. The isotype control used was M2 anti-Flag antibody which is a mouse IgG 1 .

    Article Snippet: To detect the parent antibody 100 μl/section of anti-mouse immunoglobulins coupled to horseradish peroxidase (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. To detect the Fab and isotype control, 100 μl/section of M2 anti-Flag monoclonal antibody (Sigma, St Louis, MO, USA) was applied at 10 μg/ml for 1h followed by two 5-min washes with TBS pH 7·4, then 100 μl/section of anti-mouse HRP (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. Sections were washed twice for 5 min with TBS pH 7·4 and developed with diaminobenzidine solution (BDH Chemicals, Poole, Dorset, UK) for 5 min.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

    Immunohistochemistry of AD brain sections. Brain sections were taken from the same AD brain patient for accurate comparison. The 1E8-4b recombinant Fab fragment, the 1E8 antibody and the isotype control M2 anti-Flag antibody were applied to the sections at a concentration of 20 μg/ml at x20 (a) and x60 (b) magnification. At 10 μg/ml and 20 μg/ml the 1E8-4b and 1E8 antibodies showed reactivity to amyloid associated with plaques, while the isotype control showed no reactivity. Sections 1 and 4: 1E8; sections 2 and 5: 1E8-4b; sections 3 and 6: isotype control M2 anti-Flag monoclonal antibody.

    Journal: Clinical and Experimental Immunology

    Article Title: Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related A? peptide

    doi: 10.1046/j.1365-2249.2002.01905.x

    Figure Lengend Snippet: Immunohistochemistry of AD brain sections. Brain sections were taken from the same AD brain patient for accurate comparison. The 1E8-4b recombinant Fab fragment, the 1E8 antibody and the isotype control M2 anti-Flag antibody were applied to the sections at a concentration of 20 μg/ml at x20 (a) and x60 (b) magnification. At 10 μg/ml and 20 μg/ml the 1E8-4b and 1E8 antibodies showed reactivity to amyloid associated with plaques, while the isotype control showed no reactivity. Sections 1 and 4: 1E8; sections 2 and 5: 1E8-4b; sections 3 and 6: isotype control M2 anti-Flag monoclonal antibody.

    Article Snippet: To detect the parent antibody 100 μl/section of anti-mouse immunoglobulins coupled to horseradish peroxidase (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. To detect the Fab and isotype control, 100 μl/section of M2 anti-Flag monoclonal antibody (Sigma, St Louis, MO, USA) was applied at 10 μg/ml for 1h followed by two 5-min washes with TBS pH 7·4, then 100 μl/section of anti-mouse HRP (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. Sections were washed twice for 5 min with TBS pH 7·4 and developed with diaminobenzidine solution (BDH Chemicals, Poole, Dorset, UK) for 5 min.

    Techniques: Immunohistochemistry, Recombinant, Concentration Assay

    Detection of Aβ peptides [1–40], [1–42] and [1–43] and total Aβ in FAD brain by 1E8-4b (a) and 1E8 (b) antibodies using immunoblotting followed by ECL development. Samples were electrophoresed on 14% Tris-tricine gels with 100 ng/well of Aβ peptides [1–40], [1–42] and [1–43] and APP peptides 695 and 770. A total of 5 μl of FAD brain sample was used. The recombinant Fab 1E8-4b and the isotype control M2 anti-Flag monoclonal antibody were applied to the immunoblots at 5 μg/ml, while 1E8 was used at 50 μg/ml. The isotype control displayed no reactivity to the peptides or FAD brain sample. Lane 1: Aβ peptide [1–40]; lane 2: Aβ peptide [1–42]; lane 3: Aβ peptide [1–43]; lane 4: APP peptide 695; lane 5: APP peptide 770; lane 6: FAD (PS-1mut) brain sample.

    Journal: Clinical and Experimental Immunology

    Article Title: Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related A? peptide

    doi: 10.1046/j.1365-2249.2002.01905.x

    Figure Lengend Snippet: Detection of Aβ peptides [1–40], [1–42] and [1–43] and total Aβ in FAD brain by 1E8-4b (a) and 1E8 (b) antibodies using immunoblotting followed by ECL development. Samples were electrophoresed on 14% Tris-tricine gels with 100 ng/well of Aβ peptides [1–40], [1–42] and [1–43] and APP peptides 695 and 770. A total of 5 μl of FAD brain sample was used. The recombinant Fab 1E8-4b and the isotype control M2 anti-Flag monoclonal antibody were applied to the immunoblots at 5 μg/ml, while 1E8 was used at 50 μg/ml. The isotype control displayed no reactivity to the peptides or FAD brain sample. Lane 1: Aβ peptide [1–40]; lane 2: Aβ peptide [1–42]; lane 3: Aβ peptide [1–43]; lane 4: APP peptide 695; lane 5: APP peptide 770; lane 6: FAD (PS-1mut) brain sample.

    Article Snippet: To detect the parent antibody 100 μl/section of anti-mouse immunoglobulins coupled to horseradish peroxidase (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. To detect the Fab and isotype control, 100 μl/section of M2 anti-Flag monoclonal antibody (Sigma, St Louis, MO, USA) was applied at 10 μg/ml for 1h followed by two 5-min washes with TBS pH 7·4, then 100 μl/section of anti-mouse HRP (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. Sections were washed twice for 5 min with TBS pH 7·4 and developed with diaminobenzidine solution (BDH Chemicals, Poole, Dorset, UK) for 5 min.

    Techniques: Recombinant, Western Blot

    ELISA depicting the reactivity of the parent 1E8 antibody (a) and the recombinant 1E8-4b Fab fragment (b) to Aβ[1–40], Aβ [1–42], Aβ[1–43], APP695 and APP770 peptides. The antibody is titrated from 10 μg/ml with serial doubling dilutions, while the peptide is at a constant concentration of 50 ng/well. 1E8 was probed with antimouse-HRP while 1E8-4b was probed with M2 anti-Flag antibody and then with antimouse-HRP. Detection of antibody reactivity to the peptides was via an ‘ o ’-phenylenediamine development mixture. The isotype control used was M2 anti-Flag antibody which is a mouse IgG 1 .

    Journal: Clinical and Experimental Immunology

    Article Title: Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related A? peptide

    doi: 10.1046/j.1365-2249.2002.01905.x

    Figure Lengend Snippet: ELISA depicting the reactivity of the parent 1E8 antibody (a) and the recombinant 1E8-4b Fab fragment (b) to Aβ[1–40], Aβ [1–42], Aβ[1–43], APP695 and APP770 peptides. The antibody is titrated from 10 μg/ml with serial doubling dilutions, while the peptide is at a constant concentration of 50 ng/well. 1E8 was probed with antimouse-HRP while 1E8-4b was probed with M2 anti-Flag antibody and then with antimouse-HRP. Detection of antibody reactivity to the peptides was via an ‘ o ’-phenylenediamine development mixture. The isotype control used was M2 anti-Flag antibody which is a mouse IgG 1 .

    Article Snippet: To detect the parent antibody 100 μl/section of anti-mouse immunoglobulins coupled to horseradish peroxidase (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. To detect the Fab and isotype control, 100 μl/section of M2 anti-Flag monoclonal antibody (Sigma, St Louis, MO, USA) was applied at 10 μg/ml for 1h followed by two 5-min washes with TBS pH 7·4, then 100 μl/section of anti-mouse HRP (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. Sections were washed twice for 5 min with TBS pH 7·4 and developed with diaminobenzidine solution (BDH Chemicals, Poole, Dorset, UK) for 5 min.

    Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay

    Purification of recombinant 1E8-4b Fab-EEF/FLAG conjugate from E. coli culture supernatant as analysed by SDS-PAGE under non-reducing conditions and immunoblotting (lanes 1–5) or Coomassie Brilliant Blue staining (lane 6). Immunoreactive protein bands were detected using M2 anti-Flag antibody followed by goat anti-mouse alkaline phosphatase conjugated antibody and developed with Fast Red/AS-MX Naphthol reagents. Lane 1 : Culture supernatant; lane 2 : Supernatant following 60% (v/v) SAS precipitation; lane 3 : Low-molecular-weight-protein markers (with numbers to the left in kDa); lane 4: precipitated 1E8-4b Fab protein following 60% (v/v) SAS precipitation; lane 5: pooled and concentrated M2 anti-Flag affinity gel purified 1E8-4b Fab eluates; lane 6: Coomassie stain of lane 5.

    Journal: Clinical and Experimental Immunology

    Article Title: Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related A? peptide

    doi: 10.1046/j.1365-2249.2002.01905.x

    Figure Lengend Snippet: Purification of recombinant 1E8-4b Fab-EEF/FLAG conjugate from E. coli culture supernatant as analysed by SDS-PAGE under non-reducing conditions and immunoblotting (lanes 1–5) or Coomassie Brilliant Blue staining (lane 6). Immunoreactive protein bands were detected using M2 anti-Flag antibody followed by goat anti-mouse alkaline phosphatase conjugated antibody and developed with Fast Red/AS-MX Naphthol reagents. Lane 1 : Culture supernatant; lane 2 : Supernatant following 60% (v/v) SAS precipitation; lane 3 : Low-molecular-weight-protein markers (with numbers to the left in kDa); lane 4: precipitated 1E8-4b Fab protein following 60% (v/v) SAS precipitation; lane 5: pooled and concentrated M2 anti-Flag affinity gel purified 1E8-4b Fab eluates; lane 6: Coomassie stain of lane 5.

    Article Snippet: To detect the parent antibody 100 μl/section of anti-mouse immunoglobulins coupled to horseradish peroxidase (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. To detect the Fab and isotype control, 100 μl/section of M2 anti-Flag monoclonal antibody (Sigma, St Louis, MO, USA) was applied at 10 μg/ml for 1h followed by two 5-min washes with TBS pH 7·4, then 100 μl/section of anti-mouse HRP (Dako, Carpinteria, CA, USA) was applied at 6·5 μg/ml for 1 h. Sections were washed twice for 5 min with TBS pH 7·4 and developed with diaminobenzidine solution (BDH Chemicals, Poole, Dorset, UK) for 5 min.

    Techniques: Purification, Recombinant, SDS Page, Staining, Molecular Weight

    Binding of YscY-FLAG to MBP-YscX. Cell pellet fractions from BL21(DE3) expressing MBP, MBP-YscX, MBP-YscY, and MBP-YopN were separated by SDS-PAGE and transferred to Immobilon membranes. MBP migrated slower than expected due to an in-frame fusion between malE and the vector LacZ α-peptide-encoding sequences. (A) Detection of MBP, MBP-YscX, MBP-YscY, and MBP-YopN with antiserum specific for MBP. (B) Immobilon membrane containing SDS-PAGE-separated MBP, MBP-YscX, MBP-YscY, and MBP-YopN probed with a BL21(DE3) extract containing YscY-FLAG. Bound YscY-FLAG was detected with the FLAG M2 monoclonal antibody.

    Journal: Journal of Bacteriology

    Article Title: The Yersinia pestis YscY Protein Directly Binds YscX, a Secreted Component of the Type III Secretion Machinery

    doi:

    Figure Lengend Snippet: Binding of YscY-FLAG to MBP-YscX. Cell pellet fractions from BL21(DE3) expressing MBP, MBP-YscX, MBP-YscY, and MBP-YopN were separated by SDS-PAGE and transferred to Immobilon membranes. MBP migrated slower than expected due to an in-frame fusion between malE and the vector LacZ α-peptide-encoding sequences. (A) Detection of MBP, MBP-YscX, MBP-YscY, and MBP-YopN with antiserum specific for MBP. (B) Immobilon membrane containing SDS-PAGE-separated MBP, MBP-YscX, MBP-YscY, and MBP-YopN probed with a BL21(DE3) extract containing YscY-FLAG. Bound YscY-FLAG was detected with the FLAG M2 monoclonal antibody.

    Article Snippet: FLAG-tagged proteins were detected with the FLAG M2 monoclonal antibody (Sigma).

    Techniques: Binding Assay, Expressing, SDS Page, Plasmid Preparation

    Secretion of YscX-FLAG and truncated YscX-FLAG proteins from the parent strain Y. pestis KIM8-3002. (A) Schematic representation of FLAG-tagged YscX (YscX-FLAG), YscX-FLAG Δ61–122 , YscX-FLAG Δ101–122 , and YscX-FLAG Δ1–15 . A predicted coiled-coil domain spanning residues 70 to 93 of YscX is shown (hatched box). (B) Immunoblot analysis of SDS-PAGE-separated culture supernatant (S) and cell pellet (P) fractions from Y. pestis KIM8-3002 (parent) and KIM8-3002 transformed with plasmids pFLAG-YscX, pFLAG-YscX Δ61–122 , pFLAG-YscX Δ101–122 , and pFLAG-YscX Δ1–15 . The FLAG M2 monoclonal antibody was used to detect the FLAG-tagged products. (C) Immunoblot analysis of SDS-PAGE-separated culture supernatant (S) and cell pellet (P) fractions from Y. pestis KIM8-3002 (parent) and KIM8-3002 complemented with plasmid pFLAG-YscX. Antiserum specific for YopE was used to detect this protein in the supernatant and cell pellet fractions.

    Journal: Journal of Bacteriology

    Article Title: The Yersinia pestis YscY Protein Directly Binds YscX, a Secreted Component of the Type III Secretion Machinery

    doi:

    Figure Lengend Snippet: Secretion of YscX-FLAG and truncated YscX-FLAG proteins from the parent strain Y. pestis KIM8-3002. (A) Schematic representation of FLAG-tagged YscX (YscX-FLAG), YscX-FLAG Δ61–122 , YscX-FLAG Δ101–122 , and YscX-FLAG Δ1–15 . A predicted coiled-coil domain spanning residues 70 to 93 of YscX is shown (hatched box). (B) Immunoblot analysis of SDS-PAGE-separated culture supernatant (S) and cell pellet (P) fractions from Y. pestis KIM8-3002 (parent) and KIM8-3002 transformed with plasmids pFLAG-YscX, pFLAG-YscX Δ61–122 , pFLAG-YscX Δ101–122 , and pFLAG-YscX Δ1–15 . The FLAG M2 monoclonal antibody was used to detect the FLAG-tagged products. (C) Immunoblot analysis of SDS-PAGE-separated culture supernatant (S) and cell pellet (P) fractions from Y. pestis KIM8-3002 (parent) and KIM8-3002 complemented with plasmid pFLAG-YscX. Antiserum specific for YopE was used to detect this protein in the supernatant and cell pellet fractions.

    Article Snippet: FLAG-tagged proteins were detected with the FLAG M2 monoclonal antibody (Sigma).

    Techniques: SDS Page, Transformation Assay, Plasmid Preparation

    Localization of the YscY binding domain of YscX by using truncated and internally deleted MBP-YscX hybrid proteins. (A) Schematic representation of truncated and internally deleted MBP-YscX hybrid proteins used to detect the binding of YscY-FLAG. A predicted coiled-coil domain spanning residues 70 to 93 of YscX is shown (hatched boxes). (B) Detection of MBP-YscX and derivatives with antiserum specific for MBP. MBP migrated slower than expected due to an in-frame fusion between malE and the vector LacZ α-peptide-encoding sequences. (C) Immobilon membrane containing SDS-PAGE-separated MBP-YscX, MBP-YscX Δ1–29 , MBP-YscX Δ1–49 , MBP-YscX Δ1–79 , MBP-YscX Δ111–122 , MBP-YscX Δ91–122 , and MBP-YscX Δ70–90 probed with a BL21(DE3) extract containing YscY-FLAG. Bound YscY-FLAG was detected with the FLAG M2 monoclonal antibody.

    Journal: Journal of Bacteriology

    Article Title: The Yersinia pestis YscY Protein Directly Binds YscX, a Secreted Component of the Type III Secretion Machinery

    doi:

    Figure Lengend Snippet: Localization of the YscY binding domain of YscX by using truncated and internally deleted MBP-YscX hybrid proteins. (A) Schematic representation of truncated and internally deleted MBP-YscX hybrid proteins used to detect the binding of YscY-FLAG. A predicted coiled-coil domain spanning residues 70 to 93 of YscX is shown (hatched boxes). (B) Detection of MBP-YscX and derivatives with antiserum specific for MBP. MBP migrated slower than expected due to an in-frame fusion between malE and the vector LacZ α-peptide-encoding sequences. (C) Immobilon membrane containing SDS-PAGE-separated MBP-YscX, MBP-YscX Δ1–29 , MBP-YscX Δ1–49 , MBP-YscX Δ1–79 , MBP-YscX Δ111–122 , MBP-YscX Δ91–122 , and MBP-YscX Δ70–90 probed with a BL21(DE3) extract containing YscY-FLAG. Bound YscY-FLAG was detected with the FLAG M2 monoclonal antibody.

    Article Snippet: FLAG-tagged proteins were detected with the FLAG M2 monoclonal antibody (Sigma).

    Techniques: Binding Assay, Plasmid Preparation, SDS Page

    FBW7 negatively regulates the stability of Brg1. a Immunoblot analysis (IB) of whole cell lysates (WCLs) derived from wild-type (WT) and FBW7 − /− DLD1 cells. b , c WT and FBW7 −/− DLD1 cells were treated with 20 μg/ml cycloheximide (CHX). At the indicated time points, WCLs were prepared and IB analysis was carried out with indicated antibodies ( b ). The relative Brg1 intensity was normalized to Vinculin and then normalized to the t = 0 controls ( c ). d IB analysis of WCLs derived from WT and FBW7 −/− DLD1 cells treated with MG132 (10 μM) or DMSO for 10 h. e IB analysis of WCLs derived from MKN45 cells infected with the indicated shRNA lentiviruses. f IB analysis of WCLs derived from MKN45 cells infected with the indicated shRNA lentiviruses and treated with 20 μg/ml CHX for indicated time periods. The relative intensity of Brg1 was normalized to Vinculin and then normalized to the t = 0 control (Bottom). g IB analysis of WCLs and immunoprecipitated (IPs) derived from 293T cells transfected with Flag-Brg1 together with the indicated constructs of Myc-tagged Cullin family members. h IB analysis of WCLs derived from MKN45 cells infected with the indicated lentiviral shRNA- Cullin1 constructs. i IB analysis of WCLs and IPs derived from 293T cells transfected with Flag-Brg1 together with the indicated FBW7 constructs. j Co-IP experiments in MKN45 cells were performed using anti-Brg1 antibody (sc-17796, Santa Cruz). Mouse IgG was used as a control. k IB analysis of WCLs derived from FBW7 −/− DLD1 cells infected with the indicated FBW7-expressing lentiviral vectors

    Journal: Nature Communications

    Article Title: SCFFBW7-mediated degradation of Brg1 suppresses gastric cancer metastasis

    doi: 10.1038/s41467-018-06038-y

    Figure Lengend Snippet: FBW7 negatively regulates the stability of Brg1. a Immunoblot analysis (IB) of whole cell lysates (WCLs) derived from wild-type (WT) and FBW7 − /− DLD1 cells. b , c WT and FBW7 −/− DLD1 cells were treated with 20 μg/ml cycloheximide (CHX). At the indicated time points, WCLs were prepared and IB analysis was carried out with indicated antibodies ( b ). The relative Brg1 intensity was normalized to Vinculin and then normalized to the t = 0 controls ( c ). d IB analysis of WCLs derived from WT and FBW7 −/− DLD1 cells treated with MG132 (10 μM) or DMSO for 10 h. e IB analysis of WCLs derived from MKN45 cells infected with the indicated shRNA lentiviruses. f IB analysis of WCLs derived from MKN45 cells infected with the indicated shRNA lentiviruses and treated with 20 μg/ml CHX for indicated time periods. The relative intensity of Brg1 was normalized to Vinculin and then normalized to the t = 0 control (Bottom). g IB analysis of WCLs and immunoprecipitated (IPs) derived from 293T cells transfected with Flag-Brg1 together with the indicated constructs of Myc-tagged Cullin family members. h IB analysis of WCLs derived from MKN45 cells infected with the indicated lentiviral shRNA- Cullin1 constructs. i IB analysis of WCLs and IPs derived from 293T cells transfected with Flag-Brg1 together with the indicated FBW7 constructs. j Co-IP experiments in MKN45 cells were performed using anti-Brg1 antibody (sc-17796, Santa Cruz). Mouse IgG was used as a control. k IB analysis of WCLs derived from FBW7 −/− DLD1 cells infected with the indicated FBW7-expressing lentiviral vectors

    Article Snippet: Polyclonal anti-FLAG antibody (F2425, 1:1000), monoclonal anti-FLAG antibody (F-3165, 1:1000), anti-Vinculin antibody (V9131, 1:5000), peroxidase-conjugated anti-mouse secondary antibody (A4416, 1:3000), peroxidase-conjugated anti-rabbit secondary antibody (A4914, 1:3000) and CK1 inhibitor IC261 were purchased from Sigma.

    Techniques: Derivative Assay, Infection, shRNA, Immunoprecipitation, Transfection, Construct, Co-Immunoprecipitation Assay, Expressing

    Ubiquitylation of Sec23a in Hek293T cells. (A) SDS-PAGE and Coomassie-staining of Huh7 (H) and Hek293T (K) cells transiently transfected with 3X-FLAG-Sec23a at 48 h of expression. Total extracts (Lysates), the FLAG-immunoprecipitated proteins and the unbound proteins are showed as steps of the 3xFLAG-Sec23a purification. (B) Hek293T cells were co-transfected with the pHA-Ubiquitin expression vector and the 3xFLAG-Sec23a vector. Cell lysates were immunoprecipitated and checked by Western Blot with the indicated antibodies as in (Fig. 1 ).

    Journal: The Open Biochemistry Journal

    Article Title: Identification of Cysteine Ubiquitylation Sites on the Sec23A Protein of the COPII Complex Required for Vesicle Formation from the ER

    doi: 10.2174/1874091X01711010036

    Figure Lengend Snippet: Ubiquitylation of Sec23a in Hek293T cells. (A) SDS-PAGE and Coomassie-staining of Huh7 (H) and Hek293T (K) cells transiently transfected with 3X-FLAG-Sec23a at 48 h of expression. Total extracts (Lysates), the FLAG-immunoprecipitated proteins and the unbound proteins are showed as steps of the 3xFLAG-Sec23a purification. (B) Hek293T cells were co-transfected with the pHA-Ubiquitin expression vector and the 3xFLAG-Sec23a vector. Cell lysates were immunoprecipitated and checked by Western Blot with the indicated antibodies as in (Fig. 1 ).

    Article Snippet: The immune complexes were analysed by SDS-PAGE (10%) and revealed by mouse anti-FLAG monoclonal antibody (M2, Sigma-Aldrich), mouse anti-HA antibody and mouse monoclonal anti-ʏ tubulin (Santa Cruz Biotechnology).

    Techniques: SDS Page, Staining, Transfection, Expressing, Immunoprecipitation, Purification, Plasmid Preparation, Western Blot

    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

    The γ subunits associate with Ca v 1.2 in the presence of α2/δ-1 subunit. A–D ) The α1c, β1b, and γ4 ( A ), γ6 ( B ), γ7 ( C ), and γ8 ( D ) subunits were expressed in HEK293 cells in the absence (−) or presence (+) of the α2/δ1 subunit. Lysates were prepared, and γ subunits were immunoprecipitated (IP) with anti-FLAG antibody. Negative controls were preimmune serum and cells without α2/δ-1 coexpression. Top panels: immunoblot with anti-α2 antibody. Bottom panels: immunoblot with HRP-conjugated, anti-FLAG antibody. Cont, control. E ) Same lysates as in ( B ). The α1c subunit was immunoprecipitated with an anti-α1c antibody. Top panel: immunoblot with HRP-conjugated, anti-FLAG antibody. Bottom panel: immunoblot with anti-α1c antibody. Results are representative of ≥3 similar experiments for each condition. F ) Coimmunoprecipitation of α1c and γ6 from mouse heart. The α1c immunoprecipitates of Triton X-100 extracts (1.5 mg) of mouse heart homogenates were size-fractionated on SDS-PAGE, transferred to nitrocellulose, and probed with anti-γ6 antibody. HEK cells were transfected with FLAG-γ6 and α1c. The α1c immunoprecipitates were probed with anti-γ6 antibody. The α1c subunit is truncated in heart (bottom arrow). Results are representative of 3 similar experiments.

    Journal: The FASEB Journal

    Article Title: Cardiac L-type calcium channel (Cav1.2) associates with ? subunits

    doi: 10.1096/fj.10-172353

    Figure Lengend Snippet: The γ subunits associate with Ca v 1.2 in the presence of α2/δ-1 subunit. A–D ) The α1c, β1b, and γ4 ( A ), γ6 ( B ), γ7 ( C ), and γ8 ( D ) subunits were expressed in HEK293 cells in the absence (−) or presence (+) of the α2/δ1 subunit. Lysates were prepared, and γ subunits were immunoprecipitated (IP) with anti-FLAG antibody. Negative controls were preimmune serum and cells without α2/δ-1 coexpression. Top panels: immunoblot with anti-α2 antibody. Bottom panels: immunoblot with HRP-conjugated, anti-FLAG antibody. Cont, control. E ) Same lysates as in ( B ). The α1c subunit was immunoprecipitated with an anti-α1c antibody. Top panel: immunoblot with HRP-conjugated, anti-FLAG antibody. Bottom panel: immunoblot with anti-α1c antibody. Results are representative of ≥3 similar experiments for each condition. F ) Coimmunoprecipitation of α1c and γ6 from mouse heart. The α1c immunoprecipitates of Triton X-100 extracts (1.5 mg) of mouse heart homogenates were size-fractionated on SDS-PAGE, transferred to nitrocellulose, and probed with anti-γ6 antibody. HEK cells were transfected with FLAG-γ6 and α1c. The α1c immunoprecipitates were probed with anti-γ6 antibody. The α1c subunit is truncated in heart (bottom arrow). Results are representative of 3 similar experiments.

    Article Snippet: The anti-α2 antibody (D219) and the anti-Flag antibody (A8592) were from Sigma.

    Techniques: Immunoprecipitation, SDS Page, Transfection

    The γ subunits associate with Ca v 1.2. A–D ) The α1c and β1b subunits were expressed in HEK293 cells in the absence (−) or presence (+) of the FLAG-tagged γ4 ( A ), γ6 ( B ), γ7 ( C ), and γ8 ( D ), subunits. Lysates were prepared, and α1c was immunoprecipitated (IP) with anti-α1c antibody. Negative controls were preimmune serum and cells without FLAG-γ-subunit coexpression. Top panels: immunoblot with HRP-conjugated, anti-FLAG antibody. Bottom panels: immunoblot with anti-α1c antibody. Cont, control. E–H ) FLAG-tagged γ4 ( E ), γ6 ( F ), γ7 ( G ), and γ8 ( H ) subunits and β1b subunit were expressed in the presence (+) or absence (−) of α1c in HEK293 cells; γ subunit was immunoprecipitated with anti-FLAG antibody. Top panels: immunoblot with anti-α1c antibody. Bottom panels: immunoblot with HRP-conjugated, anti-FLAG antibody. Results are representative of ≥3 similar experiments for each condition.

    Journal: The FASEB Journal

    Article Title: Cardiac L-type calcium channel (Cav1.2) associates with ? subunits

    doi: 10.1096/fj.10-172353

    Figure Lengend Snippet: The γ subunits associate with Ca v 1.2. A–D ) The α1c and β1b subunits were expressed in HEK293 cells in the absence (−) or presence (+) of the FLAG-tagged γ4 ( A ), γ6 ( B ), γ7 ( C ), and γ8 ( D ), subunits. Lysates were prepared, and α1c was immunoprecipitated (IP) with anti-α1c antibody. Negative controls were preimmune serum and cells without FLAG-γ-subunit coexpression. Top panels: immunoblot with HRP-conjugated, anti-FLAG antibody. Bottom panels: immunoblot with anti-α1c antibody. Cont, control. E–H ) FLAG-tagged γ4 ( E ), γ6 ( F ), γ7 ( G ), and γ8 ( H ) subunits and β1b subunit were expressed in the presence (+) or absence (−) of α1c in HEK293 cells; γ subunit was immunoprecipitated with anti-FLAG antibody. Top panels: immunoblot with anti-α1c antibody. Bottom panels: immunoblot with HRP-conjugated, anti-FLAG antibody. Results are representative of ≥3 similar experiments for each condition.

    Article Snippet: The anti-α2 antibody (D219) and the anti-Flag antibody (A8592) were from Sigma.

    Techniques: Immunoprecipitation

    Clusterin binds APP with intact and wild-type JH intracellularly. ( A ) Coimmunoprecipitation (CoIP) of endogenous APP and clusterin from brain homogenate. Anti-GAPDH was used as control IP antibody. ( B ) Clusterin-FLAG and APP WT without tag were either coexpressed or separately expressed in HEK293 cells. Cells with both clusterin and APP were directly lysed (coexpression), and cells with only clusterin or APP overexpression were lysed and the lysates were combined (pooled lysates). After IP with FLAG-agarose, the precipitated proteins were blotted with C20 antibody for APP and FLAG antibody for clusterin. ( C ) Schematic diagram showing the position of the JH in different CTFs. ( D ) C99, C83, and C80 were coexpressed with clusterin-FLAG in HEK293, and the lysates were subjected to anti-FLAG CoIP. C20 antibody was used to detect CTFs in the precipitates. ( E ) Clusterin-FLAG was coexpressed with wild-type C99 (C99 WT ), F615P containing C99 (C99 F615P ), and Flemish mutation containing C99 (C99 Fle ) in HEK293 cells, and cell lysates were immunoprecipitated using anti-FLAG antibody. Precipitates were Western blotted using C20 for C99 variants and anti-FLAG for clusterin. ( F ) CoIP of clusterin-FLAG with APP variants in HEK293 cells. IP was by anti-FLAG antibody, and APP detection was by C20 antibody.

    Journal: JCI Insight

    Article Title: BACE2, a conditional β-secretase, contributes to Alzheimer’s disease pathogenesis

    doi: 10.1172/jci.insight.123431

    Figure Lengend Snippet: Clusterin binds APP with intact and wild-type JH intracellularly. ( A ) Coimmunoprecipitation (CoIP) of endogenous APP and clusterin from brain homogenate. Anti-GAPDH was used as control IP antibody. ( B ) Clusterin-FLAG and APP WT without tag were either coexpressed or separately expressed in HEK293 cells. Cells with both clusterin and APP were directly lysed (coexpression), and cells with only clusterin or APP overexpression were lysed and the lysates were combined (pooled lysates). After IP with FLAG-agarose, the precipitated proteins were blotted with C20 antibody for APP and FLAG antibody for clusterin. ( C ) Schematic diagram showing the position of the JH in different CTFs. ( D ) C99, C83, and C80 were coexpressed with clusterin-FLAG in HEK293, and the lysates were subjected to anti-FLAG CoIP. C20 antibody was used to detect CTFs in the precipitates. ( E ) Clusterin-FLAG was coexpressed with wild-type C99 (C99 WT ), F615P containing C99 (C99 F615P ), and Flemish mutation containing C99 (C99 Fle ) in HEK293 cells, and cell lysates were immunoprecipitated using anti-FLAG antibody. Precipitates were Western blotted using C20 for C99 variants and anti-FLAG for clusterin. ( F ) CoIP of clusterin-FLAG with APP variants in HEK293 cells. IP was by anti-FLAG antibody, and APP detection was by C20 antibody.

    Article Snippet: The following mouse monoclonal antibodies were used: anti-Myc (9E10, Abcam), anti-FLAG (M2, Sigma-Aldrich), anti-clusterin (B-5, A-11, Santa Cruz Biotechnology), anti-BACE2 (H3, Santa Cruz Biotechnology), and anti–Aβ N-terminus (82E1, IBL).

    Techniques: Co-Immunoprecipitation Assay, Over Expression, Mutagenesis, Immunoprecipitation, Western Blot

    F615P, Flemish, and Arctic mutations abolished BACE2 cleavage of nascent APP. ( A ) APP WT and APP with the KKXX ER retention signal (APP ER ) were coexpressed with BACE1 or BACE2 in HEK293 cells. CTFs and APP were blotted with C20 and BACE1 and BACE2 were probed with anti-Myc antibody. mBACE1 and imBACE1, mature and immature BACE1, respectively. ( B ) APP ER with a FLAG tag inserted into APP after the signal peptide was coexpressed with BACE1 or BACE2 in HEK293 cells. Cell lysates were blotted for APP, CTF, and BACEs. Secreted APP (sAPP) in the conditioned media was enriched by immunoprecipitation using FLAG-agarose, and Western blotted using anti-FLAG antibody. ( C ) APP ER was coexpressed with BACE1 or BACE2 in HEK293 cells, and treated with the translation inhibitor cycloheximide (CHX, 100 μM) for the indicated times. Full-length APP ER , CTFs, and BACE2 were blotted. Immature BACE1 decreased due to ceased protein synthesis and continuous BACE1 maturation. Residual full-length APP ER relative to time 0 was plotted. Curves represent mean ± SEM. ( D ) Schematic diagram indicates domains and the first active site in BACE2. SP, signal peptide; PP, propeptide; D 110 , the first active site; TMD, transmembrane domain. APP ER was coexpressed with Myc-tagged BACE2, inactive BACE2 mutant (BACE2 D110A ), or BACE2 D110A without the propeptide (BACE2 D110A-Δpro ). BACE2 variants were immunoprecipitated (IP) with anti-Myc 9E10 antibody, and the precipitates were immunoblotted (IB) using the indicated antibodies. APP F615P ( E ) and APP Flemish and Arctic mutants ( F ) with the ER retention signal (APP F615P-ER , Flemish ER , and Arctic ER , respectively) were coexpressed with BACE2 in HEK293 cells. CTFs, APP, and BACE2 in lysates were blotted.

    Journal: JCI Insight

    Article Title: BACE2, a conditional β-secretase, contributes to Alzheimer’s disease pathogenesis

    doi: 10.1172/jci.insight.123431

    Figure Lengend Snippet: F615P, Flemish, and Arctic mutations abolished BACE2 cleavage of nascent APP. ( A ) APP WT and APP with the KKXX ER retention signal (APP ER ) were coexpressed with BACE1 or BACE2 in HEK293 cells. CTFs and APP were blotted with C20 and BACE1 and BACE2 were probed with anti-Myc antibody. mBACE1 and imBACE1, mature and immature BACE1, respectively. ( B ) APP ER with a FLAG tag inserted into APP after the signal peptide was coexpressed with BACE1 or BACE2 in HEK293 cells. Cell lysates were blotted for APP, CTF, and BACEs. Secreted APP (sAPP) in the conditioned media was enriched by immunoprecipitation using FLAG-agarose, and Western blotted using anti-FLAG antibody. ( C ) APP ER was coexpressed with BACE1 or BACE2 in HEK293 cells, and treated with the translation inhibitor cycloheximide (CHX, 100 μM) for the indicated times. Full-length APP ER , CTFs, and BACE2 were blotted. Immature BACE1 decreased due to ceased protein synthesis and continuous BACE1 maturation. Residual full-length APP ER relative to time 0 was plotted. Curves represent mean ± SEM. ( D ) Schematic diagram indicates domains and the first active site in BACE2. SP, signal peptide; PP, propeptide; D 110 , the first active site; TMD, transmembrane domain. APP ER was coexpressed with Myc-tagged BACE2, inactive BACE2 mutant (BACE2 D110A ), or BACE2 D110A without the propeptide (BACE2 D110A-Δpro ). BACE2 variants were immunoprecipitated (IP) with anti-Myc 9E10 antibody, and the precipitates were immunoblotted (IB) using the indicated antibodies. APP F615P ( E ) and APP Flemish and Arctic mutants ( F ) with the ER retention signal (APP F615P-ER , Flemish ER , and Arctic ER , respectively) were coexpressed with BACE2 in HEK293 cells. CTFs, APP, and BACE2 in lysates were blotted.

    Article Snippet: The following mouse monoclonal antibodies were used: anti-Myc (9E10, Abcam), anti-FLAG (M2, Sigma-Aldrich), anti-clusterin (B-5, A-11, Santa Cruz Biotechnology), anti-BACE2 (H3, Santa Cruz Biotechnology), and anti–Aβ N-terminus (82E1, IBL).

    Techniques: FLAG-tag, Immunoprecipitation, Western Blot, Mutagenesis

    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

    Analysis of AKT3 activity in vitro. ( A ) The primary structure of AKT3 showing the relative positions of the pleckstrin homology (PH) domain for lipid binding the catalytic kinase domain and C-terminal (C-ter) region. Mutations identified to date are shown along with the numbers of patients with these mutations in brackets. ( B ) Catalytic kinase domain and C-terminal localizing patient-derived AKT3 mutations are associated with elevated kinase activity. Ectopically expressed wild-type (WT) AKT, a kinase dead variant K177M, the E17K activating pleckstrin homology domain mutant and various patient mutants were assessed for kinase activity using a GSK3β peptide as a substrate in an ex vivo kinase assay. The upper panel shows immune detection of phosphorylated GSK3β peptide following western blotting with anti-phospho-GSK3β (Ser9/Ser21) antibody. The patient mutants all exhibit elevated phospho-activity compared to wild-type. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal (a.u. = arbitrary units). Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. ( C ) Pleckstrin homology domain localizing patient mutations are associated with elevated kinase activity and altered phospholipid-binding profile. Left panels show western blot analysis of phospho-GSK3β (Ser9/Ser21) of ectopically expressed wild-type, K177M kinase dead and three pleckstrin homology domain patient mutants; E17K, N53K and F54Y. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal. Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. The bottom panels depict PIP-membranes seeded with various lipids and phospholipids for dot blot binding analysis. Ectopically expressed FLAG-tagged wild-type and AKT3 pleckstrin homology domain mutants were incubated with the PIP Strips and bound protein detected by western blotting using anti-FLAG. All three pleckstrin homology domain mutants exhibit altered and elevated binding to specific phospholipids compared to wild-type. DMEG = dysplastic megalencephaly; HMEG = hemimegalencephaly; LPA = lysophophatidic acid; LPC = lysophosphocholine; MEG = megalencephaly; P = phosphate; PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PMG = polymicrogryria; PS = phosphatidylserine; PtdIns = phosphatidylinositol; S1P = sphingosine-1-phosphate.

    Journal: Brain

    Article Title: Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly

    doi: 10.1093/brain/awx203

    Figure Lengend Snippet: Analysis of AKT3 activity in vitro. ( A ) The primary structure of AKT3 showing the relative positions of the pleckstrin homology (PH) domain for lipid binding the catalytic kinase domain and C-terminal (C-ter) region. Mutations identified to date are shown along with the numbers of patients with these mutations in brackets. ( B ) Catalytic kinase domain and C-terminal localizing patient-derived AKT3 mutations are associated with elevated kinase activity. Ectopically expressed wild-type (WT) AKT, a kinase dead variant K177M, the E17K activating pleckstrin homology domain mutant and various patient mutants were assessed for kinase activity using a GSK3β peptide as a substrate in an ex vivo kinase assay. The upper panel shows immune detection of phosphorylated GSK3β peptide following western blotting with anti-phospho-GSK3β (Ser9/Ser21) antibody. The patient mutants all exhibit elevated phospho-activity compared to wild-type. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal (a.u. = arbitrary units). Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. ( C ) Pleckstrin homology domain localizing patient mutations are associated with elevated kinase activity and altered phospholipid-binding profile. Left panels show western blot analysis of phospho-GSK3β (Ser9/Ser21) of ectopically expressed wild-type, K177M kinase dead and three pleckstrin homology domain patient mutants; E17K, N53K and F54Y. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal. Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. The bottom panels depict PIP-membranes seeded with various lipids and phospholipids for dot blot binding analysis. Ectopically expressed FLAG-tagged wild-type and AKT3 pleckstrin homology domain mutants were incubated with the PIP Strips and bound protein detected by western blotting using anti-FLAG. All three pleckstrin homology domain mutants exhibit altered and elevated binding to specific phospholipids compared to wild-type. DMEG = dysplastic megalencephaly; HMEG = hemimegalencephaly; LPA = lysophophatidic acid; LPC = lysophosphocholine; MEG = megalencephaly; P = phosphate; PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PMG = polymicrogryria; PS = phosphatidylserine; PtdIns = phosphatidylinositol; S1P = sphingosine-1-phosphate.

    Article Snippet: FLAG immunoprecipitated AKT3 was eluted from the FLAG beads using FLAG peptide (3× FLAG® Peptide, F4799 Sigma-Aldrich).

    Techniques: Activity Assay, In Vitro, Binding Assay, Derivative Assay, Variant Assay, Mutagenesis, Ex Vivo, Kinase Assay, Western Blot, Quantitation Assay, Dot Blot, Incubation

    PBRM1 and p53 display a physical association that is enhanced after DNA damage. a U2OS cells (left) and HEK293T cells (right) were transfected with vector or Flag-PBRM1 and harvested for immunoprecipitation with Flag-M2 beads and elution with 3X Flag peptide. Inputs and eluates were analyzed by immunoblots. b , c H1299 cells were transfected with Flag-PBRM1 and Myc-p53 and treated with etoposide (50 μM, b ) or bleomycin (10 μg/ml, c ) for the indicated times. Lysates were subjected to immunoprecipitation with Flag-M2 beads. Inputs and eluates were analyzed by immunoblots. d HEK293 cells were treated with vehicle (DMSO) or etoposide (50 μM) for 8 h and harvested for immunoprecipitation with control IgG and p53 antibodies. The bound PBRM1 and p53 were examined by immunoblots. e HCT116 and HCT116 p53−/− cells were treated with DMSO or 50 μM etoposide for 24 h. Lysates were immunoprecipitated with p53 antibody. The bound PBRM1 and p53 were examined by immunoblots. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: PBRM1 acts as a p53 lysine-acetylation reader to suppress renal tumor growth

    doi: 10.1038/s41467-019-13608-1

    Figure Lengend Snippet: PBRM1 and p53 display a physical association that is enhanced after DNA damage. a U2OS cells (left) and HEK293T cells (right) were transfected with vector or Flag-PBRM1 and harvested for immunoprecipitation with Flag-M2 beads and elution with 3X Flag peptide. Inputs and eluates were analyzed by immunoblots. b , c H1299 cells were transfected with Flag-PBRM1 and Myc-p53 and treated with etoposide (50 μM, b ) or bleomycin (10 μg/ml, c ) for the indicated times. Lysates were subjected to immunoprecipitation with Flag-M2 beads. Inputs and eluates were analyzed by immunoblots. d HEK293 cells were treated with vehicle (DMSO) or etoposide (50 μM) for 8 h and harvested for immunoprecipitation with control IgG and p53 antibodies. The bound PBRM1 and p53 were examined by immunoblots. e HCT116 and HCT116 p53−/− cells were treated with DMSO or 50 μM etoposide for 24 h. Lysates were immunoprecipitated with p53 antibody. The bound PBRM1 and p53 were examined by immunoblots. Source data are provided as a Source Data file.

    Article Snippet: Beads were washed four times with EBC buffer and eluted with 3X Flag peptide (F4799, Sigma-Aldrich) overnight at 4 °C.

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

    Acetylation on K382 of p53 promotes PBRM1 binding. a Schematic depiction of the functional domains of p53 and truncated constructs. FL full-length, TAD transactivation domain, PRD proline-rich domain, DBD DNA-binding domain, TD tetramerization domain, CTD C-terminal domain. b HEK293T cells were transfected with vectors, Flag-PBRM1, Myc-p53 and Myc-p53 truncated constructs. After immunoprecipitation with Flag-M2 beads, the inputs and eluates were analyzed by immunoblots. c Vectors, Flag-PBRM1, Myc-p53 and HA-Tip60, HA-PCAF, HA-CBP or HA-p300 were transfected into HEK293T cells as indicated. Lysates were used for anti-Flag immunoprecipitation and 3X Flag peptide elution. The inputs and eluates were analyzed by immunoblots. d HEK293T cells were transfected with Flag-PBRM1, Myc-p53 and HA-p300. The amount of HA-p300 was titrated as indicated. Lysates were used in immunoprecipitations with Flag-M2 beads, and inputs and eluates were analyzed by immunoblots. e Vectors, Flag-PBRM1, HA-p300 and Myc-p53 (WT) or Myc-p53 6KR mutant were transfected into HEK293T cells as indicated. Lysates were used for anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were examined by immunoblots. 6KR: lysines mutated to arginines on p53 at K370, K372, K373, K381, K382, and K386. f , g Biotinylated p53 peptides with lysine acetylation at the indicated sites were incubated with lysates from H1299 cells ( f ) or H1299 cells transfected with full-length PBRM1 or the PBRM1 bromodomains (Flag-PBRM1 FL or Flag-PBRM1-BD1–6, respectively, g ). The peptides were pulled down with streptavidin beads and the associated proteins were analyzed by immunoblots. 6KAc: lysines acetylated on p53 at K370, K372, K373, K381, K382, and K386. h Vectors, Flag-PBRM1, HA-p300 and Myc-p53 (WT) or Myc-p53 K382R mutant were transfected into HEK293T cells as indicated. Lysates were used for anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were examined by immunoblots. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: PBRM1 acts as a p53 lysine-acetylation reader to suppress renal tumor growth

    doi: 10.1038/s41467-019-13608-1

    Figure Lengend Snippet: Acetylation on K382 of p53 promotes PBRM1 binding. a Schematic depiction of the functional domains of p53 and truncated constructs. FL full-length, TAD transactivation domain, PRD proline-rich domain, DBD DNA-binding domain, TD tetramerization domain, CTD C-terminal domain. b HEK293T cells were transfected with vectors, Flag-PBRM1, Myc-p53 and Myc-p53 truncated constructs. After immunoprecipitation with Flag-M2 beads, the inputs and eluates were analyzed by immunoblots. c Vectors, Flag-PBRM1, Myc-p53 and HA-Tip60, HA-PCAF, HA-CBP or HA-p300 were transfected into HEK293T cells as indicated. Lysates were used for anti-Flag immunoprecipitation and 3X Flag peptide elution. The inputs and eluates were analyzed by immunoblots. d HEK293T cells were transfected with Flag-PBRM1, Myc-p53 and HA-p300. The amount of HA-p300 was titrated as indicated. Lysates were used in immunoprecipitations with Flag-M2 beads, and inputs and eluates were analyzed by immunoblots. e Vectors, Flag-PBRM1, HA-p300 and Myc-p53 (WT) or Myc-p53 6KR mutant were transfected into HEK293T cells as indicated. Lysates were used for anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were examined by immunoblots. 6KR: lysines mutated to arginines on p53 at K370, K372, K373, K381, K382, and K386. f , g Biotinylated p53 peptides with lysine acetylation at the indicated sites were incubated with lysates from H1299 cells ( f ) or H1299 cells transfected with full-length PBRM1 or the PBRM1 bromodomains (Flag-PBRM1 FL or Flag-PBRM1-BD1–6, respectively, g ). The peptides were pulled down with streptavidin beads and the associated proteins were analyzed by immunoblots. 6KAc: lysines acetylated on p53 at K370, K372, K373, K381, K382, and K386. h Vectors, Flag-PBRM1, HA-p300 and Myc-p53 (WT) or Myc-p53 K382R mutant were transfected into HEK293T cells as indicated. Lysates were used for anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were examined by immunoblots. Source data are provided as a Source Data file.

    Article Snippet: Beads were washed four times with EBC buffer and eluted with 3X Flag peptide (F4799, Sigma-Aldrich) overnight at 4 °C.

    Techniques: Binding Assay, Functional Assay, Construct, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Incubation

    Bromodomain 4 of PBRM1 is required for recognition of acetylated K382 on p53. a Schematic depiction of the functional domains of PBRM1 and truncated constructs. WT: wild-type, BAH: bromo-adjacent homology domain, HMG: high-mobility group domain. b , c HEK293T cells were transfected with vectors, Flag-WT PBRM1 and Flag-PBRM1 truncated constructs ( b ), and each individual or all the PBRM1 bromodomains (BD1–6, c ). After immunoprecipitation with Flag-M2 beads, the inputs and eluates were analyzed by immunoblots. d Schematic of key amino acid residues of binding pockets in PBRM1 bromodomains 4 and 5. The critical YN residues are underlined and mutations are red. e – g H1299 PBRM1 KO cells were transfected with Flag-PBRM1 bromodomains 4 and 5 (Flag-BD45) containing mutations (BD4* or BD5*) that abolish acetyl-lysine recognition in each domain ( e ), Flag-BD45 containing tumor-derived mutations ( f ) or full-length PBRM1 containing the BD4* mutation ( g ). Lysates were incubated with biotinylated p53 peptides with lysine acetylation at the indicated sites. The peptides were pulled down with streptavidin beads and the associated proteins were immunoblotted. h Vectors, Myc-p53, HA-p300, and Flag-PBRM1 with or without the BD4* mutation were transfected into HEK293T PBRM1 knockout cells. The lysates were subjected to anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were analyzed in immunoblots. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: PBRM1 acts as a p53 lysine-acetylation reader to suppress renal tumor growth

    doi: 10.1038/s41467-019-13608-1

    Figure Lengend Snippet: Bromodomain 4 of PBRM1 is required for recognition of acetylated K382 on p53. a Schematic depiction of the functional domains of PBRM1 and truncated constructs. WT: wild-type, BAH: bromo-adjacent homology domain, HMG: high-mobility group domain. b , c HEK293T cells were transfected with vectors, Flag-WT PBRM1 and Flag-PBRM1 truncated constructs ( b ), and each individual or all the PBRM1 bromodomains (BD1–6, c ). After immunoprecipitation with Flag-M2 beads, the inputs and eluates were analyzed by immunoblots. d Schematic of key amino acid residues of binding pockets in PBRM1 bromodomains 4 and 5. The critical YN residues are underlined and mutations are red. e – g H1299 PBRM1 KO cells were transfected with Flag-PBRM1 bromodomains 4 and 5 (Flag-BD45) containing mutations (BD4* or BD5*) that abolish acetyl-lysine recognition in each domain ( e ), Flag-BD45 containing tumor-derived mutations ( f ) or full-length PBRM1 containing the BD4* mutation ( g ). Lysates were incubated with biotinylated p53 peptides with lysine acetylation at the indicated sites. The peptides were pulled down with streptavidin beads and the associated proteins were immunoblotted. h Vectors, Myc-p53, HA-p300, and Flag-PBRM1 with or without the BD4* mutation were transfected into HEK293T PBRM1 knockout cells. The lysates were subjected to anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were analyzed in immunoblots. Source data are provided as a Source Data file.

    Article Snippet: Beads were washed four times with EBC buffer and eluted with 3X Flag peptide (F4799, Sigma-Aldrich) overnight at 4 °C.

    Techniques: Functional Assay, Construct, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Derivative Assay, Mutagenesis, Incubation, Knock-Out