anti flag m2 antibody Millipore Search Results


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  • 95
    Millipore anti flag
    SH3BP5 binds to JNK through SH3BD, KIM1, and KIM2. A , structure of mouse SH3BP5 (mSH3BP5) and deletion mutants of mSH3BP5. B , 6×His-mSH3BP5 (H6-mSH3BP5) or its deletion mutant, produced in bacteria, was purified with TALON Metal Resin. Negative-control purification ( NC ) was equally done from bacteria in which the empty vector was introduced. The purified proteins were fractionated by SDS-PAGE, followed by Coomassie Blue staining. FL : SH3BP5 full-length, Δ SH3 : SH3BP5 lacking SH3BD, Δ1: SH3BP5 lacking KIM1, Δ2: SH3BP5 lacking KIM2. C , 6×His-GST, 6×His-mSH3BP5, and 6×His-mSH3BP5 deletion mutants were produced in bacteria and purified with TALON Metal Resin. Each protein (0.1 μg) was fractionated as an input for SDS-PAGE and subjected to immunoblot analysis with antibody to <t>HisG.</t> D , <t>FLAG-JNK-1a1,</t> immunoprecipitated from the cell lysates with the FLAG antibody, was mixed with indicated recombinant 6×His-GST, 6×His-mSH3BP5, or a 6×His-mSH3BP5 deletion mutant (1 μg per sample), washed extensively, and subjected to SDS-PAGE and immunoblot analysis. Immunoblot analysis was performed with the antibody to HisG for the detection of 6×His-tagged recombinant proteins and the M2 antibody to FLAG for the detection of FLAG-JNK-1a1.
    Anti Flag, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 19502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore anti flag tag
    Homo- and hetero-multimerization among the PHDs ( A ) Homo-/hetero-multimerization of PHD2 and PHD3 with PHDs. HEK-293T cells were transfected with <t>FLAG–PHD2</t> or FLAG–PHD3 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or <t>β-actin.</t> Asterisk indicates the heavy/light chains of anti-FLAG Ab. ( B ) Homo-/hetero-multimerization of PHD1 with PHDs. HEK-293T cells were transfected with FLAG–PHD1 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. Asterisk indicates the heavy chain of anti-FLAG Ab. ( C ) C-terminal portion of PHD3 is required for homomultimerization. HEK-293T cells were transfected with Myc–PHD3 together with PHD3 fragment plasmids (FLAG–F1, FLAG–F2 or FLAG–F3). Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. ( D ) Hetero-multimerization of ΔPHDs with PHD3. Myc–ΔPHDs or Myc–PHD1, Myc–PHD2, Myc–PHD3 and FLAG–PHD3 were co-transfected into HEK-293T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG Ab and the blot was probed with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or β-actin. IB, immunoblot; IP, immunoprecipitation.
    Anti Flag Tag, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore anti flag ab cross linked beads
    Homo- and hetero-multimerization among the PHDs ( A ) Homo-/hetero-multimerization of PHD2 and PHD3 with PHDs. HEK-293T cells were transfected with <t>FLAG–PHD2</t> or FLAG–PHD3 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or <t>β-actin.</t> Asterisk indicates the heavy/light chains of anti-FLAG Ab. ( B ) Homo-/hetero-multimerization of PHD1 with PHDs. HEK-293T cells were transfected with FLAG–PHD1 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. Asterisk indicates the heavy chain of anti-FLAG Ab. ( C ) C-terminal portion of PHD3 is required for homomultimerization. HEK-293T cells were transfected with Myc–PHD3 together with PHD3 fragment plasmids (FLAG–F1, FLAG–F2 or FLAG–F3). Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. ( D ) Hetero-multimerization of ΔPHDs with PHD3. Myc–ΔPHDs or Myc–PHD1, Myc–PHD2, Myc–PHD3 and FLAG–PHD3 were co-transfected into HEK-293T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG Ab and the blot was probed with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or β-actin. IB, immunoblot; IP, immunoprecipitation.
    Anti Flag Ab Cross Linked Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Millipore anti flag clone m2 ab
    Homo- and hetero-multimerization among the PHDs ( A ) Homo-/hetero-multimerization of PHD2 and PHD3 with PHDs. HEK-293T cells were transfected with <t>FLAG–PHD2</t> or FLAG–PHD3 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or <t>β-actin.</t> Asterisk indicates the heavy/light chains of anti-FLAG Ab. ( B ) Homo-/hetero-multimerization of PHD1 with PHDs. HEK-293T cells were transfected with FLAG–PHD1 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. Asterisk indicates the heavy chain of anti-FLAG Ab. ( C ) C-terminal portion of PHD3 is required for homomultimerization. HEK-293T cells were transfected with Myc–PHD3 together with PHD3 fragment plasmids (FLAG–F1, FLAG–F2 or FLAG–F3). Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. ( D ) Hetero-multimerization of ΔPHDs with PHD3. Myc–ΔPHDs or Myc–PHD1, Myc–PHD2, Myc–PHD3 and FLAG–PHD3 were co-transfected into HEK-293T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG Ab and the blot was probed with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or β-actin. IB, immunoblot; IP, immunoprecipitation.
    Anti Flag Clone M2 Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Millipore mouse anti flag tag ab
    Detection of the T. gondii mitochondrial ribosome. A. Protein immunoblot analysis of endogenously tagged TgmS35, TgbL12m and TguL3m from total cell lysate separated by SDS‐PAGE and detected using <t>anti‐HA/Strep/FLAG</t> antibodies. B. Total cell lysate separated by blue‐native PAGE and immunoblotted to detect TgmS35, TgbL12m and TguL3m with anti‐HA/Strep/FLAG antibodies. C. Validation of the promoter integration in the Tg mS15 locus via PCR analysis using primers 1, 2, 3, and 4, represented in Fig. S4 D. Western blot (top panel) of TgmS35‐3xHA in lines where Tg mS35 is under its native promoter (TgmS35‐3HA) or where Tg mS35 or Tg uS15m are under regulatable promoters (r Tg mS35‐3HA and Tg mS35‐3HA/r Tg uS15m respectively). Low panel shows instant blue staining for loading control. E. comparison of results from RT‐PCR performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (total), from IP of TgTom22 (Tom22) or from IP of TgmS35 (TgmS35). F. An example RT‐PCR experiment performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (Parental – total), from IP of TgTom22 (Tom22‐IP) or from IP of TgmS35 (TgmS35‐IP).
    Mouse Anti Flag Tag Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 562 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Millipore anti flag m2 ab agarose
    Sam68 and hnRNP L proteins associate via protein-protein interactions and are not bridged by nucleic acids . (A) LNCaP cell nuclear extracts were subjected to IP using either anti-Sam68 antisera or an anti-hnRNP L monoclonal antibody. Recovered material was subjected to Western analysis with antibodies specific to hnRNP L (left panel) or Sam68 (right panel). (B) HEK293 cells were transfected with expression vectors for <t>FLAG-Sam68</t> or FLAG alone, and hnRNP L-GST, and subjected to IP using anti-FLAG M2 agarose. Recovered material was subjected to Western analysis with antisera to GST. (C) HEK293 cell nuclear extracts were fractionated on sucrose gradients before (upper panel) or after (lower panel) treatment with MNase to digest nucleic acids. Fraction 20 (containing low molecular weight material) was taken from the top of the gradient, and fraction 1 (containing high molecular weight material) was taken from the bottom. Pelleted material is indicated as P. The migration of individual proteins in each fraction was monitored by SDS-PAGE and Western analysis. The mobility of size markers on the gradients is shown.
    Anti Flag M2 Ab Agarose, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Millipore anti flag m2 mouse ab
    Dll1IC bound to Smad2, Smad3 and Smad4. (a) Result of screening for transcription factors that bind to Dll1IC. Note that Smad binding sequences showed strong signals (boxed). (b) Signal from Smad binding sequences (boxed) were enhanced by the addition of recombinant Dll1IC protein to the nuclear extract before immunoprecipitation. Signals from Pax-5 binding sequence and mineral corticoid response element (MRE) were also enhanced by the addition of recombinant Dll1IC protein. On the other hand, signals from NF-κB binding site and NF-E2 binding sequence were disappeared by the addition of recombinant Dll1IC protein. Spots along the right and bottom side of arrays are markers for alignment. (c) COS7 cells were transiently co-transfected with each expression vector for 8 Smads and Dll1IC expression vector with or without expression vectors for constitutive activated receptor (caALK5-HA or caALK6-HA). Forty-eight hours after transfection, lysates from co-transfected COS7 cells were subjected to immunoprecipitation (IP) with the indicated antibodies, followed by western blotting (WB). αDll1IC, rabbit anti-Dll1IC antibody; αFlag, mouse monoclonal <t>anti-FLAG</t> M2 antibody. Levels of expression of Smads and Dll1IC were determined by western blotting and are shown in the bottom two panels.
    Anti Flag M2 Mouse Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore anti flag m2 hrp ab
    Dll1IC bound to Smad2, Smad3 and Smad4. (a) Result of screening for transcription factors that bind to Dll1IC. Note that Smad binding sequences showed strong signals (boxed). (b) Signal from Smad binding sequences (boxed) were enhanced by the addition of recombinant Dll1IC protein to the nuclear extract before immunoprecipitation. Signals from Pax-5 binding sequence and mineral corticoid response element (MRE) were also enhanced by the addition of recombinant Dll1IC protein. On the other hand, signals from NF-κB binding site and NF-E2 binding sequence were disappeared by the addition of recombinant Dll1IC protein. Spots along the right and bottom side of arrays are markers for alignment. (c) COS7 cells were transiently co-transfected with each expression vector for 8 Smads and Dll1IC expression vector with or without expression vectors for constitutive activated receptor (caALK5-HA or caALK6-HA). Forty-eight hours after transfection, lysates from co-transfected COS7 cells were subjected to immunoprecipitation (IP) with the indicated antibodies, followed by western blotting (WB). αDll1IC, rabbit anti-Dll1IC antibody; αFlag, mouse monoclonal <t>anti-FLAG</t> M2 antibody. Levels of expression of Smads and Dll1IC were determined by western blotting and are shown in the bottom two panels.
    Anti Flag M2 Hrp Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore monoclonal anti flag antibody
    Interactions of phosphorylated and dephosphorylated forms of CMG and Tof1 with each other as revealed by Western blotting. ( A , Left ) Zn 2+ Phos-tag gel of phosphorylated and dephosphorylated Tof1. ( A , Right ) <t>SDS/PAGE</t> profiles of both untreated Tof1-myc and that treated with λ-phosphatase. Actin loading controls are also shown. ( B ) Interaction of Tof1-myc–Csm3 complex untreated and treated with λ-phosphatase with immobilized CMG-His 6 . ( C ) WBs showing binding of Tof1-myc untreated (-) and treated with λ-phosphatase (+) to immobilized <t>FLAG-tagged</t> Mcm2–7. ( D ) Interaction of Tof1-myc with immobilized untreated CMG-His 6 ( Left ) and dephosphorylated immobilized CMG-His 6 ( Right ). ( E ) Interaction of Tof1-myc with untreated immobilized Mcm2–7-FLAG and with dephosphorylated and immobilized Mcm2–7-FLAG. ( F ) Quantification of binding of Tof1-myc untreated (-) and dephosphorylated (+) to immobilized CMG. ( G ) Binding of untreated (-) and dephosphorylated Tof1-myc to immobilized Mcm2–7-FLAG (not dephosphorylated with λ-phosphatase). ( H ) Binding of untreated Tof1-myc to immobilized untreated CMG-His 6 and to the same after dephosphorylation with λ-phosphatase. ( I ) Quantification of binding of Tof1-myc to untreated immobilized Mcm2–7-FLAG and to the same after dephosphorylation with λ-phosphatase. ( J ) Quantification of binding of Cdc45 in solution to untreated immobilized Tof1-myc or to the same after treatment with λ-phosphatase. ( K ) Quantification of the binding of immobilized GINS complex to Tof1-myc in solution before (-) and after (+) treatment with λ-phosphatase. Error bars represent standard error.
    Monoclonal Anti Flag Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1347 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore anti flag m2 magnetic beads
    RIF1 promotes PP1 to dephosphorylate AXIN. a , b Immunoprecipitation was performed with PP1 antibody and the precipitated protein complexes were analyzed by western blots with antibodies to RIF1 and AXIN in H1299 ( a ) and SK-MES-1 ( b ) cells. c , d RIF1-silenced H1299 ( c ) and SK-MES-1 ( d ) cells and the scrambled control cells were transfected with Flag-tagged AXIN plasmid. Immunoprecipitation was done with anti-FLAG <t>M2</t> magnetic beads and the precipitated complexes, which were pull down by Flag-tagged AXIN were subjected to western blot with antibodies to RIF1, PP1 and p-Ser. e, f H1299 ( e ) and SK-MES-1 ( f ) cells were co-transfected Flag-tagged AXIN plasmid with control vector or RIF1 overexpression plasmid. Immunoprecipitation was performed with anti-FLAG M2 magnetic beads and the precipitated complexes were analyzed by western blots with antibodies to RIF1, PP1 and p-Ser (mouse IgG conjugated with magnetic beads being the negative control)
    Anti Flag M2 Magnetic Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 6538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Millipore anti flag m2 ab agarose beads
    RIF1 promotes PP1 to dephosphorylate AXIN. a , b Immunoprecipitation was performed with PP1 antibody and the precipitated protein complexes were analyzed by western blots with antibodies to RIF1 and AXIN in H1299 ( a ) and SK-MES-1 ( b ) cells. c , d RIF1-silenced H1299 ( c ) and SK-MES-1 ( d ) cells and the scrambled control cells were transfected with Flag-tagged AXIN plasmid. Immunoprecipitation was done with anti-FLAG <t>M2</t> magnetic beads and the precipitated complexes, which were pull down by Flag-tagged AXIN were subjected to western blot with antibodies to RIF1, PP1 and p-Ser. e, f H1299 ( e ) and SK-MES-1 ( f ) cells were co-transfected Flag-tagged AXIN plasmid with control vector or RIF1 overexpression plasmid. Immunoprecipitation was performed with anti-FLAG M2 magnetic beads and the precipitated complexes were analyzed by western blots with antibodies to RIF1, PP1 and p-Ser (mouse IgG conjugated with magnetic beads being the negative control)
    Anti Flag M2 Ab Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 82/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore monoclonal anti flag m2 antibody
    Ezh2 is threonine-phosphorylated. A , Ezh2 is phosphorylated in sites other than Ser-21. HEK293T cells exogenously expressing <t>FLAG-tagged</t> wild-type Ezh2 or S21A were radiolabeled with [γ- 32 P]ATP. Following immunoprecipitation using FLAG <t>antibodies,</t> bound proteins were treated with no phosphatase, with PP1, or with λ-PPase. Autoradiography ( Autorad ) confirms that both wild-type and S21A mutant Ezh2 are phosphorylated and that the signal diminishes upon phosphatase treatment. Bottom panel verifies that equal amounts FLAG-Ezh2 were used in the assay. Ctrl , control. B , Ezh2 is threonine-phosphorylated. FLAG-Ezh2 was transfected into HEK293T cells and immunoprecipitated using FLAG antibodies. Bound proteins were treated with or without λ-PPase and subjected to Western blot analysis using antibodies specific for phospho-serine (α -pSer ), phospho-threonine (α -pThr ), and phospho-tyrosine (α -pTyr ).
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 30447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore anti flag m2 affinity gel
    SRSF2 binding to the RBM25 Lys-77 peptide is regulated by methylation. A , immobilized non-methyl or methyl peptides centered on RBM25 Lys-77 were used to capture proteins from nuclear extract of 293T cells prepared with SILAC. Bound proteins were identified and quantified using LC/MS-MS. B , relative binding to methyl over non-methyl peptide was measured in two experiments with isotopic labels reversed. Each axis represents one experiment with the ratio of bound protein shown on a log 2 scale. Several SR proteins are highlighted in red. C , recombinant SRSF2 or the indicated domain was expressed with N-terminal GST in E. coli and tested for binding to immobilized peptide. Bound proteins were visualized by Western blotting for the GST tag. 3xMBT was used as a positive control for mono- and dimethylated peptides. D , SRSF2 was tested for binding to a panel of non-methylated peptides from proteins involved in transcription, splicing, and translation. E , RBM25 and SRSF2 were expressed by transient transfection in 293T cells with <t>FLAG</t> and Myc tags, respectively. Following FLAG, co-IP bound SRSF2 was measured by Western blotting. F , a panel of SR proteins was tested for binding to immobilized RBM25 peptides. G , SRSF1, SRSF2, and 3xMBT were incubated with varying concentrations of the indicated immobilized RBM25 peptide, and the amount of bound protein was measured by quantitative Western blotting for GST with near-IR fluorescent secondary <t>antibodies.</t> Error bars indicate mean ± S.E. ( n = 3).
    Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 21354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore m2 anti flag ab conjugated sepharose
    SRSF2 binding to the RBM25 Lys-77 peptide is regulated by methylation. A , immobilized non-methyl or methyl peptides centered on RBM25 Lys-77 were used to capture proteins from nuclear extract of 293T cells prepared with SILAC. Bound proteins were identified and quantified using LC/MS-MS. B , relative binding to methyl over non-methyl peptide was measured in two experiments with isotopic labels reversed. Each axis represents one experiment with the ratio of bound protein shown on a log 2 scale. Several SR proteins are highlighted in red. C , recombinant SRSF2 or the indicated domain was expressed with N-terminal GST in E. coli and tested for binding to immobilized peptide. Bound proteins were visualized by Western blotting for the GST tag. 3xMBT was used as a positive control for mono- and dimethylated peptides. D , SRSF2 was tested for binding to a panel of non-methylated peptides from proteins involved in transcription, splicing, and translation. E , RBM25 and SRSF2 were expressed by transient transfection in 293T cells with <t>FLAG</t> and Myc tags, respectively. Following FLAG, co-IP bound SRSF2 was measured by Western blotting. F , a panel of SR proteins was tested for binding to immobilized RBM25 peptides. G , SRSF1, SRSF2, and 3xMBT were incubated with varying concentrations of the indicated immobilized RBM25 peptide, and the amount of bound protein was measured by quantitative Western blotting for GST with near-IR fluorescent secondary <t>antibodies.</t> Error bars indicate mean ± S.E. ( n = 3).
    M2 Anti Flag Ab Conjugated Sepharose, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore cross linking mouse anti flag ab m2
    SRSF2 binding to the RBM25 Lys-77 peptide is regulated by methylation. A , immobilized non-methyl or methyl peptides centered on RBM25 Lys-77 were used to capture proteins from nuclear extract of 293T cells prepared with SILAC. Bound proteins were identified and quantified using LC/MS-MS. B , relative binding to methyl over non-methyl peptide was measured in two experiments with isotopic labels reversed. Each axis represents one experiment with the ratio of bound protein shown on a log 2 scale. Several SR proteins are highlighted in red. C , recombinant SRSF2 or the indicated domain was expressed with N-terminal GST in E. coli and tested for binding to immobilized peptide. Bound proteins were visualized by Western blotting for the GST tag. 3xMBT was used as a positive control for mono- and dimethylated peptides. D , SRSF2 was tested for binding to a panel of non-methylated peptides from proteins involved in transcription, splicing, and translation. E , RBM25 and SRSF2 were expressed by transient transfection in 293T cells with <t>FLAG</t> and Myc tags, respectively. Following FLAG, co-IP bound SRSF2 was measured by Western blotting. F , a panel of SR proteins was tested for binding to immobilized RBM25 peptides. G , SRSF1, SRSF2, and 3xMBT were incubated with varying concentrations of the indicated immobilized RBM25 peptide, and the amount of bound protein was measured by quantitative Western blotting for GST with near-IR fluorescent secondary <t>antibodies.</t> Error bars indicate mean ± S.E. ( n = 3).
    Cross Linking Mouse Anti Flag Ab M2, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore hrp conjugated anti flag m2 ab
    Analysis of the Ab3 anti-anti-Id scFv 69 response in sera of BALB/c mice (immunisation protocol 1) by inhibition of the binding of anti-Id scFv 69 (Ab2) on immobilised trastuzumab F(ab′) 2 fragments (Ab1) by inhibition ELISA. Serial dilutions of preimmune sera or sera from mice each of the three groups: primed with HER-2/neu ECD-Fc fusion protein or with PBS or with anti-Id scFv 69 were preincubated with soluble anti-Id scFv 69. This solution was subsequently incubated for 2 h on trastuzumab F(ab′) 2 fragments followed by the detection of bound scFv by <t>HRP-conjugated</t> M2 <t>anti-FLAG</t> mAb. The results obtained are expressed as percent inhibition at each serum dilution.
    Hrp Conjugated Anti Flag M2 Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti flag high sensitivity m2 coated 96 well plates
    WRS is secreted in response to virus infection. (A) ELISA of WRS levels in the supernatant of U-937 and THP-1 cells infected with VSV-GFP at a multiplicity of infection (MOI) of 3 at indicated time points. (B) mRNA expression level of WRS shown in panel A was determined by qRT-PCR. (C) <t>Flag-tagged</t> mWRS expressing stable Raw264.7 cells and control cells were infected with VSV-GFP (upper, left) or HSV-GFP (upper, right) at an MOI of 1. Secreted levels of Flag-tagged mWRS were assessed by <t>anti-Flag</t> ELISA in the supernatant at the indicated time points. Shown is anti-Flag ELISA of the supernatant in the same cell line with treatment of 40 μg poly(I-C) (lower left) or transfection of 1 μg poly(dA-dT) (lower right). OD, optical density. (D) ELISA of WRS levels in the supernatant of HeLa cells infected with VSV-GFP at an MOI of 0.1 or 1 for indicated time points. ND, not determined. (E) ELISA of WRS levels in the supernatant of HEK293T cells infected with VSV-GFP at an MOI of 0.01 or 3 for indicated time points. (F) mRNA expression level of WRS in HEK293T cells infected with VSV-GFP at an MOI of 0.01, as determined by qRT-PCR. NS, not significant. (G) THP-1 cells were treated with 100 or 1,000 U of recombinant IFN-β. Secreted levels of WRS were assessed by ELISA; 1,000 ng/ml of recombinant WRS standard was used as a positive control for the experiment. ND, not detected; NS, not significant. Error bars, means ± SD.
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    WRS is secreted in response to virus infection. (A) ELISA of WRS levels in the supernatant of U-937 and THP-1 cells infected with VSV-GFP at a multiplicity of infection (MOI) of 3 at indicated time points. (B) mRNA expression level of WRS shown in panel A was determined by qRT-PCR. (C) <t>Flag-tagged</t> mWRS expressing stable Raw264.7 cells and control cells were infected with VSV-GFP (upper, left) or HSV-GFP (upper, right) at an MOI of 1. Secreted levels of Flag-tagged mWRS were assessed by <t>anti-Flag</t> ELISA in the supernatant at the indicated time points. Shown is anti-Flag ELISA of the supernatant in the same cell line with treatment of 40 μg poly(I-C) (lower left) or transfection of 1 μg poly(dA-dT) (lower right). OD, optical density. (D) ELISA of WRS levels in the supernatant of HeLa cells infected with VSV-GFP at an MOI of 0.1 or 1 for indicated time points. ND, not determined. (E) ELISA of WRS levels in the supernatant of HEK293T cells infected with VSV-GFP at an MOI of 0.01 or 3 for indicated time points. (F) mRNA expression level of WRS in HEK293T cells infected with VSV-GFP at an MOI of 0.01, as determined by qRT-PCR. NS, not significant. (G) THP-1 cells were treated with 100 or 1,000 U of recombinant IFN-β. Secreted levels of WRS were assessed by ELISA; 1,000 ng/ml of recombinant WRS standard was used as a positive control for the experiment. ND, not detected; NS, not significant. Error bars, means ± SD.
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    <t>RNF11</t> disrupts the interaction between TRAF3 and TBK1/IKKi. (A, C) 293T cells were transfected with 1 μg of either HA-TRAF3, Flag-IKKi, Flag-TBK1 or RNF11-GFP. Co-IPs were conducted using anti-Flag followed by immunoblotting with anti-HA and anti-Flag. Immunoblotting was performed with lysates using anti-Flag, anti-HA, anti-GFP and anti-Actin. (B, D) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), and 1 µg of RNF11, TRAF3, IKKi or TBK1. Dual luciferase assays were performed with protein lysates 24 h later. *, p
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    <t>Sortilin</t> forms homodimers on the cell surface of HEK293 cells. A , schematic of <t>FLAG-sortilin</t> and His 6 -sortilin. FLAG tag and His 6 tag were inserted following propeptide and 3 amino acids (Ser 78 -Ala 79 -Pro 80 ) in sortilin. SP , signal peptide; PP , propeptide. B , overexpression of FLAG-sortilin and His 6 -sortilin in HEK293 cells was validated by Western blotting. C and D , detection of binding of FLAG-sortilin and His 6 -sortilin on the cell surface of HEK293 in TR-FRET assay ( C ) and HTRF assay ( D ). Change of FRET signal by expression of His 6 -sortilin is indicated by percent change (mean ± S.D., three independent experiments). Error bars represent S.D. *, p
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    Smed-β-catenin-2 interacts with the cadherin complex. A , HEK293T cells were transfected with the indicated plasmids, and lysates were immunoprecipitated ( IP ) with <t>FLAG-M2</t> beads. Western blotting ( IB ) with anti-GFP revealed co-IP of Smed-β-catenin-2
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    Smed-β-catenin-2 interacts with the cadherin complex. A , HEK293T cells were transfected with the indicated plasmids, and lysates were immunoprecipitated ( IP ) with <t>FLAG-M2</t> beads. Western blotting ( IB ) with anti-GFP revealed co-IP of Smed-β-catenin-2
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    SOCS-6 associates with elongins B and C. (A) Endogenous SOCS-6 binds to endogenous elongins B and C. M1 cells were lysed and subjected to immunoprecipitation with either the 1C3 anti-SOCS-6 antibody, a control isotype matched (IgG2B) antibody, or a monoclonal antibody that recognizes both elongins B and C. This was followed by Western blotting with the polyclonal antibody that recognizes both elongins B and C (top). To confirm that SOCS-6 was immunoprecipitated, 1/10 of each immunoprecipitate was subjected to Western blotting with the 3A7 anti-SOCS-6 antibody (bottom). (B) SOCS-6 binds to elongins B and C through its SOCS box. 293T cells were transfected with cDNAs encoding <t>FLAG-S6,</t> N, N+SH2, or SH2+SB proteins or with the empty vector (V). Lysates were prepared and subjected to immunoprecipitation using <t>M2</t> anti-FLAG resin followed by Western blotting with a polyclonal antibody that recognizes both elongins B and C (top). The filter was then stripped and reprobed with anti-FLAG antibodies (bottom). Molecular masses (kilodaltons) are on the left. IPPT, immunoprecipitate; S6, SOCS-6; El, elongin; C, control antibody; IgH, immunoglobulin heavy chain.
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    MMG8 associates with <t>γTuCs</t> and is required for γTuC attachment to the Golgi. (A,B) Anti-MMG8 (A) and anti-GCP3 (B) immunoprecipitations (IPs) were performed using HeLa extracts. The immunoprecipitates and inputs were immunoblotted. (C) <t>FLAG–MMG8</t> and FLAG–MMG8 (389–1116) transiently expressed in HEK293T cells were immunoprecipitated in RIPA buffer. The beads were then used in a pull-down assay together with RPE1 lysates of cells that were transfected with control or AKAP450 siRNA. The pull-downs were examined on immunoblots. (D) RPE1 cells were double-stained for γ-tubulin and MMG8. (E) Cells transfected with siRNAs were immunostained for γ-tubulin and mannosidase II (Man II). Arrows indicate centrosomes. Images shown in D,E are representative ( > 90%) of 200 cells analyzed for each sample. Scale bars: 5 µm. (F) Golgi membranes were isolated from siRNA-transfected cells. Both the Golgi fractions and the whole cell lysates (WCLs) were analyzed on the immunoblots. The graph shows the protein quantification of the isolated Golgi membranes as the mean±s.d. from three independent experiments; ** P
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    MMG8 associates with <t>γTuCs</t> and is required for γTuC attachment to the Golgi. (A,B) Anti-MMG8 (A) and anti-GCP3 (B) immunoprecipitations (IPs) were performed using HeLa extracts. The immunoprecipitates and inputs were immunoblotted. (C) <t>FLAG–MMG8</t> and FLAG–MMG8 (389–1116) transiently expressed in HEK293T cells were immunoprecipitated in RIPA buffer. The beads were then used in a pull-down assay together with RPE1 lysates of cells that were transfected with control or AKAP450 siRNA. The pull-downs were examined on immunoblots. (D) RPE1 cells were double-stained for γ-tubulin and MMG8. (E) Cells transfected with siRNAs were immunostained for γ-tubulin and mannosidase II (Man II). Arrows indicate centrosomes. Images shown in D,E are representative ( > 90%) of 200 cells analyzed for each sample. Scale bars: 5 µm. (F) Golgi membranes were isolated from siRNA-transfected cells. Both the Golgi fractions and the whole cell lysates (WCLs) were analyzed on the immunoblots. The graph shows the protein quantification of the isolated Golgi membranes as the mean±s.d. from three independent experiments; ** P
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    MMG8 associates with <t>γTuCs</t> and is required for γTuC attachment to the Golgi. (A,B) Anti-MMG8 (A) and anti-GCP3 (B) immunoprecipitations (IPs) were performed using HeLa extracts. The immunoprecipitates and inputs were immunoblotted. (C) <t>FLAG–MMG8</t> and FLAG–MMG8 (389–1116) transiently expressed in HEK293T cells were immunoprecipitated in RIPA buffer. The beads were then used in a pull-down assay together with RPE1 lysates of cells that were transfected with control or AKAP450 siRNA. The pull-downs were examined on immunoblots. (D) RPE1 cells were double-stained for γ-tubulin and MMG8. (E) Cells transfected with siRNAs were immunostained for γ-tubulin and mannosidase II (Man II). Arrows indicate centrosomes. Images shown in D,E are representative ( > 90%) of 200 cells analyzed for each sample. Scale bars: 5 µm. (F) Golgi membranes were isolated from siRNA-transfected cells. Both the Golgi fractions and the whole cell lysates (WCLs) were analyzed on the immunoblots. The graph shows the protein quantification of the isolated Golgi membranes as the mean±s.d. from three independent experiments; ** P
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    MMG8 associates with <t>γTuCs</t> and is required for γTuC attachment to the Golgi. (A,B) Anti-MMG8 (A) and anti-GCP3 (B) immunoprecipitations (IPs) were performed using HeLa extracts. The immunoprecipitates and inputs were immunoblotted. (C) <t>FLAG–MMG8</t> and FLAG–MMG8 (389–1116) transiently expressed in HEK293T cells were immunoprecipitated in RIPA buffer. The beads were then used in a pull-down assay together with RPE1 lysates of cells that were transfected with control or AKAP450 siRNA. The pull-downs were examined on immunoblots. (D) RPE1 cells were double-stained for γ-tubulin and MMG8. (E) Cells transfected with siRNAs were immunostained for γ-tubulin and mannosidase II (Man II). Arrows indicate centrosomes. Images shown in D,E are representative ( > 90%) of 200 cells analyzed for each sample. Scale bars: 5 µm. (F) Golgi membranes were isolated from siRNA-transfected cells. Both the Golgi fractions and the whole cell lysates (WCLs) were analyzed on the immunoblots. The graph shows the protein quantification of the isolated Golgi membranes as the mean±s.d. from three independent experiments; ** P
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    PHD-induced hydroxylation of wild type and mutant HIF-1α-(531–652) A , in vitro translated, 35 S-labeled wild type or mutant GAL4-HIF-1α-(531–652) was incubated with 0.5 μg of His 6 -PHD1, His 6 -PHD2, or His 6 -PHD3 for the indicated times. Hydroxylated reaction products were then isolated by first incubating with His 6 <t>-FLAG-VBC</t> and then by <t>immunoprecipitating</t> with anti-FLAG antibodies coupled to agarose. The immunoprecipitated reaction products were subjected to SDS-PAGE and autoradiography. B , same as in A except that His 6 -PHD2 and either HA-HIF-1α or GAL4-HIF-1α-(531–652) were employed. Input ( In ) designates 20% of the substrate analyzed at a given time point. One of three representative results is shown.
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    PHD-induced hydroxylation of wild type and mutant HIF-1α-(531–652) A , in vitro translated, 35 S-labeled wild type or mutant GAL4-HIF-1α-(531–652) was incubated with 0.5 μg of His 6 -PHD1, His 6 -PHD2, or His 6 -PHD3 for the indicated times. Hydroxylated reaction products were then isolated by first incubating with His 6 <t>-FLAG-VBC</t> and then by <t>immunoprecipitating</t> with anti-FLAG antibodies coupled to agarose. The immunoprecipitated reaction products were subjected to SDS-PAGE and autoradiography. B , same as in A except that His 6 -PHD2 and either HA-HIF-1α or GAL4-HIF-1α-(531–652) were employed. Input ( In ) designates 20% of the substrate analyzed at a given time point. One of three representative results is shown.
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    PHD-induced hydroxylation of wild type and mutant HIF-1α-(531–652) A , in vitro translated, 35 S-labeled wild type or mutant GAL4-HIF-1α-(531–652) was incubated with 0.5 μg of His 6 -PHD1, His 6 -PHD2, or His 6 -PHD3 for the indicated times. Hydroxylated reaction products were then isolated by first incubating with His 6 <t>-FLAG-VBC</t> and then by <t>immunoprecipitating</t> with anti-FLAG antibodies coupled to agarose. The immunoprecipitated reaction products were subjected to SDS-PAGE and autoradiography. B , same as in A except that His 6 -PHD2 and either HA-HIF-1α or GAL4-HIF-1α-(531–652) were employed. Input ( In ) designates 20% of the substrate analyzed at a given time point. One of three representative results is shown.
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    PHD-induced hydroxylation of wild type and mutant HIF-1α-(531–652) A , in vitro translated, 35 S-labeled wild type or mutant GAL4-HIF-1α-(531–652) was incubated with 0.5 μg of His 6 -PHD1, His 6 -PHD2, or His 6 -PHD3 for the indicated times. Hydroxylated reaction products were then isolated by first incubating with His 6 <t>-FLAG-VBC</t> and then by <t>immunoprecipitating</t> with anti-FLAG antibodies coupled to agarose. The immunoprecipitated reaction products were subjected to SDS-PAGE and autoradiography. B , same as in A except that His 6 -PHD2 and either HA-HIF-1α or GAL4-HIF-1α-(531–652) were employed. Input ( In ) designates 20% of the substrate analyzed at a given time point. One of three representative results is shown.
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    Image Search Results


    SH3BP5 binds to JNK through SH3BD, KIM1, and KIM2. A , structure of mouse SH3BP5 (mSH3BP5) and deletion mutants of mSH3BP5. B , 6×His-mSH3BP5 (H6-mSH3BP5) or its deletion mutant, produced in bacteria, was purified with TALON Metal Resin. Negative-control purification ( NC ) was equally done from bacteria in which the empty vector was introduced. The purified proteins were fractionated by SDS-PAGE, followed by Coomassie Blue staining. FL : SH3BP5 full-length, Δ SH3 : SH3BP5 lacking SH3BD, Δ1: SH3BP5 lacking KIM1, Δ2: SH3BP5 lacking KIM2. C , 6×His-GST, 6×His-mSH3BP5, and 6×His-mSH3BP5 deletion mutants were produced in bacteria and purified with TALON Metal Resin. Each protein (0.1 μg) was fractionated as an input for SDS-PAGE and subjected to immunoblot analysis with antibody to HisG. D , FLAG-JNK-1a1, immunoprecipitated from the cell lysates with the FLAG antibody, was mixed with indicated recombinant 6×His-GST, 6×His-mSH3BP5, or a 6×His-mSH3BP5 deletion mutant (1 μg per sample), washed extensively, and subjected to SDS-PAGE and immunoblot analysis. Immunoblot analysis was performed with the antibody to HisG for the detection of 6×His-tagged recombinant proteins and the M2 antibody to FLAG for the detection of FLAG-JNK-1a1.

    Journal: The Journal of Biological Chemistry

    Article Title: SH3-binding Protein 5 Mediates the Neuroprotective Effect of the Secreted Bioactive Peptide Humanin by Inhibiting c-Jun NH2-terminal Kinase *

    doi: 10.1074/jbc.M113.469692

    Figure Lengend Snippet: SH3BP5 binds to JNK through SH3BD, KIM1, and KIM2. A , structure of mouse SH3BP5 (mSH3BP5) and deletion mutants of mSH3BP5. B , 6×His-mSH3BP5 (H6-mSH3BP5) or its deletion mutant, produced in bacteria, was purified with TALON Metal Resin. Negative-control purification ( NC ) was equally done from bacteria in which the empty vector was introduced. The purified proteins were fractionated by SDS-PAGE, followed by Coomassie Blue staining. FL : SH3BP5 full-length, Δ SH3 : SH3BP5 lacking SH3BD, Δ1: SH3BP5 lacking KIM1, Δ2: SH3BP5 lacking KIM2. C , 6×His-GST, 6×His-mSH3BP5, and 6×His-mSH3BP5 deletion mutants were produced in bacteria and purified with TALON Metal Resin. Each protein (0.1 μg) was fractionated as an input for SDS-PAGE and subjected to immunoblot analysis with antibody to HisG. D , FLAG-JNK-1a1, immunoprecipitated from the cell lysates with the FLAG antibody, was mixed with indicated recombinant 6×His-GST, 6×His-mSH3BP5, or a 6×His-mSH3BP5 deletion mutant (1 μg per sample), washed extensively, and subjected to SDS-PAGE and immunoblot analysis. Immunoblot analysis was performed with the antibody to HisG for the detection of 6×His-tagged recombinant proteins and the M2 antibody to FLAG for the detection of FLAG-JNK-1a1.

    Article Snippet: Ready-made antibodies were purchased from following companies: anti-Myc and anti-HisG-HRP from Invitrogen; anti-FLAG (clone M2) from Sigma-Aldrich; anti-APP (clone 22C11) from Chemicon; anti-total JNK from Santa Cruz Biotechnology; anti-phospho-SAPK/JNK from Cell Signaling Technology; anti-total JNK from Santa Cruz Biotechnology; anti-HA (clone 3F10) from Roche Diagnostics.

    Techniques: Mutagenesis, Produced, Purification, Negative Control, Plasmid Preparation, SDS Page, Staining, Immunoprecipitation, Recombinant

    Both KIM1 and KIM2 of SH3BP5 are involved in inhibiting JNK. A , FLAG-JNK-1a1, immunoprecipitated from the cell lysates with the FLAG antibody, was mixed with indicated recombinant 6×His-GST, 6×His-mSH3BP5, or a 6×His-mSH3BP5 deletion mutant (1 μg per sample), and subjected to in vitro JNK kinase assays using recombinant c-Jun as a substrate ( top panel ) and to SDS-PAGE and immunoblot analysis ( middle and bottom panels ). Immunoblot analysis was performed with the antibody to phospho c-Jun, the antibody to HisG for the detection of 6×His-tagged recombinant proteins, and the antibody to FLAG for the detection of FLAG-JNK-1a1. B , F11 cells, cotransfected with the empty pFLAG vector ( vector ) or a pFLAG vector encoding mSH3BP5 or an mSH3BP5 deletion mutant together with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP). At 72 h after transfection, they were harvested for Trypan Blue cell death assays. Cell lysates were subjected to SDS-PAGE and immunoblot analysis with the M2 antibody to FLAG and the APP antibody (22C11). ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: SH3-binding Protein 5 Mediates the Neuroprotective Effect of the Secreted Bioactive Peptide Humanin by Inhibiting c-Jun NH2-terminal Kinase *

    doi: 10.1074/jbc.M113.469692

    Figure Lengend Snippet: Both KIM1 and KIM2 of SH3BP5 are involved in inhibiting JNK. A , FLAG-JNK-1a1, immunoprecipitated from the cell lysates with the FLAG antibody, was mixed with indicated recombinant 6×His-GST, 6×His-mSH3BP5, or a 6×His-mSH3BP5 deletion mutant (1 μg per sample), and subjected to in vitro JNK kinase assays using recombinant c-Jun as a substrate ( top panel ) and to SDS-PAGE and immunoblot analysis ( middle and bottom panels ). Immunoblot analysis was performed with the antibody to phospho c-Jun, the antibody to HisG for the detection of 6×His-tagged recombinant proteins, and the antibody to FLAG for the detection of FLAG-JNK-1a1. B , F11 cells, cotransfected with the empty pFLAG vector ( vector ) or a pFLAG vector encoding mSH3BP5 or an mSH3BP5 deletion mutant together with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP). At 72 h after transfection, they were harvested for Trypan Blue cell death assays. Cell lysates were subjected to SDS-PAGE and immunoblot analysis with the M2 antibody to FLAG and the APP antibody (22C11). ***, p

    Article Snippet: Ready-made antibodies were purchased from following companies: anti-Myc and anti-HisG-HRP from Invitrogen; anti-FLAG (clone M2) from Sigma-Aldrich; anti-APP (clone 22C11) from Chemicon; anti-total JNK from Santa Cruz Biotechnology; anti-phospho-SAPK/JNK from Cell Signaling Technology; anti-total JNK from Santa Cruz Biotechnology; anti-HA (clone 3F10) from Roche Diagnostics.

    Techniques: Immunoprecipitation, Recombinant, Mutagenesis, In Vitro, SDS Page, Plasmid Preparation, Transfection

    Overexpression of SH3BP5 inhibits V642I-APP-induced death of mouse neuronal cells. A–D , F11 cells were cotransfected with the empty pFLAG vector ( vector ) or the pFLAG vector encoding mouse SH3BP5 (mSH3BP5) together with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP). At 72 h after transfection, they were harvested for microscopic analysis ( A ), Trypan Blue cell death assays ( B ), and WST-8 cell viability assays ( C ). Cell lysates were subjected to SDS-PAGE and immunoblot analysis with FLAG antibody ( M2 ) and APP antibody (22C11) ( D ). E , PHNs were cotransfected with the empty pFLAG vector ( vector ) or the pFLAG vector encoding mSH3BP5 together with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP). At 48 h after transfection, the cells were immunostained with both the APP and FLAG antibodies. Nuclei were stained with DAPI. For the calculation of cell-death percentages in the cells that had been transfected with the two empty vectors, the percentages of morphologically apoptotic cells in total cells were counted. For the calculation of cell-death percentages in the cells that had been cotransfected with the empty vector and the V642I-APP-encoding vector, with the mSH3BP5-encoding vector and the empty vector, or with the V642I-APP- and mSH3BP5-FLAG-encoding vectors, the percentages of apoptotic cells in the cells that overexpressed V642I-APP, SH3BP5-FLAG, and both V642I-APP and mouse SH3BP5-FLAG, were counted, respectively. This study was performed with n = 3; 50–100 cells were counted per well. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: SH3-binding Protein 5 Mediates the Neuroprotective Effect of the Secreted Bioactive Peptide Humanin by Inhibiting c-Jun NH2-terminal Kinase *

    doi: 10.1074/jbc.M113.469692

    Figure Lengend Snippet: Overexpression of SH3BP5 inhibits V642I-APP-induced death of mouse neuronal cells. A–D , F11 cells were cotransfected with the empty pFLAG vector ( vector ) or the pFLAG vector encoding mouse SH3BP5 (mSH3BP5) together with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP). At 72 h after transfection, they were harvested for microscopic analysis ( A ), Trypan Blue cell death assays ( B ), and WST-8 cell viability assays ( C ). Cell lysates were subjected to SDS-PAGE and immunoblot analysis with FLAG antibody ( M2 ) and APP antibody (22C11) ( D ). E , PHNs were cotransfected with the empty pFLAG vector ( vector ) or the pFLAG vector encoding mSH3BP5 together with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP). At 48 h after transfection, the cells were immunostained with both the APP and FLAG antibodies. Nuclei were stained with DAPI. For the calculation of cell-death percentages in the cells that had been transfected with the two empty vectors, the percentages of morphologically apoptotic cells in total cells were counted. For the calculation of cell-death percentages in the cells that had been cotransfected with the empty vector and the V642I-APP-encoding vector, with the mSH3BP5-encoding vector and the empty vector, or with the V642I-APP- and mSH3BP5-FLAG-encoding vectors, the percentages of apoptotic cells in the cells that overexpressed V642I-APP, SH3BP5-FLAG, and both V642I-APP and mouse SH3BP5-FLAG, were counted, respectively. This study was performed with n = 3; 50–100 cells were counted per well. ***, p

    Article Snippet: Ready-made antibodies were purchased from following companies: anti-Myc and anti-HisG-HRP from Invitrogen; anti-FLAG (clone M2) from Sigma-Aldrich; anti-APP (clone 22C11) from Chemicon; anti-total JNK from Santa Cruz Biotechnology; anti-phospho-SAPK/JNK from Cell Signaling Technology; anti-total JNK from Santa Cruz Biotechnology; anti-HA (clone 3F10) from Roche Diagnostics.

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

    Simultaneous interaction of myocardin with p300 and HDAC5. COS cells were transiently transfected with expression vectors encoding hemagglutinin (HA)-tagged p300, FLAG-tagged myocardin, and Myc-tagged HDAC5. FLAG-tagged myocardin was immunoprecipitated

    Journal:

    Article Title: Modulation of Smooth Muscle Gene Expression by Association of Histone Acetyltransferases and Deacetylases with Myocardin

    doi: 10.1128/MCB.25.1.364-376.2005

    Figure Lengend Snippet: Simultaneous interaction of myocardin with p300 and HDAC5. COS cells were transiently transfected with expression vectors encoding hemagglutinin (HA)-tagged p300, FLAG-tagged myocardin, and Myc-tagged HDAC5. FLAG-tagged myocardin was immunoprecipitated

    Article Snippet: To determine the cellular localization of myocardin and HDAC5, COS cells were transfected with FLAG-tagged myocardin and Myc-tagged HDAC5 and stained with anti-FLAG (mouse monoclonal M2; Sigma) and anti-Myc (rabbit polyclonal; Santa Cruz) antibodies.

    Techniques: Transfection, Expressing, Immunoprecipitation

    Acetylation of histones associated with smooth muscle gene promoters in response to myocardin and modulation of myocardin myogenic activity by p300 and HDAC5. (A) 10T1/2 cells were transiently transfected with a FLAG-tagged myocardin expression vector

    Journal:

    Article Title: Modulation of Smooth Muscle Gene Expression by Association of Histone Acetyltransferases and Deacetylases with Myocardin

    doi: 10.1128/MCB.25.1.364-376.2005

    Figure Lengend Snippet: Acetylation of histones associated with smooth muscle gene promoters in response to myocardin and modulation of myocardin myogenic activity by p300 and HDAC5. (A) 10T1/2 cells were transiently transfected with a FLAG-tagged myocardin expression vector

    Article Snippet: To determine the cellular localization of myocardin and HDAC5, COS cells were transfected with FLAG-tagged myocardin and Myc-tagged HDAC5 and stained with anti-FLAG (mouse monoclonal M2; Sigma) and anti-Myc (rabbit polyclonal; Santa Cruz) antibodies.

    Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation

    Spartin interacts with AIP4 and AIP5 ( A ) Western blotting (WB) against endogenous AIP4 detects an AIP4-sized band after immunoprecipitation (IP) of HeLa cell lysate with α-spartin, but not with pre-immune (PI) serum. ( B ) Immunoblotting against endogenous AIP5 detects an AIP5-sized band after immunoprecipitation of HeLa cell lysate with α-spartin, but not with pre-immune serum. ( C ) Endogenous spartin partially co-localizes with endogenous AIP4, in small puncta. Arrows indicate puncta showing co-localization. The inset shows a magnified image of the boxed area. Scale bar=10 μm. ( D ) Endogenous spartin was immunoprecipitated from HeLa cells transfected with FLAG-tagged wild-type AIP4 or catalytically inactive AIP4 (C803A). Immunoblotting with anti-FLAG demonstrated that wild-type and catalytically inactive AIP4 were co-immunoprecipitated with spartin. Neither spartin nor the AIP4 proteins were immunoprecipitated by pre-immune serum. UT, untransfected cells. ( E ) Endogenous spartin was immunoprecipitated from HeLa cells transfected with wild-type (wt) or catalytically inactive (C962S; mut.) Myc-tagged Nedd4.2. Immunoblotting with anti-Myc showed that the Nedd4.2 proteins had not been co-immunoprecipitated with spartin. ( F ) Sequence of the first 210 amino acids of spartin. The MIT (microtubule-interacting and transport) domain is shown in bold, the PPXY motif is boxed. ( G ) HeLa cells were transfected with FLAG-AIP4 and either wild-type Myc-tagged spartin (WT) or Myc-tagged spartin in which the PPXY motif had been mutated to AAAA. After immunoprecipitation with anti-Myc, FLAG-AIP4 co-immunoprecipitated with WT Myc–spartin, but not Myc–spartinAAAA. ( H ) HeLa cells were transfected with wild-type Myc–tagged spartin (left panels) or Myc–spartinAAAA (right panels). After immunoprecipitation with anti-Myc, endogenous AIP5 co-immunoprecipited with wild-type Myc-spartin, but not with Myc–spartinAAAA. An additional short exposure is shown so that immunoblotting results for the input lanes can be properly visualized.

    Journal: Biochemical Journal

    Article Title: Endogenous spartin (SPG20) is recruited to endosomes and lipid droplets and interacts with the ubiquitin E3 ligases AIP4 and AIP5

    doi: 10.1042/BJ20082398

    Figure Lengend Snippet: Spartin interacts with AIP4 and AIP5 ( A ) Western blotting (WB) against endogenous AIP4 detects an AIP4-sized band after immunoprecipitation (IP) of HeLa cell lysate with α-spartin, but not with pre-immune (PI) serum. ( B ) Immunoblotting against endogenous AIP5 detects an AIP5-sized band after immunoprecipitation of HeLa cell lysate with α-spartin, but not with pre-immune serum. ( C ) Endogenous spartin partially co-localizes with endogenous AIP4, in small puncta. Arrows indicate puncta showing co-localization. The inset shows a magnified image of the boxed area. Scale bar=10 μm. ( D ) Endogenous spartin was immunoprecipitated from HeLa cells transfected with FLAG-tagged wild-type AIP4 or catalytically inactive AIP4 (C803A). Immunoblotting with anti-FLAG demonstrated that wild-type and catalytically inactive AIP4 were co-immunoprecipitated with spartin. Neither spartin nor the AIP4 proteins were immunoprecipitated by pre-immune serum. UT, untransfected cells. ( E ) Endogenous spartin was immunoprecipitated from HeLa cells transfected with wild-type (wt) or catalytically inactive (C962S; mut.) Myc-tagged Nedd4.2. Immunoblotting with anti-Myc showed that the Nedd4.2 proteins had not been co-immunoprecipitated with spartin. ( F ) Sequence of the first 210 amino acids of spartin. The MIT (microtubule-interacting and transport) domain is shown in bold, the PPXY motif is boxed. ( G ) HeLa cells were transfected with FLAG-AIP4 and either wild-type Myc-tagged spartin (WT) or Myc-tagged spartin in which the PPXY motif had been mutated to AAAA. After immunoprecipitation with anti-Myc, FLAG-AIP4 co-immunoprecipitated with WT Myc–spartin, but not Myc–spartinAAAA. ( H ) HeLa cells were transfected with wild-type Myc–tagged spartin (left panels) or Myc–spartinAAAA (right panels). After immunoprecipitation with anti-Myc, endogenous AIP5 co-immunoprecipited with wild-type Myc-spartin, but not with Myc–spartinAAAA. An additional short exposure is shown so that immunoblotting results for the input lanes can be properly visualized.

    Article Snippet: Antibodies and markers Mouse monoclonal anti-α-tubulin (T-9026) and anti-FLAG (A-9594) and rabbit polyclonal anti-actin (A-2066) were from Sigma–Aldrich.

    Techniques: Western Blot, Immunoprecipitation, Transfection, Sequencing

    Homo- and hetero-multimerization among the PHDs ( A ) Homo-/hetero-multimerization of PHD2 and PHD3 with PHDs. HEK-293T cells were transfected with FLAG–PHD2 or FLAG–PHD3 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or β-actin. Asterisk indicates the heavy/light chains of anti-FLAG Ab. ( B ) Homo-/hetero-multimerization of PHD1 with PHDs. HEK-293T cells were transfected with FLAG–PHD1 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. Asterisk indicates the heavy chain of anti-FLAG Ab. ( C ) C-terminal portion of PHD3 is required for homomultimerization. HEK-293T cells were transfected with Myc–PHD3 together with PHD3 fragment plasmids (FLAG–F1, FLAG–F2 or FLAG–F3). Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. ( D ) Hetero-multimerization of ΔPHDs with PHD3. Myc–ΔPHDs or Myc–PHD1, Myc–PHD2, Myc–PHD3 and FLAG–PHD3 were co-transfected into HEK-293T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG Ab and the blot was probed with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or β-actin. IB, immunoblot; IP, immunoprecipitation.

    Journal: Biochemical Journal

    Article Title: Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2

    doi: 10.1042/BJ20061135

    Figure Lengend Snippet: Homo- and hetero-multimerization among the PHDs ( A ) Homo-/hetero-multimerization of PHD2 and PHD3 with PHDs. HEK-293T cells were transfected with FLAG–PHD2 or FLAG–PHD3 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or β-actin. Asterisk indicates the heavy/light chains of anti-FLAG Ab. ( B ) Homo-/hetero-multimerization of PHD1 with PHDs. HEK-293T cells were transfected with FLAG–PHD1 together with Myc–PHD1, Myc–PHD2 or Myc–PHD3. Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. Asterisk indicates the heavy chain of anti-FLAG Ab. ( C ) C-terminal portion of PHD3 is required for homomultimerization. HEK-293T cells were transfected with Myc–PHD3 together with PHD3 fragment plasmids (FLAG–F1, FLAG–F2 or FLAG–F3). Cell lysates immunoprecipitated with anti-FLAG Ab were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. ( D ) Hetero-multimerization of ΔPHDs with PHD3. Myc–ΔPHDs or Myc–PHD1, Myc–PHD2, Myc–PHD3 and FLAG–PHD3 were co-transfected into HEK-293T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG Ab and the blot was probed with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or β-actin. IB, immunoblot; IP, immunoprecipitation.

    Article Snippet: Anti-FLAG tag (monoclonal and polyclonal M2) and anti-β-actin Abs were purchased from Sigma.

    Techniques: Transfection, Immunoprecipitation, Western Blot

    ΔPHDs homo- and hetero-multimerize with PHDs ( A ) Homo-/hetero-multimerization of ΔPHDs. FLAG–ΔPHD1 or FLAG–ΔPHD2 and Myc–PHD3, Myc–ΔPHD1 or Myc–ΔPHD2 were co-transfected into HEK-293T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG Ab and the blot was probed with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or β-actin. Asterisk indicates the light chain of anti-FLAG Ab. ( B ) Heteromultimerization of ΔPHDs with PHD1, PHD2 and PHD3. FLAG–ΔPHD1 or FLAG–ΔPHD2 and Myc–PHD1, Myc–PHD2 or Myc–PHD3 were co-transfected into HEK-293T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG Ab, and the blot was probed with anti-Myc Ab, anti-FLAG Ab or β-actin. Asterisk indicates the heavy chain of anti-FLAG Ab. IB, immunoblot; IP, immuno precipitation.

    Journal: Biochemical Journal

    Article Title: Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2

    doi: 10.1042/BJ20061135

    Figure Lengend Snippet: ΔPHDs homo- and hetero-multimerize with PHDs ( A ) Homo-/hetero-multimerization of ΔPHDs. FLAG–ΔPHD1 or FLAG–ΔPHD2 and Myc–PHD3, Myc–ΔPHD1 or Myc–ΔPHD2 were co-transfected into HEK-293T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG Ab and the blot was probed with anti-Myc Ab (second panel: dark exposure), anti-FLAG Ab or β-actin. Asterisk indicates the light chain of anti-FLAG Ab. ( B ) Heteromultimerization of ΔPHDs with PHD1, PHD2 and PHD3. FLAG–ΔPHD1 or FLAG–ΔPHD2 and Myc–PHD1, Myc–PHD2 or Myc–PHD3 were co-transfected into HEK-293T cells. Cell lysates were subjected to immunoprecipitation with anti-FLAG Ab, and the blot was probed with anti-Myc Ab, anti-FLAG Ab or β-actin. Asterisk indicates the heavy chain of anti-FLAG Ab. IB, immunoblot; IP, immuno precipitation.

    Article Snippet: Anti-FLAG tag (monoclonal and polyclonal M2) and anti-β-actin Abs were purchased from Sigma.

    Techniques: Transfection, Immunoprecipitation

    Degradation of PHD3 by Siah2 is dependent on its C-terminal region ( A ) Degradation of PHD3 deletion mutants by Siah2. Schematic diagrams of PHD3 deletion mutants are shown in the upper panel. Amino acid numbers are indicated for each fragment. Siah2 and PHD3 fragment plasmids were co-transfected into HEK-293T cells. The total cell lysate was subjected to SDS/PAGE and the membrane was probed with anti-FLAG Ab (top panel) and anti-β-actin Ab (bottom panel). ( B ) Competition of PHD3 and its deleted form for Siah. FLAG–PHD3 (2 μg) together with each of the FLAG–PHD3 fragments (0.5 and 2 μg) were co-transfected with Siah2 (2 μg) into HEK-293T cells. The total cell lysate was subjected to Western blotting and the membrane was probed with anti-FLAG Ab (top and middle panels) and anti-β-actin Ab (bottom panel). The numbers below the top panel indicate the relative expression levels of PHD3. ( C ) Effect of PHD3 fragments on PHD3–Siah2 interaction. The RING-mutant form of HA–Siah2 (Siah2Rm) was co-transfected with Myc-PHD3 plasmid together with FLAG–PHD3 fragments in HEK-293T cells, and the cell lysate was subjected to immunoprecipitation (IP) with anti-Myc Ab. Western blotting with anti-HA Ab shows the interaction of Siah2Rm with PHD3. Immunoprecipitated Myc–PHD3 (second from top panel), expression of HA–Siah2Rm, FLAG-tagged PHD3 fragments and β-actin control in cell lysate are shown in the lower panels. ( D ) Exogenous expression of F3 alters HIF-1α expression. Different amounts of PHD3-F3 (indicated on the top of the panel) were transfected into HeLa cells, and 48 h later, the cells were exposed to hypoxia (1%) for 5 h and the cell lysate was subjected to Western blotting. Expression of HIF-1α (top panel), F3 (middle panel) and β-actin (bottom panel) is shown.

    Journal: Biochemical Journal

    Article Title: Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2

    doi: 10.1042/BJ20061135

    Figure Lengend Snippet: Degradation of PHD3 by Siah2 is dependent on its C-terminal region ( A ) Degradation of PHD3 deletion mutants by Siah2. Schematic diagrams of PHD3 deletion mutants are shown in the upper panel. Amino acid numbers are indicated for each fragment. Siah2 and PHD3 fragment plasmids were co-transfected into HEK-293T cells. The total cell lysate was subjected to SDS/PAGE and the membrane was probed with anti-FLAG Ab (top panel) and anti-β-actin Ab (bottom panel). ( B ) Competition of PHD3 and its deleted form for Siah. FLAG–PHD3 (2 μg) together with each of the FLAG–PHD3 fragments (0.5 and 2 μg) were co-transfected with Siah2 (2 μg) into HEK-293T cells. The total cell lysate was subjected to Western blotting and the membrane was probed with anti-FLAG Ab (top and middle panels) and anti-β-actin Ab (bottom panel). The numbers below the top panel indicate the relative expression levels of PHD3. ( C ) Effect of PHD3 fragments on PHD3–Siah2 interaction. The RING-mutant form of HA–Siah2 (Siah2Rm) was co-transfected with Myc-PHD3 plasmid together with FLAG–PHD3 fragments in HEK-293T cells, and the cell lysate was subjected to immunoprecipitation (IP) with anti-Myc Ab. Western blotting with anti-HA Ab shows the interaction of Siah2Rm with PHD3. Immunoprecipitated Myc–PHD3 (second from top panel), expression of HA–Siah2Rm, FLAG-tagged PHD3 fragments and β-actin control in cell lysate are shown in the lower panels. ( D ) Exogenous expression of F3 alters HIF-1α expression. Different amounts of PHD3-F3 (indicated on the top of the panel) were transfected into HeLa cells, and 48 h later, the cells were exposed to hypoxia (1%) for 5 h and the cell lysate was subjected to Western blotting. Expression of HIF-1α (top panel), F3 (middle panel) and β-actin (bottom panel) is shown.

    Article Snippet: Anti-FLAG tag (monoclonal and polyclonal M2) and anti-β-actin Abs were purchased from Sigma.

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

    Activities of PHD3 to hydroxylate HIF-1α in different molecular mass complexes ( A ) An in vitro hydroxylation assay using HIF-ODD was performed with the fractions obtained from the gel filtration. After the reaction, samples were separated on gels and the blot was probed with anti-HIF-1α hydroxylated P564 Ab, anti-FLAG Ab and GST Ab. ( B ) Profile of PHD3 hydroxylase activity in the different complexes. In vitro PHD3 hydroxylase activities are shown by circles and plotted against the y -axis (open: normoxia; filled: hypoxia). The graph represents one of three experiments that were performed independently. ( C ) PHD2–PHD3 homo-/hetero-multimerization in normoxia and hypoxia. HEK-293T cells were co-transfected with Myc–PHD3 and FLAG–PHD1, FLAG–PHD2, FLAG–PHD3 or FLAG–F3. After 48 h, cells were exposed to normoxia or hypoxia (1%, 5 h) and harvested. Hypoxia-treated cells were harvested in a hypoxia workstation and the following procedures were performed in the workstation. Cell lysates were subjected to immunoprecipitation (IP) at 4 °C with anti-FLAG Ab and immunoprecipitates were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. IB, immunoblot.

    Journal: Biochemical Journal

    Article Title: Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2

    doi: 10.1042/BJ20061135

    Figure Lengend Snippet: Activities of PHD3 to hydroxylate HIF-1α in different molecular mass complexes ( A ) An in vitro hydroxylation assay using HIF-ODD was performed with the fractions obtained from the gel filtration. After the reaction, samples were separated on gels and the blot was probed with anti-HIF-1α hydroxylated P564 Ab, anti-FLAG Ab and GST Ab. ( B ) Profile of PHD3 hydroxylase activity in the different complexes. In vitro PHD3 hydroxylase activities are shown by circles and plotted against the y -axis (open: normoxia; filled: hypoxia). The graph represents one of three experiments that were performed independently. ( C ) PHD2–PHD3 homo-/hetero-multimerization in normoxia and hypoxia. HEK-293T cells were co-transfected with Myc–PHD3 and FLAG–PHD1, FLAG–PHD2, FLAG–PHD3 or FLAG–F3. After 48 h, cells were exposed to normoxia or hypoxia (1%, 5 h) and harvested. Hypoxia-treated cells were harvested in a hypoxia workstation and the following procedures were performed in the workstation. Cell lysates were subjected to immunoprecipitation (IP) at 4 °C with anti-FLAG Ab and immunoprecipitates were subjected to Western blotting probing with anti-Myc Ab, anti-FLAG Ab or β-actin. IB, immunoblot.

    Article Snippet: Anti-FLAG tag (monoclonal and polyclonal M2) and anti-β-actin Abs were purchased from Sigma.

    Techniques: In Vitro, Filtration, Activity Assay, Transfection, Immunoprecipitation, Western Blot

    Efficient degradation of ΔPHDs by Siah2 ( A ) Schematic diagram of three mouse PHDs and their deletion mutants. Positions of the deletions are indicated by amino acid number. ( B ) Siah2-dependent degradation of ΔPHDs. FLAG–PHD and Siah2 plasmid (2 μg each) were co-transfected into HEK-293T cells. Total cell lysates were subjected to SDS/PAGE, and the membrane was probed with anti-FLAG Ab (top panel) and anti-β-actin Ab (bottom panel) as an internal control. ( C ) Inhibition of Siah2-dependent ΔPHDs degradation by lactacystin (lact). FLAG–PHDs and Siah2 plasmid (2 μg each) were co-transfected into HEK-293T cells. Cells were treated with lactacystin (10 μM for 5 h) and harvested. Total cell lysates were subjected to SDS/PAGE, and the membrane was probed with anti-FLAG Ab (top panel) and anti-β-actin Ab (bottom panel). ( D ) Interaction of ΔPHDs with Siah2. The RING-mutant form of HA–Siah2 (Siah2Rm) was co-transfected with FLAG-tagged PHD plasmids in HEK-293T cells and cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG Ab. Western blotting with anti-HA Ab shows the interaction of Siah2Rm with PHDs. Immunoprecipitated PHDs (second panel from top) and expression of HA–Siah2Rm and FLAG–PHDs as well as β-actin control in total cell lysate are shown in the three lower panels.

    Journal: Biochemical Journal

    Article Title: Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2

    doi: 10.1042/BJ20061135

    Figure Lengend Snippet: Efficient degradation of ΔPHDs by Siah2 ( A ) Schematic diagram of three mouse PHDs and their deletion mutants. Positions of the deletions are indicated by amino acid number. ( B ) Siah2-dependent degradation of ΔPHDs. FLAG–PHD and Siah2 plasmid (2 μg each) were co-transfected into HEK-293T cells. Total cell lysates were subjected to SDS/PAGE, and the membrane was probed with anti-FLAG Ab (top panel) and anti-β-actin Ab (bottom panel) as an internal control. ( C ) Inhibition of Siah2-dependent ΔPHDs degradation by lactacystin (lact). FLAG–PHDs and Siah2 plasmid (2 μg each) were co-transfected into HEK-293T cells. Cells were treated with lactacystin (10 μM for 5 h) and harvested. Total cell lysates were subjected to SDS/PAGE, and the membrane was probed with anti-FLAG Ab (top panel) and anti-β-actin Ab (bottom panel). ( D ) Interaction of ΔPHDs with Siah2. The RING-mutant form of HA–Siah2 (Siah2Rm) was co-transfected with FLAG-tagged PHD plasmids in HEK-293T cells and cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG Ab. Western blotting with anti-HA Ab shows the interaction of Siah2Rm with PHDs. Immunoprecipitated PHDs (second panel from top) and expression of HA–Siah2Rm and FLAG–PHDs as well as β-actin control in total cell lysate are shown in the three lower panels.

    Article Snippet: Anti-FLAG tag (monoclonal and polyclonal M2) and anti-β-actin Abs were purchased from Sigma.

    Techniques: Plasmid Preparation, Transfection, SDS Page, Inhibition, Mutagenesis, Immunoprecipitation, Western Blot, Expressing

    Detection of the T. gondii mitochondrial ribosome. A. Protein immunoblot analysis of endogenously tagged TgmS35, TgbL12m and TguL3m from total cell lysate separated by SDS‐PAGE and detected using anti‐HA/Strep/FLAG antibodies. B. Total cell lysate separated by blue‐native PAGE and immunoblotted to detect TgmS35, TgbL12m and TguL3m with anti‐HA/Strep/FLAG antibodies. C. Validation of the promoter integration in the Tg mS15 locus via PCR analysis using primers 1, 2, 3, and 4, represented in Fig. S4 D. Western blot (top panel) of TgmS35‐3xHA in lines where Tg mS35 is under its native promoter (TgmS35‐3HA) or where Tg mS35 or Tg uS15m are under regulatable promoters (r Tg mS35‐3HA and Tg mS35‐3HA/r Tg uS15m respectively). Low panel shows instant blue staining for loading control. E. comparison of results from RT‐PCR performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (total), from IP of TgTom22 (Tom22) or from IP of TgmS35 (TgmS35). F. An example RT‐PCR experiment performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (Parental – total), from IP of TgTom22 (Tom22‐IP) or from IP of TgmS35 (TgmS35‐IP).

    Journal: Molecular Microbiology

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

    doi: 10.1111/mmi.14357

    Figure Lengend Snippet: Detection of the T. gondii mitochondrial ribosome. A. Protein immunoblot analysis of endogenously tagged TgmS35, TgbL12m and TguL3m from total cell lysate separated by SDS‐PAGE and detected using anti‐HA/Strep/FLAG antibodies. B. Total cell lysate separated by blue‐native PAGE and immunoblotted to detect TgmS35, TgbL12m and TguL3m with anti‐HA/Strep/FLAG antibodies. C. Validation of the promoter integration in the Tg mS15 locus via PCR analysis using primers 1, 2, 3, and 4, represented in Fig. S4 D. Western blot (top panel) of TgmS35‐3xHA in lines where Tg mS35 is under its native promoter (TgmS35‐3HA) or where Tg mS35 or Tg uS15m are under regulatable promoters (r Tg mS35‐3HA and Tg mS35‐3HA/r Tg uS15m respectively). Low panel shows instant blue staining for loading control. E. comparison of results from RT‐PCR performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (total), from IP of TgTom22 (Tom22) or from IP of TgmS35 (TgmS35). F. An example RT‐PCR experiment performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (Parental – total), from IP of TgTom22 (Tom22‐IP) or from IP of TgmS35 (TgmS35‐IP).

    Article Snippet: Cells were then labelled with different sets of primary and secondary antibodies: mouse anti‐HA antibody (1:1000, Sigma), rabbit anti‐TgMys (1:1000) (Ovciarikova et al. , ), mouse anti‐Myc (1:1000, Cell Signalling), rabbit anti‐TgTOM40 (1:2000)(van Dooren et al. , ); mouse anti‐Strep (1:1000, StrepMAB‐Classic, IBA), mouse anti‐FLAG (1:1000, Monoclonal ANTI‐FLAG® M2, Sigma‐Aldrich) coupled with goat anti‐mouse or anti‐rabbit fluorescent antibody (AlexaFluor 594 or 488 1:1000 (Invitrogen)).

    Techniques: Western Blot, SDS Page, Blue Native PAGE, Polymerase Chain Reaction, Staining, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Mitochondrial localisation of putative mitochondrial ribosomal proteins by epitope tagging. A. Localisation through fluorescence microscopy analysis of 4 gene‐products predicted to encode for mitoribosomal proteins identified in our search. B. Localisation of 2 additional gene‐products predicted to be mitochondrial ribosomal proteins not found in our search. For both panels, mitochondria are marked with anti‐TgMys or anti‐TOM40; the mentioned GOI (genes of interest) are marked with anti‐HA, anti‐FLAG or anti‐Strep. Scale bar 1 μm. Images in A also appear in Fig. S2 . [Colour figure can be viewed at https://www.wileyonlinelibrary.com ]

    Journal: Molecular Microbiology

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

    doi: 10.1111/mmi.14357

    Figure Lengend Snippet: Mitochondrial localisation of putative mitochondrial ribosomal proteins by epitope tagging. A. Localisation through fluorescence microscopy analysis of 4 gene‐products predicted to encode for mitoribosomal proteins identified in our search. B. Localisation of 2 additional gene‐products predicted to be mitochondrial ribosomal proteins not found in our search. For both panels, mitochondria are marked with anti‐TgMys or anti‐TOM40; the mentioned GOI (genes of interest) are marked with anti‐HA, anti‐FLAG or anti‐Strep. Scale bar 1 μm. Images in A also appear in Fig. S2 . [Colour figure can be viewed at https://www.wileyonlinelibrary.com ]

    Article Snippet: Cells were then labelled with different sets of primary and secondary antibodies: mouse anti‐HA antibody (1:1000, Sigma), rabbit anti‐TgMys (1:1000) (Ovciarikova et al. , ), mouse anti‐Myc (1:1000, Cell Signalling), rabbit anti‐TgTOM40 (1:2000)(van Dooren et al. , ); mouse anti‐Strep (1:1000, StrepMAB‐Classic, IBA), mouse anti‐FLAG (1:1000, Monoclonal ANTI‐FLAG® M2, Sigma‐Aldrich) coupled with goat anti‐mouse or anti‐rabbit fluorescent antibody (AlexaFluor 594 or 488 1:1000 (Invitrogen)).

    Techniques: Fluorescence, Microscopy

    Transient expression of Cas9 results in mitochondria morphology defects. A. Representative immunofluorescence micrographs of parasites transiently expressing CAS9 and sgRNA for the mentioned GOI. Mitochondria in red are marked by anti‐TgMys. CAS9‐FLAG in green is marked with anti‐FLAG. Merge shows DAPI, FLAG and TgMys. Green boxes highlight parasites with wild‐type looking mitochondria. Red boxes highlight parasites with mitochondria that appear to have a morphological defect herein named ‘abnormal mitochondria.’ B. Quantification of vacuole with abnormal mitochondria morphologies for each condition. Bars represent the mean ± SEM ( n = 3), * p

    Journal: Molecular Microbiology

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

    doi: 10.1111/mmi.14357

    Figure Lengend Snippet: Transient expression of Cas9 results in mitochondria morphology defects. A. Representative immunofluorescence micrographs of parasites transiently expressing CAS9 and sgRNA for the mentioned GOI. Mitochondria in red are marked by anti‐TgMys. CAS9‐FLAG in green is marked with anti‐FLAG. Merge shows DAPI, FLAG and TgMys. Green boxes highlight parasites with wild‐type looking mitochondria. Red boxes highlight parasites with mitochondria that appear to have a morphological defect herein named ‘abnormal mitochondria.’ B. Quantification of vacuole with abnormal mitochondria morphologies for each condition. Bars represent the mean ± SEM ( n = 3), * p

    Article Snippet: Cells were then labelled with different sets of primary and secondary antibodies: mouse anti‐HA antibody (1:1000, Sigma), rabbit anti‐TgMys (1:1000) (Ovciarikova et al. , ), mouse anti‐Myc (1:1000, Cell Signalling), rabbit anti‐TgTOM40 (1:2000)(van Dooren et al. , ); mouse anti‐Strep (1:1000, StrepMAB‐Classic, IBA), mouse anti‐FLAG (1:1000, Monoclonal ANTI‐FLAG® M2, Sigma‐Aldrich) coupled with goat anti‐mouse or anti‐rabbit fluorescent antibody (AlexaFluor 594 or 488 1:1000 (Invitrogen)).

    Techniques: Expressing, Immunofluorescence

    Human SCP1 as a substrate target for O-GlcNAcylation. The immunologically defined epitope-tagged hSCP1s (Wt and D 96 N) were induced in NIH/3T3/hSCP1-V5 (A) and NIH/3T3/M2-hSCP1 (B) cells for the indicated periods and hSCP1 from equal amount of total cell lysate (2 mg) were immunoprecipitated with either α-Flag antibody or α-V5 epitope antibody. O -GlcNAcylated hSCP1 was detected using the α- O -GlcNAc antibody. For reprobing the blots, the blots were washed, incubated with BlotFresh Western Blot Stripping Reagent, and remonitored the input hSCP1 proteins. (B) At 72 h of the postinduction, total cell lysates (2 mg) from the noninduced and induced cells for the indicated periods were incubated with sWGA Agarose beads. The affinity-purified hSCP1 in the precipitate was resolved onto SDS-PAGE and probed with either α-DYKDDDDK or α-V5 epitope antibody.

    Journal: BMB Reports

    Article Title: In vivo putative O-GlcNAcylation of human SCP1 and evidence for possible role of its N-terminal disordered structure

    doi: 10.5483/BMBRep.2014.47.10.144

    Figure Lengend Snippet: Human SCP1 as a substrate target for O-GlcNAcylation. The immunologically defined epitope-tagged hSCP1s (Wt and D 96 N) were induced in NIH/3T3/hSCP1-V5 (A) and NIH/3T3/M2-hSCP1 (B) cells for the indicated periods and hSCP1 from equal amount of total cell lysate (2 mg) were immunoprecipitated with either α-Flag antibody or α-V5 epitope antibody. O -GlcNAcylated hSCP1 was detected using the α- O -GlcNAc antibody. For reprobing the blots, the blots were washed, incubated with BlotFresh Western Blot Stripping Reagent, and remonitored the input hSCP1 proteins. (B) At 72 h of the postinduction, total cell lysates (2 mg) from the noninduced and induced cells for the indicated periods were incubated with sWGA Agarose beads. The affinity-purified hSCP1 in the precipitate was resolved onto SDS-PAGE and probed with either α-DYKDDDDK or α-V5 epitope antibody.

    Article Snippet: Primary antibodies used are as follows: mouse α-Flag (M2 clone, Sigma-Aldrich), rat α-DYKDDDDK (BioLegend, San Diego, CA, USA), mouse α-V5 epitope (Santa Cruz Biotech., Santa Cruz, CA, USA), mouse α-O -GlcNAc (CTD110.6 clone, Covance, Prinston, NJ, USA), and rabbit a α-tubulin (AbClon, Seoul, Korea).

    Techniques: Immunoprecipitation, Incubation, Western Blot, Stripping, Affinity Purification, SDS Page

    Determination of O -GlcNAcylation site on hSCP1. (A) The Flag-tagged hSCP1 (Wt) from NIH/3T3/M2-hSCP1 cells (20 mg of total cell lysate) was subjected to the immunoprecipitation with α-Flag-Agarose. The immunoprecipitates were subjected to 6-15% SDS-PAGE and stained with CBB. Arrow indicates hSCP1. (B) A small portion of immunoprecipitate was subjected to Western blot analysis with α-DYKDDDDK antibody. (C) The Q-TOF spectrum and the sequencing results of a GlcNAc-modified peptide corresponding to residues 36-50 are shown. S 41 residue in hSCP1 is an O -GlcNAcylation site. The expected increase in mass by the O -GlcNAc modification is 203.2 Da. (D) Whole cell lysates of NIH/3T3 transfected with V5-tagged hSCP1 (empty vector (pcDNA3), Wt and S 41 A) were immunoprecipitated with V5-epitope Agarose beads and characterized by Western blot analysis with α O -GlcNAc antibody. The protein input was monitored with α-V5 antibody after stripping the blot. Arrow head indicates IgG light chain.

    Journal: BMB Reports

    Article Title: In vivo putative O-GlcNAcylation of human SCP1 and evidence for possible role of its N-terminal disordered structure

    doi: 10.5483/BMBRep.2014.47.10.144

    Figure Lengend Snippet: Determination of O -GlcNAcylation site on hSCP1. (A) The Flag-tagged hSCP1 (Wt) from NIH/3T3/M2-hSCP1 cells (20 mg of total cell lysate) was subjected to the immunoprecipitation with α-Flag-Agarose. The immunoprecipitates were subjected to 6-15% SDS-PAGE and stained with CBB. Arrow indicates hSCP1. (B) A small portion of immunoprecipitate was subjected to Western blot analysis with α-DYKDDDDK antibody. (C) The Q-TOF spectrum and the sequencing results of a GlcNAc-modified peptide corresponding to residues 36-50 are shown. S 41 residue in hSCP1 is an O -GlcNAcylation site. The expected increase in mass by the O -GlcNAc modification is 203.2 Da. (D) Whole cell lysates of NIH/3T3 transfected with V5-tagged hSCP1 (empty vector (pcDNA3), Wt and S 41 A) were immunoprecipitated with V5-epitope Agarose beads and characterized by Western blot analysis with α O -GlcNAc antibody. The protein input was monitored with α-V5 antibody after stripping the blot. Arrow head indicates IgG light chain.

    Article Snippet: Primary antibodies used are as follows: mouse α-Flag (M2 clone, Sigma-Aldrich), rat α-DYKDDDDK (BioLegend, San Diego, CA, USA), mouse α-V5 epitope (Santa Cruz Biotech., Santa Cruz, CA, USA), mouse α-O -GlcNAc (CTD110.6 clone, Covance, Prinston, NJ, USA), and rabbit a α-tubulin (AbClon, Seoul, Korea).

    Techniques: Immunoprecipitation, SDS Page, Staining, Western Blot, Sequencing, Modification, Transfection, Plasmid Preparation, Stripping Membranes

    Sam68 and hnRNP L proteins associate via protein-protein interactions and are not bridged by nucleic acids . (A) LNCaP cell nuclear extracts were subjected to IP using either anti-Sam68 antisera or an anti-hnRNP L monoclonal antibody. Recovered material was subjected to Western analysis with antibodies specific to hnRNP L (left panel) or Sam68 (right panel). (B) HEK293 cells were transfected with expression vectors for FLAG-Sam68 or FLAG alone, and hnRNP L-GST, and subjected to IP using anti-FLAG M2 agarose. Recovered material was subjected to Western analysis with antisera to GST. (C) HEK293 cell nuclear extracts were fractionated on sucrose gradients before (upper panel) or after (lower panel) treatment with MNase to digest nucleic acids. Fraction 20 (containing low molecular weight material) was taken from the top of the gradient, and fraction 1 (containing high molecular weight material) was taken from the bottom. Pelleted material is indicated as P. The migration of individual proteins in each fraction was monitored by SDS-PAGE and Western analysis. The mobility of size markers on the gradients is shown.

    Journal: BMC Cell Biology

    Article Title: Proteomic identification of heterogeneous nuclear ribonucleoprotein L as a novel component of SLM/Sam68 Nuclear Bodies

    doi: 10.1186/1471-2121-10-82

    Figure Lengend Snippet: Sam68 and hnRNP L proteins associate via protein-protein interactions and are not bridged by nucleic acids . (A) LNCaP cell nuclear extracts were subjected to IP using either anti-Sam68 antisera or an anti-hnRNP L monoclonal antibody. Recovered material was subjected to Western analysis with antibodies specific to hnRNP L (left panel) or Sam68 (right panel). (B) HEK293 cells were transfected with expression vectors for FLAG-Sam68 or FLAG alone, and hnRNP L-GST, and subjected to IP using anti-FLAG M2 agarose. Recovered material was subjected to Western analysis with antisera to GST. (C) HEK293 cell nuclear extracts were fractionated on sucrose gradients before (upper panel) or after (lower panel) treatment with MNase to digest nucleic acids. Fraction 20 (containing low molecular weight material) was taken from the top of the gradient, and fraction 1 (containing high molecular weight material) was taken from the bottom. Pelleted material is indicated as P. The migration of individual proteins in each fraction was monitored by SDS-PAGE and Western analysis. The mobility of size markers on the gradients is shown.

    Article Snippet: Immunoprecipitations were performed using Dynabeads Protein A (Invitrogen) or anti-FLAG M2 agarose (Sigma) according to manufacturers' instructions.

    Techniques: Western Blot, Transfection, Expressing, Molecular Weight, Migration, SDS Page

    (A) OPN and Stat1 protein expression in 4T1 and 4T07 cell lysates and culture media. The mouse mammary tumor cell lines 4T1 and 4T07 were cultured. Cells were lysed and centrifuged. For media analysis, serum-free DMEM was centrifuged to remove cellular materials and was concentrated 100-fold. Cell lysates (50 µg/lane) were separated by SDS-12% polyacrylamide gel electrophoresis, and products were electrotransferred to polyvinylidene difluoride membrane. After blocking and washing, membranes were incubated with primary antibody, washed, and incubated with horse-radish peroxidase-conjugated secondary antibody. Bound peroxidase activity was detected. The blot is representative of three experiments. (B) Steady-state Stat1 mRNA expression in 4T1 and 4T07 cells. Total RNA was isolated and reverse-transcribed into cDNA. cDNA were used in subsequent PCRs; the primers for Stat1 were 5′-CTTATTCCATGGACAAGGTTTTG-3′ (forward) and 5′-GGTGCTTCTTAATGAGCTCTAGG-3′ (reverse). The gel is representative of three experiments. (C) co-IP of Ub-Stat1 in 4T1 and 4T07 cells. 4T1 and 4T07 cells were transfected with FLAG/Stat1 expression plasmid, HA/Ub expression plasmid, and/or empty expression plasmids. Full-length Stat1 cDNA (U06924) was obtained by RT-PCR with the following primers: Stat1-F 5′-ACGAAGCTTATGTCACAGTGGTTCGAGCTTCAG-3′ and Stat1-R 5′-ACGAAGCTTTTACACTTCAGACACAGAAATCAAC-3′; the 2250-bp fragment was inserted into the pCMV-FLAG2 vector (Sigma-Aldrich). HA/Ub plasmid was kindly provided by Dr. Bohmann (University of Rochester). After 36 hours of transfection, cells were stimulated with 10 µM MG132, an inhibitor of 26S proteasome function, or DMSO control for 2 hours; washed; and lysed for 15 minutes on ice in RIPA lysis buffer. Immunoprecipitation were performed with 1000 µg of cell lysate and 40 µl of M2 anti-FLAG agarose (Sigma-Aldrich). Immunoblotting was performed with HA or Ub antibody, as described. Blots are representative of three experiments.

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

    Article Title: Osteopontin Regulates Ubiquitin-Dependent Degradation of Stat1 in Murine Mammary Epithelial Tumor Cells 1

    doi:

    Figure Lengend Snippet: (A) OPN and Stat1 protein expression in 4T1 and 4T07 cell lysates and culture media. The mouse mammary tumor cell lines 4T1 and 4T07 were cultured. Cells were lysed and centrifuged. For media analysis, serum-free DMEM was centrifuged to remove cellular materials and was concentrated 100-fold. Cell lysates (50 µg/lane) were separated by SDS-12% polyacrylamide gel electrophoresis, and products were electrotransferred to polyvinylidene difluoride membrane. After blocking and washing, membranes were incubated with primary antibody, washed, and incubated with horse-radish peroxidase-conjugated secondary antibody. Bound peroxidase activity was detected. The blot is representative of three experiments. (B) Steady-state Stat1 mRNA expression in 4T1 and 4T07 cells. Total RNA was isolated and reverse-transcribed into cDNA. cDNA were used in subsequent PCRs; the primers for Stat1 were 5′-CTTATTCCATGGACAAGGTTTTG-3′ (forward) and 5′-GGTGCTTCTTAATGAGCTCTAGG-3′ (reverse). The gel is representative of three experiments. (C) co-IP of Ub-Stat1 in 4T1 and 4T07 cells. 4T1 and 4T07 cells were transfected with FLAG/Stat1 expression plasmid, HA/Ub expression plasmid, and/or empty expression plasmids. Full-length Stat1 cDNA (U06924) was obtained by RT-PCR with the following primers: Stat1-F 5′-ACGAAGCTTATGTCACAGTGGTTCGAGCTTCAG-3′ and Stat1-R 5′-ACGAAGCTTTTACACTTCAGACACAGAAATCAAC-3′; the 2250-bp fragment was inserted into the pCMV-FLAG2 vector (Sigma-Aldrich). HA/Ub plasmid was kindly provided by Dr. Bohmann (University of Rochester). After 36 hours of transfection, cells were stimulated with 10 µM MG132, an inhibitor of 26S proteasome function, or DMSO control for 2 hours; washed; and lysed for 15 minutes on ice in RIPA lysis buffer. Immunoprecipitation were performed with 1000 µg of cell lysate and 40 µl of M2 anti-FLAG agarose (Sigma-Aldrich). Immunoblotting was performed with HA or Ub antibody, as described. Blots are representative of three experiments.

    Article Snippet: Immunoprecipitation was performed with 1000 µg of cell lysate and 40 µl of M2 anti-FLAG agarose, according the method provided by the manufacturer (Sigma-Aldrich).

    Techniques: Expressing, Cell Culture, Polyacrylamide Gel Electrophoresis, Blocking Assay, Incubation, Activity Assay, Isolation, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Lysis, Immunoprecipitation

    SC1 forms a complex with HDACs 1, 2, and 3, and its repression is TSA sensitive.  (A) Repression of the Gal4-luc promoter is abolished by the addition of 50 ng/ml TSA to the transfected HEK293 cells. Relative luciferase activity was measured as described in   Fig. 2  A. Black bars represent the control measurement with Gal4 only as an effector; open bars represent the measurement with Gal4SC1 as an effector. (B) SC1 coprecipitates with HDACs 1, 2, and 3 when cotransfected in HEK293 cells. Proteins from transfected cell lysates were immunoprecipitated using anti-Flag M2 Sepharose, separated by SDS-PAGE, and immunoblotted with either anti-Flag M2 antibody for SC1 or anti-HA antibody for HDACs. The bottom panel shows input of HDACs.

    Journal: The Journal of Cell Biology

    Article Title: The p75NTR-interacting protein SC1 inhibits cell cycle progression by transcriptional repression of cyclin E

    doi: 10.1083/jcb.200301106

    Figure Lengend Snippet: SC1 forms a complex with HDACs 1, 2, and 3, and its repression is TSA sensitive. (A) Repression of the Gal4-luc promoter is abolished by the addition of 50 ng/ml TSA to the transfected HEK293 cells. Relative luciferase activity was measured as described in Fig. 2 A. Black bars represent the control measurement with Gal4 only as an effector; open bars represent the measurement with Gal4SC1 as an effector. (B) SC1 coprecipitates with HDACs 1, 2, and 3 when cotransfected in HEK293 cells. Proteins from transfected cell lysates were immunoprecipitated using anti-Flag M2 Sepharose, separated by SDS-PAGE, and immunoblotted with either anti-Flag M2 antibody for SC1 or anti-HA antibody for HDACs. The bottom panel shows input of HDACs.

    Article Snippet: Lysates were then precleared using protein A/G beads (Amersham Biosciences) and Flag-tagged proteins were precipitated using anti-Flag M2 Sepharose (Sigma-Aldrich).

    Techniques: Transfection, Luciferase, Activity Assay, Immunoprecipitation, SDS Page

    PXR exhibits a ligand-dependent interaction with HNF4α. ( A ) Huh7 cells were reverse transfected with indicated expression plasmids for 30 h and then treated with RIF or DMSO (DM) for another 2 h in FBS-free MEM, from which whole cell extracts were prepared and subjected to immunoprecipitation with FLAG-M2 agarose. Immunoprecipitated proteins were detected by western blotting with anti-FLAG-M2 HRP-conjugated antibody or anti-V5 HRP-conjugated antibody. ( B ) Model of mechanism for the PXR-mediated repression of the SULT1E1 gene.

    Journal: Nucleic Acids Research

    Article Title: Liganded pregnane X receptor represses the human sulfotransferase SULT1E1 promoter through disrupting its chromatin structure

    doi: 10.1093/nar/gkr458

    Figure Lengend Snippet: PXR exhibits a ligand-dependent interaction with HNF4α. ( A ) Huh7 cells were reverse transfected with indicated expression plasmids for 30 h and then treated with RIF or DMSO (DM) for another 2 h in FBS-free MEM, from which whole cell extracts were prepared and subjected to immunoprecipitation with FLAG-M2 agarose. Immunoprecipitated proteins were detected by western blotting with anti-FLAG-M2 HRP-conjugated antibody or anti-V5 HRP-conjugated antibody. ( B ) Model of mechanism for the PXR-mediated repression of the SULT1E1 gene.

    Article Snippet: Materials RIF, SR12813, cycloheximide (CHX), TCPOBOP, anti-FLAG-M2 HRP-conjugated antibody and FLAG-M2 agarose were purchased from Sigma-Aldrich (St Louis, MO, USA); CITCO from BioMol (Plymouth Meeting, PA, USA); restriction endonucleases and DNA-modifying enzymes from New England Biolabs, Inc. (Beverly, MA, USA); antibody to human PXR from Perseus Proteomics Inc. (Tokyo, Japan); mouse, goat and rabbit normal IgGs, antibodies against HNF3β (M-20), HNF4α (H-171) and β-actin (C4) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-V5 HRP-conjugated antibody from Invitrogen (Carlsbad, CA, USA); anti-H3K9K14ac (06–599) from Millipore (Billerica, MA, USA); [32 P]dATP from GE Healthcare (Piscataway, NJ, USA) and [35 S]methionine from PerkinElmer (Waltham, MA, USA).

    Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

    Dll1IC bound to Smad2, Smad3 and Smad4. (a) Result of screening for transcription factors that bind to Dll1IC. Note that Smad binding sequences showed strong signals (boxed). (b) Signal from Smad binding sequences (boxed) were enhanced by the addition of recombinant Dll1IC protein to the nuclear extract before immunoprecipitation. Signals from Pax-5 binding sequence and mineral corticoid response element (MRE) were also enhanced by the addition of recombinant Dll1IC protein. On the other hand, signals from NF-κB binding site and NF-E2 binding sequence were disappeared by the addition of recombinant Dll1IC protein. Spots along the right and bottom side of arrays are markers for alignment. (c) COS7 cells were transiently co-transfected with each expression vector for 8 Smads and Dll1IC expression vector with or without expression vectors for constitutive activated receptor (caALK5-HA or caALK6-HA). Forty-eight hours after transfection, lysates from co-transfected COS7 cells were subjected to immunoprecipitation (IP) with the indicated antibodies, followed by western blotting (WB). αDll1IC, rabbit anti-Dll1IC antibody; αFlag, mouse monoclonal anti-FLAG M2 antibody. Levels of expression of Smads and Dll1IC were determined by western blotting and are shown in the bottom two panels.

    Journal: Nucleic Acids Research

    Article Title: The Delta intracellular domain mediates TGF-?/Activin signaling through binding to Smads and has an important bi-directional function in the Notch-Delta signaling pathway

    doi: 10.1093/nar/gkl1128

    Figure Lengend Snippet: Dll1IC bound to Smad2, Smad3 and Smad4. (a) Result of screening for transcription factors that bind to Dll1IC. Note that Smad binding sequences showed strong signals (boxed). (b) Signal from Smad binding sequences (boxed) were enhanced by the addition of recombinant Dll1IC protein to the nuclear extract before immunoprecipitation. Signals from Pax-5 binding sequence and mineral corticoid response element (MRE) were also enhanced by the addition of recombinant Dll1IC protein. On the other hand, signals from NF-κB binding site and NF-E2 binding sequence were disappeared by the addition of recombinant Dll1IC protein. Spots along the right and bottom side of arrays are markers for alignment. (c) COS7 cells were transiently co-transfected with each expression vector for 8 Smads and Dll1IC expression vector with or without expression vectors for constitutive activated receptor (caALK5-HA or caALK6-HA). Forty-eight hours after transfection, lysates from co-transfected COS7 cells were subjected to immunoprecipitation (IP) with the indicated antibodies, followed by western blotting (WB). αDll1IC, rabbit anti-Dll1IC antibody; αFlag, mouse monoclonal anti-FLAG M2 antibody. Levels of expression of Smads and Dll1IC were determined by western blotting and are shown in the bottom two panels.

    Article Snippet: Forty-eight hours after transfection, cells were disrupted and aliquots of 20 μg of cell lysates were incubated with 1 μg of rabbit anti-Dll1IC antibody or 1 μg of mouse monoclonal anti-FLAG M2 antibody (Sigma), followed by immunoprecipitation and western blotting as described ( ).

    Techniques: Binding Assay, Recombinant, Immunoprecipitation, Sequencing, Transfection, Expressing, Plasmid Preparation, Western Blot

    Western immunoblot analysis of L-flag epitope tag fusions. Lysates were obtained from BSR-T7 cells transfected with pTM-TΔC180 (lane 1) (A) or pTM-1 (empty vector) (lane 2); pTM-TΔN1062 (lane 1) (B), pTM-TΔC759 (lane 2), pTM-TΔN394 (lane 3), or pTM-1 (empty vector) (lane 4); or pTM-1 (empty vector) (lane 1) (C) or pTM-TL (lane 2). Blots were probed with anti-flag M2 murine primary antibody (Sigma) and goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Sigma). Arrows indicate protein from: pTM-TΔC180 (a), pTM-TΔN394 (b), pTM-TΔC759 (c), pTM-TΔN1062 (d), or pTM-TL (e).

    Journal: Journal of Virology

    Article Title: Characterization of the Nuclear Localization Signal of the Borna Disease Virus Polymerase

    doi: 10.1128/JVI.76.16.8460-8467.2002

    Figure Lengend Snippet: Western immunoblot analysis of L-flag epitope tag fusions. Lysates were obtained from BSR-T7 cells transfected with pTM-TΔC180 (lane 1) (A) or pTM-1 (empty vector) (lane 2); pTM-TΔN1062 (lane 1) (B), pTM-TΔC759 (lane 2), pTM-TΔN394 (lane 3), or pTM-1 (empty vector) (lane 4); or pTM-1 (empty vector) (lane 1) (C) or pTM-TL (lane 2). Blots were probed with anti-flag M2 murine primary antibody (Sigma) and goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Sigma). Arrows indicate protein from: pTM-TΔC180 (a), pTM-TΔN394 (b), pTM-TΔC759 (c), pTM-TΔN1062 (d), or pTM-TL (e).

    Article Snippet: Indirect immunofluorescence with murine anti-flag M2 antibody (Sigma) on BSR-T7 cells transfected with expression plasmids showed clear nuclear staining when residues 759 to 1062 were present (Fig. ).

    Techniques: Western Blot, FLAG-tag, Transfection, Plasmid Preparation

    Subcellular localization of L-flag epitope tag fusion proteins. (A) Description of expression plasmids and summary of results. Amino acid sequence of L, with a diagram of full-length and deletion mutant L-flag epitope tag fusion expression plasmids. Plasmid names and subcellular localization of fusion proteins are listed. (B through G) Subcellular localization of full-length or deletion mutants of L-flag epitope tag by indirect immunofluorescence in BSR-T7 cells transfected with expression plasmids. (B) pTM-TL; (C) pTM-TΔC759; (D) pTM-TΔC180; (E) pTM-TΔN1062; (F) pTM-TΔN394; (G) pTM1. Cells were stained with anti-flag M2 murine antibody (Sigma) and goat anti-mouse IgG fluorescein isothiocyanate (Caltag).

    Journal: Journal of Virology

    Article Title: Characterization of the Nuclear Localization Signal of the Borna Disease Virus Polymerase

    doi: 10.1128/JVI.76.16.8460-8467.2002

    Figure Lengend Snippet: Subcellular localization of L-flag epitope tag fusion proteins. (A) Description of expression plasmids and summary of results. Amino acid sequence of L, with a diagram of full-length and deletion mutant L-flag epitope tag fusion expression plasmids. Plasmid names and subcellular localization of fusion proteins are listed. (B through G) Subcellular localization of full-length or deletion mutants of L-flag epitope tag by indirect immunofluorescence in BSR-T7 cells transfected with expression plasmids. (B) pTM-TL; (C) pTM-TΔC759; (D) pTM-TΔC180; (E) pTM-TΔN1062; (F) pTM-TΔN394; (G) pTM1. Cells were stained with anti-flag M2 murine antibody (Sigma) and goat anti-mouse IgG fluorescein isothiocyanate (Caltag).

    Article Snippet: Indirect immunofluorescence with murine anti-flag M2 antibody (Sigma) on BSR-T7 cells transfected with expression plasmids showed clear nuclear staining when residues 759 to 1062 were present (Fig. ).

    Techniques: FLAG-tag, Expressing, Sequencing, Mutagenesis, Plasmid Preparation, Immunofluorescence, Transfection, Staining

    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: The proteins were transferred onto nitrocellulose (Biorad, Hercules, CA, USA) in a Biorad Mini Trans-Blot apparatus at 300 mA for 1 h. The recombinant antibody, containing C-terminal flag and tubulin tags, was detected with mouse M2 anti-Flag (Sigma, St Louis, MO, USA) or rat antitubulin antibodies (Serotec, Oxford, UK), respectively, followed by goat anti-mouse immunoglobulin coupled to alkaline phosphatase (Promega, Madison, WI, USA) or goat anti-rat immunoglobulin coupled to alkaline phosphatase (Southern Biotechnology Associates), respectively, and developed with a substrate solution containing fast red and naphthol phosphate AS-MX reagents (Sigma, St Louis, MO, USA).

    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: The proteins were transferred onto nitrocellulose (Biorad, Hercules, CA, USA) in a Biorad Mini Trans-Blot apparatus at 300 mA for 1 h. The recombinant antibody, containing C-terminal flag and tubulin tags, was detected with mouse M2 anti-Flag (Sigma, St Louis, MO, USA) or rat antitubulin antibodies (Serotec, Oxford, UK), respectively, followed by goat anti-mouse immunoglobulin coupled to alkaline phosphatase (Promega, Madison, WI, USA) or goat anti-rat immunoglobulin coupled to alkaline phosphatase (Southern Biotechnology Associates), respectively, and developed with a substrate solution containing fast red and naphthol phosphate AS-MX reagents (Sigma, St Louis, MO, USA).

    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: The proteins were transferred onto nitrocellulose (Biorad, Hercules, CA, USA) in a Biorad Mini Trans-Blot apparatus at 300 mA for 1 h. The recombinant antibody, containing C-terminal flag and tubulin tags, was detected with mouse M2 anti-Flag (Sigma, St Louis, MO, USA) or rat antitubulin antibodies (Serotec, Oxford, UK), respectively, followed by goat anti-mouse immunoglobulin coupled to alkaline phosphatase (Promega, Madison, WI, USA) or goat anti-rat immunoglobulin coupled to alkaline phosphatase (Southern Biotechnology Associates), respectively, and developed with a substrate solution containing fast red and naphthol phosphate AS-MX reagents (Sigma, St Louis, MO, USA).

    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: The proteins were transferred onto nitrocellulose (Biorad, Hercules, CA, USA) in a Biorad Mini Trans-Blot apparatus at 300 mA for 1 h. The recombinant antibody, containing C-terminal flag and tubulin tags, was detected with mouse M2 anti-Flag (Sigma, St Louis, MO, USA) or rat antitubulin antibodies (Serotec, Oxford, UK), respectively, followed by goat anti-mouse immunoglobulin coupled to alkaline phosphatase (Promega, Madison, WI, USA) or goat anti-rat immunoglobulin coupled to alkaline phosphatase (Southern Biotechnology Associates), respectively, and developed with a substrate solution containing fast red and naphthol phosphate AS-MX reagents (Sigma, St Louis, MO, USA).

    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: The proteins were transferred onto nitrocellulose (Biorad, Hercules, CA, USA) in a Biorad Mini Trans-Blot apparatus at 300 mA for 1 h. The recombinant antibody, containing C-terminal flag and tubulin tags, was detected with mouse M2 anti-Flag (Sigma, St Louis, MO, USA) or rat antitubulin antibodies (Serotec, Oxford, UK), respectively, followed by goat anti-mouse immunoglobulin coupled to alkaline phosphatase (Promega, Madison, WI, USA) or goat anti-rat immunoglobulin coupled to alkaline phosphatase (Southern Biotechnology Associates), respectively, and developed with a substrate solution containing fast red and naphthol phosphate AS-MX reagents (Sigma, St Louis, MO, USA).

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

    Interactions of phosphorylated and dephosphorylated forms of CMG and Tof1 with each other as revealed by Western blotting. ( A , Left ) Zn 2+ Phos-tag gel of phosphorylated and dephosphorylated Tof1. ( A , Right ) SDS/PAGE profiles of both untreated Tof1-myc and that treated with λ-phosphatase. Actin loading controls are also shown. ( B ) Interaction of Tof1-myc–Csm3 complex untreated and treated with λ-phosphatase with immobilized CMG-His 6 . ( C ) WBs showing binding of Tof1-myc untreated (-) and treated with λ-phosphatase (+) to immobilized FLAG-tagged Mcm2–7. ( D ) Interaction of Tof1-myc with immobilized untreated CMG-His 6 ( Left ) and dephosphorylated immobilized CMG-His 6 ( Right ). ( E ) Interaction of Tof1-myc with untreated immobilized Mcm2–7-FLAG and with dephosphorylated and immobilized Mcm2–7-FLAG. ( F ) Quantification of binding of Tof1-myc untreated (-) and dephosphorylated (+) to immobilized CMG. ( G ) Binding of untreated (-) and dephosphorylated Tof1-myc to immobilized Mcm2–7-FLAG (not dephosphorylated with λ-phosphatase). ( H ) Binding of untreated Tof1-myc to immobilized untreated CMG-His 6 and to the same after dephosphorylation with λ-phosphatase. ( I ) Quantification of binding of Tof1-myc to untreated immobilized Mcm2–7-FLAG and to the same after dephosphorylation with λ-phosphatase. ( J ) Quantification of binding of Cdc45 in solution to untreated immobilized Tof1-myc or to the same after treatment with λ-phosphatase. ( K ) Quantification of the binding of immobilized GINS complex to Tof1-myc in solution before (-) and after (+) treatment with λ-phosphatase. Error bars represent standard error.

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

    Article Title: Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

    doi: 10.1073/pnas.1607552113

    Figure Lengend Snippet: Interactions of phosphorylated and dephosphorylated forms of CMG and Tof1 with each other as revealed by Western blotting. ( A , Left ) Zn 2+ Phos-tag gel of phosphorylated and dephosphorylated Tof1. ( A , Right ) SDS/PAGE profiles of both untreated Tof1-myc and that treated with λ-phosphatase. Actin loading controls are also shown. ( B ) Interaction of Tof1-myc–Csm3 complex untreated and treated with λ-phosphatase with immobilized CMG-His 6 . ( C ) WBs showing binding of Tof1-myc untreated (-) and treated with λ-phosphatase (+) to immobilized FLAG-tagged Mcm2–7. ( D ) Interaction of Tof1-myc with immobilized untreated CMG-His 6 ( Left ) and dephosphorylated immobilized CMG-His 6 ( Right ). ( E ) Interaction of Tof1-myc with untreated immobilized Mcm2–7-FLAG and with dephosphorylated and immobilized Mcm2–7-FLAG. ( F ) Quantification of binding of Tof1-myc untreated (-) and dephosphorylated (+) to immobilized CMG. ( G ) Binding of untreated (-) and dephosphorylated Tof1-myc to immobilized Mcm2–7-FLAG (not dephosphorylated with λ-phosphatase). ( H ) Binding of untreated Tof1-myc to immobilized untreated CMG-His 6 and to the same after dephosphorylation with λ-phosphatase. ( I ) Quantification of binding of Tof1-myc to untreated immobilized Mcm2–7-FLAG and to the same after dephosphorylation with λ-phosphatase. ( J ) Quantification of binding of Cdc45 in solution to untreated immobilized Tof1-myc or to the same after treatment with λ-phosphatase. ( K ) Quantification of the binding of immobilized GINS complex to Tof1-myc in solution before (-) and after (+) treatment with λ-phosphatase. Error bars represent standard error.

    Article Snippet: Proteins were analyzed by electrophoresis on 6% SDS/PAGE, followed by Western blotting using monoclonal anti-FLAG antibody (M2; Sigma-Aldrich) as the primary antibody.

    Techniques: Western Blot, SDS Page, Binding Assay, De-Phosphorylation Assay

    RIF1 promotes PP1 to dephosphorylate AXIN. a , b Immunoprecipitation was performed with PP1 antibody and the precipitated protein complexes were analyzed by western blots with antibodies to RIF1 and AXIN in H1299 ( a ) and SK-MES-1 ( b ) cells. c , d RIF1-silenced H1299 ( c ) and SK-MES-1 ( d ) cells and the scrambled control cells were transfected with Flag-tagged AXIN plasmid. Immunoprecipitation was done with anti-FLAG M2 magnetic beads and the precipitated complexes, which were pull down by Flag-tagged AXIN were subjected to western blot with antibodies to RIF1, PP1 and p-Ser. e, f H1299 ( e ) and SK-MES-1 ( f ) cells were co-transfected Flag-tagged AXIN plasmid with control vector or RIF1 overexpression plasmid. Immunoprecipitation was performed with anti-FLAG M2 magnetic beads and the precipitated complexes were analyzed by western blots with antibodies to RIF1, PP1 and p-Ser (mouse IgG conjugated with magnetic beads being the negative control)

    Journal: Cell Death & Disease

    Article Title: RIF1 promotes tumor growth and cancer stem cell-like traits in NSCLC by protein phosphatase 1-mediated activation of Wnt/β-catenin signaling

    doi: 10.1038/s41419-018-0972-4

    Figure Lengend Snippet: RIF1 promotes PP1 to dephosphorylate AXIN. a , b Immunoprecipitation was performed with PP1 antibody and the precipitated protein complexes were analyzed by western blots with antibodies to RIF1 and AXIN in H1299 ( a ) and SK-MES-1 ( b ) cells. c , d RIF1-silenced H1299 ( c ) and SK-MES-1 ( d ) cells and the scrambled control cells were transfected with Flag-tagged AXIN plasmid. Immunoprecipitation was done with anti-FLAG M2 magnetic beads and the precipitated complexes, which were pull down by Flag-tagged AXIN were subjected to western blot with antibodies to RIF1, PP1 and p-Ser. e, f H1299 ( e ) and SK-MES-1 ( f ) cells were co-transfected Flag-tagged AXIN plasmid with control vector or RIF1 overexpression plasmid. Immunoprecipitation was performed with anti-FLAG M2 magnetic beads and the precipitated complexes were analyzed by western blots with antibodies to RIF1, PP1 and p-Ser (mouse IgG conjugated with magnetic beads being the negative control)

    Article Snippet: Subsequently, the lysate was cleared by centrifugation at 14,000 g for 15 min. Immunoprecipitation was carried out with indicated antibody together with protein A/G agarose magnetic beads (GE Healthcare) or anti-Flag M2 magnetic beads (Sigma) with normal mouse IgG or mouse IgG conjugated with magnetic beads as the negative control, respectively.

    Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Magnetic Beads, Over Expression, Negative Control

    Ezh2 is threonine-phosphorylated. A , Ezh2 is phosphorylated in sites other than Ser-21. HEK293T cells exogenously expressing FLAG-tagged wild-type Ezh2 or S21A were radiolabeled with [γ- 32 P]ATP. Following immunoprecipitation using FLAG antibodies, bound proteins were treated with no phosphatase, with PP1, or with λ-PPase. Autoradiography ( Autorad ) confirms that both wild-type and S21A mutant Ezh2 are phosphorylated and that the signal diminishes upon phosphatase treatment. Bottom panel verifies that equal amounts FLAG-Ezh2 were used in the assay. Ctrl , control. B , Ezh2 is threonine-phosphorylated. FLAG-Ezh2 was transfected into HEK293T cells and immunoprecipitated using FLAG antibodies. Bound proteins were treated with or without λ-PPase and subjected to Western blot analysis using antibodies specific for phospho-serine (α -pSer ), phospho-threonine (α -pThr ), and phospho-tyrosine (α -pTyr ).

    Journal: The Journal of Biological Chemistry

    Article Title: Cyclin-dependent Kinase 1 (CDK1)-mediated Phosphorylation of Enhancer of Zeste 2 (Ezh2) Regulates Its Stability *

    doi: 10.1074/jbc.M111.240515

    Figure Lengend Snippet: Ezh2 is threonine-phosphorylated. A , Ezh2 is phosphorylated in sites other than Ser-21. HEK293T cells exogenously expressing FLAG-tagged wild-type Ezh2 or S21A were radiolabeled with [γ- 32 P]ATP. Following immunoprecipitation using FLAG antibodies, bound proteins were treated with no phosphatase, with PP1, or with λ-PPase. Autoradiography ( Autorad ) confirms that both wild-type and S21A mutant Ezh2 are phosphorylated and that the signal diminishes upon phosphatase treatment. Bottom panel verifies that equal amounts FLAG-Ezh2 were used in the assay. Ctrl , control. B , Ezh2 is threonine-phosphorylated. FLAG-Ezh2 was transfected into HEK293T cells and immunoprecipitated using FLAG antibodies. Bound proteins were treated with or without λ-PPase and subjected to Western blot analysis using antibodies specific for phospho-serine (α -pSer ), phospho-threonine (α -pThr ), and phospho-tyrosine (α -pTyr ).

    Article Snippet: Antibodies The following antibodies were used in this study: FLAG M2 mouse monoclonal (Sigma catalog number F3165), mouse phospho-serine (Chemicon catalog number AB1603), mouse phospho-threonine (Cell Signaling catalog number 9386), mouse phospho-tyrosine 4G10 (gift from Weiguo Zhang, Duke University), rabbit Ezh2 (Cell Signaling catalog number 4905), rabbit SLBP (gift from William Marzluff, University of North Carolina), mouse cyclin B1 (Santa Cruz Biotechnology), mouse α-tubulin (Sigma catalog number T6199), lamin B (Santa Cruz Biotechnology catalog number sc-6217), mouse GST (Santa Cruz Biotechnology catalog number sc-138), rabbit H3K27me3 (Millipore catalog number 07-449), rabbit H3 (Abcam catalog number ab1791-100), and rabbit Suz12 (as described in Ref. ).

    Techniques: Expressing, Immunoprecipitation, Autoradiography, Mutagenesis, Transfection, Western Blot

    Phosphorylated Ezh2 is ubiquitinated and degraded via the proteasome pathway. A , phospho-Ezh2 exhibits a shorter half-life when compared with total Ezh2. Asynchronous HEK293T cells were transfected with FLAG-tagged Ezh2 and treated with 100 μg/ml cycloheximide ( CHX ) for the indicated times. Western blot analysis was performed using the indicated antibodies ( top panel ). Western blot signals were quantified using ImageJ ( bottom panel ). α -pT345 , Thr(P)-345 antibody; α -pT487 , Thr(P)-487 antibody. B , mutant Ezh2 degrades more slowly after mitosis. HeLa cells stably expressing FLAG-tagged wild-type Ezh2 ( WT ) or T345A/T487A ( Mut-A ) were synchronized at mitosis by thymidine-nocodazole block and then released into the cell cycle for the times indicated. Protein levels of Ezh2 were detected by FLAG Western blot ( top panel ). Cyclin B1 is a marker for mitosis, and tubulin was used as a loading control. Western blot signals were quantified using ImageJ ( bottom panel ). C , phospho-Ezh2 is ubiquitinated. Phospho-Ezh2 was immunoprecipitated from HeLa extracts using antibodies against Thr(P)-345 and Thr(P)-487. Total Ezh2 was immunoprecipitated using Ezh2 antibodies. Western blot analysis of bound proteins was performed using the antibodies indicated. D , ubiquitination status of Ezh2 in mitotic cells. HeLa cells stably expressing empty vector ( Ctrl ), FLAG-tagged wild-type Ezh2 ( WT ), T345A/T487A ( Mut-A ), and T345E/T487E ( Mut-E ) were generated by lentiviral infection and transfected with HA-tagged ubiquitin. Cells were synchronized and arrested at mitosis by thymidine-nocodazole block. 4 h prior to harvesting, cells were treated with or without 25 μ m MG132. Lysates were prepared under denaturing conditions and subjected to immunoprecipitation ( IP ) using FLAG antibodies. Bound proteins were analyzed by Western blot using the indicated antibodies.

    Journal: The Journal of Biological Chemistry

    Article Title: Cyclin-dependent Kinase 1 (CDK1)-mediated Phosphorylation of Enhancer of Zeste 2 (Ezh2) Regulates Its Stability *

    doi: 10.1074/jbc.M111.240515

    Figure Lengend Snippet: Phosphorylated Ezh2 is ubiquitinated and degraded via the proteasome pathway. A , phospho-Ezh2 exhibits a shorter half-life when compared with total Ezh2. Asynchronous HEK293T cells were transfected with FLAG-tagged Ezh2 and treated with 100 μg/ml cycloheximide ( CHX ) for the indicated times. Western blot analysis was performed using the indicated antibodies ( top panel ). Western blot signals were quantified using ImageJ ( bottom panel ). α -pT345 , Thr(P)-345 antibody; α -pT487 , Thr(P)-487 antibody. B , mutant Ezh2 degrades more slowly after mitosis. HeLa cells stably expressing FLAG-tagged wild-type Ezh2 ( WT ) or T345A/T487A ( Mut-A ) were synchronized at mitosis by thymidine-nocodazole block and then released into the cell cycle for the times indicated. Protein levels of Ezh2 were detected by FLAG Western blot ( top panel ). Cyclin B1 is a marker for mitosis, and tubulin was used as a loading control. Western blot signals were quantified using ImageJ ( bottom panel ). C , phospho-Ezh2 is ubiquitinated. Phospho-Ezh2 was immunoprecipitated from HeLa extracts using antibodies against Thr(P)-345 and Thr(P)-487. Total Ezh2 was immunoprecipitated using Ezh2 antibodies. Western blot analysis of bound proteins was performed using the antibodies indicated. D , ubiquitination status of Ezh2 in mitotic cells. HeLa cells stably expressing empty vector ( Ctrl ), FLAG-tagged wild-type Ezh2 ( WT ), T345A/T487A ( Mut-A ), and T345E/T487E ( Mut-E ) were generated by lentiviral infection and transfected with HA-tagged ubiquitin. Cells were synchronized and arrested at mitosis by thymidine-nocodazole block. 4 h prior to harvesting, cells were treated with or without 25 μ m MG132. Lysates were prepared under denaturing conditions and subjected to immunoprecipitation ( IP ) using FLAG antibodies. Bound proteins were analyzed by Western blot using the indicated antibodies.

    Article Snippet: Antibodies The following antibodies were used in this study: FLAG M2 mouse monoclonal (Sigma catalog number F3165), mouse phospho-serine (Chemicon catalog number AB1603), mouse phospho-threonine (Cell Signaling catalog number 9386), mouse phospho-tyrosine 4G10 (gift from Weiguo Zhang, Duke University), rabbit Ezh2 (Cell Signaling catalog number 4905), rabbit SLBP (gift from William Marzluff, University of North Carolina), mouse cyclin B1 (Santa Cruz Biotechnology), mouse α-tubulin (Sigma catalog number T6199), lamin B (Santa Cruz Biotechnology catalog number sc-6217), mouse GST (Santa Cruz Biotechnology catalog number sc-138), rabbit H3K27me3 (Millipore catalog number 07-449), rabbit H3 (Abcam catalog number ab1791-100), and rabbit Suz12 (as described in Ref. ).

    Techniques: Transfection, Western Blot, Mutagenesis, Stable Transfection, Expressing, Blocking Assay, Marker, Immunoprecipitation, Plasmid Preparation, Generated, Infection

    Thr-345 and Thr-487 of Ezh2 are important for cell proliferation. Western blot analysis demonstrates that wild type and T345A/T487A mutant are expressed at similar levels. A and B , PC3 ( A ) and HeLa ( B ) cells were infected with lentiviruses overexpressing empty vector ( Ctrl ), wild-type Ezh2 ( WT ), or T345A/T487A ( Mut-A ). Exogenous Ezh2 was detected using FLAG antibodies, whereas tubulin served as a loading control. C and D , PC3 ( C ) and HeLa ( D ) cells expressing T345A/T487A proliferate more slowly when compared with wild type. Approximately 5000 cells were seeded in triplicate in a 96-well plate, and cell proliferation was monitored by absorbance using the MTS assay at the indicated times. The measured absorbance was converted to the number of cells using a standard curve. For each cell line, the number of cells at day 0 was set to 1. Error bars in panels C and D indicate S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Cyclin-dependent Kinase 1 (CDK1)-mediated Phosphorylation of Enhancer of Zeste 2 (Ezh2) Regulates Its Stability *

    doi: 10.1074/jbc.M111.240515

    Figure Lengend Snippet: Thr-345 and Thr-487 of Ezh2 are important for cell proliferation. Western blot analysis demonstrates that wild type and T345A/T487A mutant are expressed at similar levels. A and B , PC3 ( A ) and HeLa ( B ) cells were infected with lentiviruses overexpressing empty vector ( Ctrl ), wild-type Ezh2 ( WT ), or T345A/T487A ( Mut-A ). Exogenous Ezh2 was detected using FLAG antibodies, whereas tubulin served as a loading control. C and D , PC3 ( C ) and HeLa ( D ) cells expressing T345A/T487A proliferate more slowly when compared with wild type. Approximately 5000 cells were seeded in triplicate in a 96-well plate, and cell proliferation was monitored by absorbance using the MTS assay at the indicated times. The measured absorbance was converted to the number of cells using a standard curve. For each cell line, the number of cells at day 0 was set to 1. Error bars in panels C and D indicate S.D.

    Article Snippet: Antibodies The following antibodies were used in this study: FLAG M2 mouse monoclonal (Sigma catalog number F3165), mouse phospho-serine (Chemicon catalog number AB1603), mouse phospho-threonine (Cell Signaling catalog number 9386), mouse phospho-tyrosine 4G10 (gift from Weiguo Zhang, Duke University), rabbit Ezh2 (Cell Signaling catalog number 4905), rabbit SLBP (gift from William Marzluff, University of North Carolina), mouse cyclin B1 (Santa Cruz Biotechnology), mouse α-tubulin (Sigma catalog number T6199), lamin B (Santa Cruz Biotechnology catalog number sc-6217), mouse GST (Santa Cruz Biotechnology catalog number sc-138), rabbit H3K27me3 (Millipore catalog number 07-449), rabbit H3 (Abcam catalog number ab1791-100), and rabbit Suz12 (as described in Ref. ).

    Techniques: Western Blot, Mutagenesis, Infection, Plasmid Preparation, Expressing, MTS Assay

    Ezh2 is phosphorylated at threonines 345 and 487. A , threonines 345 and 487 are conserved across species. Human, mouse, zebrafish, and fly Ezh2 were aligned using MultAlin. Relevant regions are shown. Threonines 345 and 487 are underlined , whereas conserved amino acids are highlighted in bold. B , specificity test of the phospho-Ezh2 antibodies. Dot blot analysis was performed using 2-fold dilutions of the unmodified ( Unmod. ) and phosphorylated ( Phos. ) peptides corresponding to amino acids surrounding Thr-345 or Thr-487. C , exogenously expressed Ezh2 is phosphorylated at Thr-345 ( pT345 ) and Thr-487 ( pT487 ). HEK293T cells were transfected with empty vector ( Ctrl ) or FLAG-tagged wild-type ( F-WT ), T345A ( F-T345A ), or T487A mutant Ezh2 ( F-T487A ). Following FLAG immunoprecipitation, bound proteins were analyzed by Western blot analysis using the indicated antibodies. D , endogenous Ezh2 is phosphorylated at Thr-345 and Thr-487. Western blot analysis of NIH3T3 and HeLa cell extracts using the phospho-Ezh2 antibodies was performed. For NIH3T3 extracts, 100 μg of nuclear extract ( NE ) and the equivalent volume of cytoplasmic extract ( CYT ) were loaded. For HeLa extracts, 100 μg of each fraction was loaded. Recombinant PRC2 complex was used as a positive control. Subcellular fractionation was confirmed using the following controls: tubulin (cytoplasmic), Ezh2 (nuclear), and lamin B (nuclear). NP , nuclear pellet. E , knockdown ( KD ) of Ezh2 results in decreased levels of phospho-Ezh2. HeLa cells were infected with lentiviruses expressing either control shRNA or Ezh2 shRNA. Top panel , quantitative RT-PCR confirming Ezh2 knockdown. Bottom panel , lysates were subjected to Western blot analysis using the indicated antibodies. Error bars indicate S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Cyclin-dependent Kinase 1 (CDK1)-mediated Phosphorylation of Enhancer of Zeste 2 (Ezh2) Regulates Its Stability *

    doi: 10.1074/jbc.M111.240515

    Figure Lengend Snippet: Ezh2 is phosphorylated at threonines 345 and 487. A , threonines 345 and 487 are conserved across species. Human, mouse, zebrafish, and fly Ezh2 were aligned using MultAlin. Relevant regions are shown. Threonines 345 and 487 are underlined , whereas conserved amino acids are highlighted in bold. B , specificity test of the phospho-Ezh2 antibodies. Dot blot analysis was performed using 2-fold dilutions of the unmodified ( Unmod. ) and phosphorylated ( Phos. ) peptides corresponding to amino acids surrounding Thr-345 or Thr-487. C , exogenously expressed Ezh2 is phosphorylated at Thr-345 ( pT345 ) and Thr-487 ( pT487 ). HEK293T cells were transfected with empty vector ( Ctrl ) or FLAG-tagged wild-type ( F-WT ), T345A ( F-T345A ), or T487A mutant Ezh2 ( F-T487A ). Following FLAG immunoprecipitation, bound proteins were analyzed by Western blot analysis using the indicated antibodies. D , endogenous Ezh2 is phosphorylated at Thr-345 and Thr-487. Western blot analysis of NIH3T3 and HeLa cell extracts using the phospho-Ezh2 antibodies was performed. For NIH3T3 extracts, 100 μg of nuclear extract ( NE ) and the equivalent volume of cytoplasmic extract ( CYT ) were loaded. For HeLa extracts, 100 μg of each fraction was loaded. Recombinant PRC2 complex was used as a positive control. Subcellular fractionation was confirmed using the following controls: tubulin (cytoplasmic), Ezh2 (nuclear), and lamin B (nuclear). NP , nuclear pellet. E , knockdown ( KD ) of Ezh2 results in decreased levels of phospho-Ezh2. HeLa cells were infected with lentiviruses expressing either control shRNA or Ezh2 shRNA. Top panel , quantitative RT-PCR confirming Ezh2 knockdown. Bottom panel , lysates were subjected to Western blot analysis using the indicated antibodies. Error bars indicate S.D.

    Article Snippet: Antibodies The following antibodies were used in this study: FLAG M2 mouse monoclonal (Sigma catalog number F3165), mouse phospho-serine (Chemicon catalog number AB1603), mouse phospho-threonine (Cell Signaling catalog number 9386), mouse phospho-tyrosine 4G10 (gift from Weiguo Zhang, Duke University), rabbit Ezh2 (Cell Signaling catalog number 4905), rabbit SLBP (gift from William Marzluff, University of North Carolina), mouse cyclin B1 (Santa Cruz Biotechnology), mouse α-tubulin (Sigma catalog number T6199), lamin B (Santa Cruz Biotechnology catalog number sc-6217), mouse GST (Santa Cruz Biotechnology catalog number sc-138), rabbit H3K27me3 (Millipore catalog number 07-449), rabbit H3 (Abcam catalog number ab1791-100), and rabbit Suz12 (as described in Ref. ).

    Techniques: Dot Blot, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Western Blot, Recombinant, Positive Control, Fractionation, Infection, Expressing, shRNA, Quantitative RT-PCR

    CDK1 mediates Ezh2 phosphorylation at Thr-345 and Thr-487. A , CDK1 phosphorylates Ezh2 at Thr-345 and Thr-487 ( pT487 ) in vitro . Recombinant GST-tagged Ezh2 was subjected to a cold kinase assay using CDK1-cyclin B kinase followed by Western blot analysis using phospho-Ezh2 antibodies specific for Thr(P)-345 (α -pT345 ) and Thr(P)-487 (α -pT487 ), respectively. B , phosphorylation of Ezh2 by CDKs can be inhibited by roscovitine ( Ros ). HEK293T cells were transfected with empty vector ( Ctrl ) or FLAG-tagged Ezh2 ( F-Ezh2 ). 24 h after transfection, cells were treated with 50 μ m roscovitine for 24 h to inhibit CDKs. Following FLAG immunoprecipitation, bound proteins were subjected to Western blot analysis using the indicated antibodies. C , inhibition of CDKs results in loss of endogenous phospho-Ezh2. HeLa cells were treated with 50 μ m roscovitine for the indicated times ( hr Ros ). Lysates were analyzed by Western blot analysis using the indicated antibodies. DMSO , dimethyl sulfoxide. D , specific inhibition of CDK1 results in loss of endogenous phospho-Ezh2. HeLa cells were treated with 2 μ m CGP74514A ( CGP ) to specifically inhibit CDK1. Lysates were analyzed by Western blot analysis using the indicated antibodies. E , phospho-Ezh2 is enriched in cells arrested at mitosis. HeLa cells were arrested at S-phase ( S ) and M-phase ( M ) by double thymidine block and thymidine-nocodazole block, respectively. Extracts were analyzed by Western blot using the indicated antibodies. SLBP, S-phase control; cyclin B1, M-phase control; tubulin, loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Cyclin-dependent Kinase 1 (CDK1)-mediated Phosphorylation of Enhancer of Zeste 2 (Ezh2) Regulates Its Stability *

    doi: 10.1074/jbc.M111.240515

    Figure Lengend Snippet: CDK1 mediates Ezh2 phosphorylation at Thr-345 and Thr-487. A , CDK1 phosphorylates Ezh2 at Thr-345 and Thr-487 ( pT487 ) in vitro . Recombinant GST-tagged Ezh2 was subjected to a cold kinase assay using CDK1-cyclin B kinase followed by Western blot analysis using phospho-Ezh2 antibodies specific for Thr(P)-345 (α -pT345 ) and Thr(P)-487 (α -pT487 ), respectively. B , phosphorylation of Ezh2 by CDKs can be inhibited by roscovitine ( Ros ). HEK293T cells were transfected with empty vector ( Ctrl ) or FLAG-tagged Ezh2 ( F-Ezh2 ). 24 h after transfection, cells were treated with 50 μ m roscovitine for 24 h to inhibit CDKs. Following FLAG immunoprecipitation, bound proteins were subjected to Western blot analysis using the indicated antibodies. C , inhibition of CDKs results in loss of endogenous phospho-Ezh2. HeLa cells were treated with 50 μ m roscovitine for the indicated times ( hr Ros ). Lysates were analyzed by Western blot analysis using the indicated antibodies. DMSO , dimethyl sulfoxide. D , specific inhibition of CDK1 results in loss of endogenous phospho-Ezh2. HeLa cells were treated with 2 μ m CGP74514A ( CGP ) to specifically inhibit CDK1. Lysates were analyzed by Western blot analysis using the indicated antibodies. E , phospho-Ezh2 is enriched in cells arrested at mitosis. HeLa cells were arrested at S-phase ( S ) and M-phase ( M ) by double thymidine block and thymidine-nocodazole block, respectively. Extracts were analyzed by Western blot using the indicated antibodies. SLBP, S-phase control; cyclin B1, M-phase control; tubulin, loading control.

    Article Snippet: Antibodies The following antibodies were used in this study: FLAG M2 mouse monoclonal (Sigma catalog number F3165), mouse phospho-serine (Chemicon catalog number AB1603), mouse phospho-threonine (Cell Signaling catalog number 9386), mouse phospho-tyrosine 4G10 (gift from Weiguo Zhang, Duke University), rabbit Ezh2 (Cell Signaling catalog number 4905), rabbit SLBP (gift from William Marzluff, University of North Carolina), mouse cyclin B1 (Santa Cruz Biotechnology), mouse α-tubulin (Sigma catalog number T6199), lamin B (Santa Cruz Biotechnology catalog number sc-6217), mouse GST (Santa Cruz Biotechnology catalog number sc-138), rabbit H3K27me3 (Millipore catalog number 07-449), rabbit H3 (Abcam catalog number ab1791-100), and rabbit Suz12 (as described in Ref. ).

    Techniques: In Vitro, Recombinant, Kinase Assay, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Inhibition, Blocking Assay

    SRSF2 binding to the RBM25 Lys-77 peptide is regulated by methylation. A , immobilized non-methyl or methyl peptides centered on RBM25 Lys-77 were used to capture proteins from nuclear extract of 293T cells prepared with SILAC. Bound proteins were identified and quantified using LC/MS-MS. B , relative binding to methyl over non-methyl peptide was measured in two experiments with isotopic labels reversed. Each axis represents one experiment with the ratio of bound protein shown on a log 2 scale. Several SR proteins are highlighted in red. C , recombinant SRSF2 or the indicated domain was expressed with N-terminal GST in E. coli and tested for binding to immobilized peptide. Bound proteins were visualized by Western blotting for the GST tag. 3xMBT was used as a positive control for mono- and dimethylated peptides. D , SRSF2 was tested for binding to a panel of non-methylated peptides from proteins involved in transcription, splicing, and translation. E , RBM25 and SRSF2 were expressed by transient transfection in 293T cells with FLAG and Myc tags, respectively. Following FLAG, co-IP bound SRSF2 was measured by Western blotting. F , a panel of SR proteins was tested for binding to immobilized RBM25 peptides. G , SRSF1, SRSF2, and 3xMBT were incubated with varying concentrations of the indicated immobilized RBM25 peptide, and the amount of bound protein was measured by quantitative Western blotting for GST with near-IR fluorescent secondary antibodies. Error bars indicate mean ± S.E. ( n = 3).

    Journal: The Journal of Biological Chemistry

    Article Title: RBM25 is a global splicing factor promoting inclusion of alternatively spliced exons and is itself regulated by lysine mono-methylation

    doi: 10.1074/jbc.M117.784371

    Figure Lengend Snippet: SRSF2 binding to the RBM25 Lys-77 peptide is regulated by methylation. A , immobilized non-methyl or methyl peptides centered on RBM25 Lys-77 were used to capture proteins from nuclear extract of 293T cells prepared with SILAC. Bound proteins were identified and quantified using LC/MS-MS. B , relative binding to methyl over non-methyl peptide was measured in two experiments with isotopic labels reversed. Each axis represents one experiment with the ratio of bound protein shown on a log 2 scale. Several SR proteins are highlighted in red. C , recombinant SRSF2 or the indicated domain was expressed with N-terminal GST in E. coli and tested for binding to immobilized peptide. Bound proteins were visualized by Western blotting for the GST tag. 3xMBT was used as a positive control for mono- and dimethylated peptides. D , SRSF2 was tested for binding to a panel of non-methylated peptides from proteins involved in transcription, splicing, and translation. E , RBM25 and SRSF2 were expressed by transient transfection in 293T cells with FLAG and Myc tags, respectively. Following FLAG, co-IP bound SRSF2 was measured by Western blotting. F , a panel of SR proteins was tested for binding to immobilized RBM25 peptides. G , SRSF1, SRSF2, and 3xMBT were incubated with varying concentrations of the indicated immobilized RBM25 peptide, and the amount of bound protein was measured by quantitative Western blotting for GST with near-IR fluorescent secondary antibodies. Error bars indicate mean ± S.E. ( n = 3).

    Article Snippet: Antibodies used were as follows: RBM25 (Bethyl Labs, A301-068A), FLAG M2 for Western blotting (Sigma-Aldrich, F1804), immobilized FLAG M2 (Sigma-Aldrich, A2220), glutathione S -transferase (custom rabbit polyclonal), and Myc tag (Pierce Fisher, MA1-21316).

    Techniques: Binding Assay, Methylation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Recombinant, Western Blot, Positive Control, Transfection, Co-Immunoprecipitation Assay, Incubation

    Analysis of the Ab3 anti-anti-Id scFv 69 response in sera of BALB/c mice (immunisation protocol 1) by inhibition of the binding of anti-Id scFv 69 (Ab2) on immobilised trastuzumab F(ab′) 2 fragments (Ab1) by inhibition ELISA. Serial dilutions of preimmune sera or sera from mice each of the three groups: primed with HER-2/neu ECD-Fc fusion protein or with PBS or with anti-Id scFv 69 were preincubated with soluble anti-Id scFv 69. This solution was subsequently incubated for 2 h on trastuzumab F(ab′) 2 fragments followed by the detection of bound scFv by HRP-conjugated M2 anti-FLAG mAb. The results obtained are expressed as percent inhibition at each serum dilution.

    Journal: British Journal of Cancer

    Article Title: Isolation and characterisation of a human anti-idiotypic scFv used as a surrogate tumour antigen to elicit an anti-HER-2/neu humoral response in mice

    doi: 10.1038/sj.bjc.6601825

    Figure Lengend Snippet: Analysis of the Ab3 anti-anti-Id scFv 69 response in sera of BALB/c mice (immunisation protocol 1) by inhibition of the binding of anti-Id scFv 69 (Ab2) on immobilised trastuzumab F(ab′) 2 fragments (Ab1) by inhibition ELISA. Serial dilutions of preimmune sera or sera from mice each of the three groups: primed with HER-2/neu ECD-Fc fusion protein or with PBS or with anti-Id scFv 69 were preincubated with soluble anti-Id scFv 69. This solution was subsequently incubated for 2 h on trastuzumab F(ab′) 2 fragments followed by the detection of bound scFv by HRP-conjugated M2 anti-FLAG mAb. The results obtained are expressed as percent inhibition at each serum dilution.

    Article Snippet: The ELISA was performed as described above except that after incubation of E. coli HB2151 supernatants, bound soluble scFv was detected using HRP-conjugated anti-FLAG-tag M2 Ab (Sigma) for 1.5 h. For Western blot analysis, protein extracts were size fractionated by SDS–PAGE (12.5%) and electroblotted onto nitrocellulose.

    Techniques: Mouse Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

    Inhibition of purified soluble scFv 39, 40 and 69 binding on trastuzumab F(ab′) 2 fragments by ( A ) HER-2/neu ECD-Fc fusion protein and ( B ) rhCEA, using a competitive ELISA. Increasing amounts of inhibitor were mixed with either anti-Id scFv 39, 40 or 69 used at a dilution giving an A 490 ranging from 1 to 1.5 in ELISA (corresponding to a concentration of about 20 μ g ml −1 ). The incubation on trastuzumab F(ab′)2 fragments for 1.5 h was followed by the detection of scFv binding with HRP-conjugated M2 anti-FLAG mAb (1 : 2000). The results obtained are expressed as percent inhibition at each concentration of inhibitor.

    Journal: British Journal of Cancer

    Article Title: Isolation and characterisation of a human anti-idiotypic scFv used as a surrogate tumour antigen to elicit an anti-HER-2/neu humoral response in mice

    doi: 10.1038/sj.bjc.6601825

    Figure Lengend Snippet: Inhibition of purified soluble scFv 39, 40 and 69 binding on trastuzumab F(ab′) 2 fragments by ( A ) HER-2/neu ECD-Fc fusion protein and ( B ) rhCEA, using a competitive ELISA. Increasing amounts of inhibitor were mixed with either anti-Id scFv 39, 40 or 69 used at a dilution giving an A 490 ranging from 1 to 1.5 in ELISA (corresponding to a concentration of about 20 μ g ml −1 ). The incubation on trastuzumab F(ab′)2 fragments for 1.5 h was followed by the detection of scFv binding with HRP-conjugated M2 anti-FLAG mAb (1 : 2000). The results obtained are expressed as percent inhibition at each concentration of inhibitor.

    Article Snippet: The ELISA was performed as described above except that after incubation of E. coli HB2151 supernatants, bound soluble scFv was detected using HRP-conjugated anti-FLAG-tag M2 Ab (Sigma) for 1.5 h. For Western blot analysis, protein extracts were size fractionated by SDS–PAGE (12.5%) and electroblotted onto nitrocellulose.

    Techniques: Inhibition, Purification, Binding Assay, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation

    Analysis of antibody-containing periplasmic fractions containing antibodies ( A ) After induction of single ampicillin-resistant infected E. coli HB2151 colonies with 1 m M IPTG. Lane 1: clone 39; lane 2: clone 40; lane 3: clone 92; lane 4: clone 69. After size fractionation on 12.5% SDS–PAGE, protein extracts were blotted onto nitrocellulose. The immunoblot was developed with HRP-conjugated M2 anti-FLAG mAb (1 : 2000) followed by addition of the 4-chloro-1-naphtol substrate. ( B ) Before and after purification. The purity was controlled on 12.5% SDS–PAGE gel followed by silver staining. Lane a: nonpurified periplasmic fraction. Lane b: Periplasmic fraction purified on a Hitrap Ni-activated chelating column. Lane c: Periplasmic fraction purified on a Hitrap Ni-activated chelating column followed by gel filtration on Superdex 75. Lane d: Standard molecular mass markers.

    Journal: British Journal of Cancer

    Article Title: Isolation and characterisation of a human anti-idiotypic scFv used as a surrogate tumour antigen to elicit an anti-HER-2/neu humoral response in mice

    doi: 10.1038/sj.bjc.6601825

    Figure Lengend Snippet: Analysis of antibody-containing periplasmic fractions containing antibodies ( A ) After induction of single ampicillin-resistant infected E. coli HB2151 colonies with 1 m M IPTG. Lane 1: clone 39; lane 2: clone 40; lane 3: clone 92; lane 4: clone 69. After size fractionation on 12.5% SDS–PAGE, protein extracts were blotted onto nitrocellulose. The immunoblot was developed with HRP-conjugated M2 anti-FLAG mAb (1 : 2000) followed by addition of the 4-chloro-1-naphtol substrate. ( B ) Before and after purification. The purity was controlled on 12.5% SDS–PAGE gel followed by silver staining. Lane a: nonpurified periplasmic fraction. Lane b: Periplasmic fraction purified on a Hitrap Ni-activated chelating column. Lane c: Periplasmic fraction purified on a Hitrap Ni-activated chelating column followed by gel filtration on Superdex 75. Lane d: Standard molecular mass markers.

    Article Snippet: The ELISA was performed as described above except that after incubation of E. coli HB2151 supernatants, bound soluble scFv was detected using HRP-conjugated anti-FLAG-tag M2 Ab (Sigma) for 1.5 h. For Western blot analysis, protein extracts were size fractionated by SDS–PAGE (12.5%) and electroblotted onto nitrocellulose.

    Techniques: Infection, Fractionation, SDS Page, Purification, Silver Staining, Filtration

    WRS is secreted in response to virus infection. (A) ELISA of WRS levels in the supernatant of U-937 and THP-1 cells infected with VSV-GFP at a multiplicity of infection (MOI) of 3 at indicated time points. (B) mRNA expression level of WRS shown in panel A was determined by qRT-PCR. (C) Flag-tagged mWRS expressing stable Raw264.7 cells and control cells were infected with VSV-GFP (upper, left) or HSV-GFP (upper, right) at an MOI of 1. Secreted levels of Flag-tagged mWRS were assessed by anti-Flag ELISA in the supernatant at the indicated time points. Shown is anti-Flag ELISA of the supernatant in the same cell line with treatment of 40 μg poly(I-C) (lower left) or transfection of 1 μg poly(dA-dT) (lower right). OD, optical density. (D) ELISA of WRS levels in the supernatant of HeLa cells infected with VSV-GFP at an MOI of 0.1 or 1 for indicated time points. ND, not determined. (E) ELISA of WRS levels in the supernatant of HEK293T cells infected with VSV-GFP at an MOI of 0.01 or 3 for indicated time points. (F) mRNA expression level of WRS in HEK293T cells infected with VSV-GFP at an MOI of 0.01, as determined by qRT-PCR. NS, not significant. (G) THP-1 cells were treated with 100 or 1,000 U of recombinant IFN-β. Secreted levels of WRS were assessed by ELISA; 1,000 ng/ml of recombinant WRS standard was used as a positive control for the experiment. ND, not detected; NS, not significant. Error bars, means ± SD.

    Journal: Journal of Virology

    Article Title: Released Tryptophanyl-tRNA Synthetase Stimulates Innate Immune Responses against Viral Infection

    doi: 10.1128/JVI.01291-18

    Figure Lengend Snippet: WRS is secreted in response to virus infection. (A) ELISA of WRS levels in the supernatant of U-937 and THP-1 cells infected with VSV-GFP at a multiplicity of infection (MOI) of 3 at indicated time points. (B) mRNA expression level of WRS shown in panel A was determined by qRT-PCR. (C) Flag-tagged mWRS expressing stable Raw264.7 cells and control cells were infected with VSV-GFP (upper, left) or HSV-GFP (upper, right) at an MOI of 1. Secreted levels of Flag-tagged mWRS were assessed by anti-Flag ELISA in the supernatant at the indicated time points. Shown is anti-Flag ELISA of the supernatant in the same cell line with treatment of 40 μg poly(I-C) (lower left) or transfection of 1 μg poly(dA-dT) (lower right). OD, optical density. (D) ELISA of WRS levels in the supernatant of HeLa cells infected with VSV-GFP at an MOI of 0.1 or 1 for indicated time points. ND, not determined. (E) ELISA of WRS levels in the supernatant of HEK293T cells infected with VSV-GFP at an MOI of 0.01 or 3 for indicated time points. (F) mRNA expression level of WRS in HEK293T cells infected with VSV-GFP at an MOI of 0.01, as determined by qRT-PCR. NS, not significant. (G) THP-1 cells were treated with 100 or 1,000 U of recombinant IFN-β. Secreted levels of WRS were assessed by ELISA; 1,000 ng/ml of recombinant WRS standard was used as a positive control for the experiment. ND, not detected; NS, not significant. Error bars, means ± SD.

    Article Snippet: Flag ELISA was conducted using anti-Flag-coated plates (P2983; Sigma).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Transfection, Recombinant, Positive Control

    RNF11 disrupts the interaction between TRAF3 and TBK1/IKKi. (A, C) 293T cells were transfected with 1 μg of either HA-TRAF3, Flag-IKKi, Flag-TBK1 or RNF11-GFP. Co-IPs were conducted using anti-Flag followed by immunoblotting with anti-HA and anti-Flag. Immunoblotting was performed with lysates using anti-Flag, anti-HA, anti-GFP and anti-Actin. (B, D) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), and 1 µg of RNF11, TRAF3, IKKi or TBK1. Dual luciferase assays were performed with protein lysates 24 h later. *, p

    Journal: PLoS ONE

    Article Title: RING Finger Protein 11 Targets TBK1/IKKi Kinases to Inhibit Antiviral Signaling

    doi: 10.1371/journal.pone.0053717

    Figure Lengend Snippet: RNF11 disrupts the interaction between TRAF3 and TBK1/IKKi. (A, C) 293T cells were transfected with 1 μg of either HA-TRAF3, Flag-IKKi, Flag-TBK1 or RNF11-GFP. Co-IPs were conducted using anti-Flag followed by immunoblotting with anti-HA and anti-Flag. Immunoblotting was performed with lysates using anti-Flag, anti-HA, anti-GFP and anti-Actin. (B, D) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), and 1 µg of RNF11, TRAF3, IKKi or TBK1. Dual luciferase assays were performed with protein lysates 24 h later. *, p

    Article Snippet: Antibodies and siRNAs Flag M2 and RNF11 antibodies were purchased from Sigma.

    Techniques: Transfection, Luciferase

    RNF11 is a negative regulator of virus-induced IFN-β production. (A) Micrographs of 293T cells transfected with either empty vector or Myc-RNF11 and then infected with VSV-GFP (MOI of 0.1) 24 h later. Pictures were taken 24 h post-infection. Immunoblotting was conducted with protein lysates using anti-Myc, anti-GFP and anti-Actin (right panel). (B) MEFs were transfected with either empty vector or Myc-RNF11 and were transfected again with poly(I:C) (15 μg) 24 h later. An IFN-β ELISA was performed 16 h later using supernatants. Immunoblotting was conducted with anti-Myc and anti-Actin. (C) 293T cells were transfected with an IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), empty vector (1 µg) or RNF11-GFP (1 µg). Cells were transfected 24 h later with poly(I:C) (15 µg) and dual luciferase assays were performed after 16 h. Immunoblotting was conducted with protein lysates using anti-GFP and anti-Actin. (D) 293T cells were transfected with RNF11-GFP (1 μg) and Myc-A20 (1 μg) and then transfected 24 h later with poly(I:C) (20 µg). Immunoblotting was performed with anti-p-IRF3, anti-Myc, anti-GFP, anti-IRF3 and anti-Actin.

    Journal: PLoS ONE

    Article Title: RING Finger Protein 11 Targets TBK1/IKKi Kinases to Inhibit Antiviral Signaling

    doi: 10.1371/journal.pone.0053717

    Figure Lengend Snippet: RNF11 is a negative regulator of virus-induced IFN-β production. (A) Micrographs of 293T cells transfected with either empty vector or Myc-RNF11 and then infected with VSV-GFP (MOI of 0.1) 24 h later. Pictures were taken 24 h post-infection. Immunoblotting was conducted with protein lysates using anti-Myc, anti-GFP and anti-Actin (right panel). (B) MEFs were transfected with either empty vector or Myc-RNF11 and were transfected again with poly(I:C) (15 μg) 24 h later. An IFN-β ELISA was performed 16 h later using supernatants. Immunoblotting was conducted with anti-Myc and anti-Actin. (C) 293T cells were transfected with an IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), empty vector (1 µg) or RNF11-GFP (1 µg). Cells were transfected 24 h later with poly(I:C) (15 µg) and dual luciferase assays were performed after 16 h. Immunoblotting was conducted with protein lysates using anti-GFP and anti-Actin. (D) 293T cells were transfected with RNF11-GFP (1 μg) and Myc-A20 (1 μg) and then transfected 24 h later with poly(I:C) (20 µg). Immunoblotting was performed with anti-p-IRF3, anti-Myc, anti-GFP, anti-IRF3 and anti-Actin.

    Article Snippet: Antibodies and siRNAs Flag M2 and RNF11 antibodies were purchased from Sigma.

    Techniques: Transfection, Plasmid Preparation, Infection, Enzyme-linked Immunosorbent Assay, Luciferase

    RNF11 inhibits IFN-β production at the level of TBK1/IKKi. (A–E) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), empty vector (1 μg), RNF11-GFP (1 μg) and either 0.5 μg of ΔRIG-I (A), MDA5 (B), IPS-1 (C), TBK1 (D) or IRF3-SA (E). Dual luciferase assays were performed with protein lysates 24 h later. (F, G) 293T cells were transfected with either control scrambled or RNF11 siRNA (60 pmol). After 24 h, cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), and either Flag-TBK1 or Flag-IKKi (0.5 μg) and dual luciferase assays were performed 24 h later. (H) 293T cells were transfected with either control scrambled or RNF11 siRNA (60 pmol). After 48 h, RT-PCR was performed to detect RNF11 and Actin transcripts. *, p

    Journal: PLoS ONE

    Article Title: RING Finger Protein 11 Targets TBK1/IKKi Kinases to Inhibit Antiviral Signaling

    doi: 10.1371/journal.pone.0053717

    Figure Lengend Snippet: RNF11 inhibits IFN-β production at the level of TBK1/IKKi. (A–E) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), empty vector (1 μg), RNF11-GFP (1 μg) and either 0.5 μg of ΔRIG-I (A), MDA5 (B), IPS-1 (C), TBK1 (D) or IRF3-SA (E). Dual luciferase assays were performed with protein lysates 24 h later. (F, G) 293T cells were transfected with either control scrambled or RNF11 siRNA (60 pmol). After 24 h, cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), and either Flag-TBK1 or Flag-IKKi (0.5 μg) and dual luciferase assays were performed 24 h later. (H) 293T cells were transfected with either control scrambled or RNF11 siRNA (60 pmol). After 48 h, RT-PCR was performed to detect RNF11 and Actin transcripts. *, p

    Article Snippet: Antibodies and siRNAs Flag M2 and RNF11 antibodies were purchased from Sigma.

    Techniques: Transfection, Luciferase, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    RNF11 interacts with TBK1/IKKi and blocks their Lys63-linked polyubiquitination. (A) 293T cells were transfected with 1 µg of RNF11-GFP, Flag-IKKi and Flag-TBK1. Co-IPs were conducted using anti-Flag for IP followed by immunoblotting with anti-GFP and anti-Flag. Immunoblotting was performed with lysates using anti-GFP, anti-Flag and anti-Actin. (B) 293T cells were transfected with poly(I:C) (20 μg) and co-IPs were performed with anti-RNF11 or isotype control IgG followed by immunoblotting with anti-IKKi (top panel) or anti-TBK1 (lower panel). Immunoblots were also performed with lysates using anti-IKKi, anti-RNF11, anti-TBK1 and anti-Actin. (C) 293T cells were transfected with empty vector, Flag-RNF11 or RNF11-GFP (1 µg) and HA-Ub-Lys63-only (500 ng). Cells were transfected again 24 h later with poly(I:C) (20 μg) and co-IPs were conducted the next day using anti-TBK1 (left panel) or anti-IKKi (right panel) followed by immunoblotting with anti-HA, anti-TBK1 (left panel) and anti-IKKi (right panel). Immunoblotting was performed with lysates with anti-Flag, anti-GFP and anti-Actin.

    Journal: PLoS ONE

    Article Title: RING Finger Protein 11 Targets TBK1/IKKi Kinases to Inhibit Antiviral Signaling

    doi: 10.1371/journal.pone.0053717

    Figure Lengend Snippet: RNF11 interacts with TBK1/IKKi and blocks their Lys63-linked polyubiquitination. (A) 293T cells were transfected with 1 µg of RNF11-GFP, Flag-IKKi and Flag-TBK1. Co-IPs were conducted using anti-Flag for IP followed by immunoblotting with anti-GFP and anti-Flag. Immunoblotting was performed with lysates using anti-GFP, anti-Flag and anti-Actin. (B) 293T cells were transfected with poly(I:C) (20 μg) and co-IPs were performed with anti-RNF11 or isotype control IgG followed by immunoblotting with anti-IKKi (top panel) or anti-TBK1 (lower panel). Immunoblots were also performed with lysates using anti-IKKi, anti-RNF11, anti-TBK1 and anti-Actin. (C) 293T cells were transfected with empty vector, Flag-RNF11 or RNF11-GFP (1 µg) and HA-Ub-Lys63-only (500 ng). Cells were transfected again 24 h later with poly(I:C) (20 μg) and co-IPs were conducted the next day using anti-TBK1 (left panel) or anti-IKKi (right panel) followed by immunoblotting with anti-HA, anti-TBK1 (left panel) and anti-IKKi (right panel). Immunoblotting was performed with lysates with anti-Flag, anti-GFP and anti-Actin.

    Article Snippet: Antibodies and siRNAs Flag M2 and RNF11 antibodies were purchased from Sigma.

    Techniques: Transfection, Western Blot, Plasmid Preparation

    RNF11 requires TAX1BP1 to inhibit antiviral signaling. (A) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), RNF11 (1 µg) and either control scrambled or TAX1BP1 siRNA (60 pmol). After 24 h, cells were transfected with poly(I:C) (15 µg) and dual luciferase assays were performed 16 h later. The data is presented as percent inhibition by RNF11 of poly(I:C)-induced IFN-β promoter induction with either control scrambled or TAX1BP1 siRNA (left panel). Knockdown of TAX1BP1 was confirmed by immunoblotting using anti-TAX1BP1 and anti-Actin (right panel). (B) Tax1bp1 +/– and Tax1bp1 –/– MEFS were transfected with poly(I:C) (20 μg), and co-IPs were performed with anti-RNF11 followed by immunoblotting with anti-IKKi (left panel) or anti-TBK1 (right panel). Immunoblots were performed with lysates using anti-IKKi, anti-RNF11, anti-TBK1 and anti-Actin. *, p

    Journal: PLoS ONE

    Article Title: RING Finger Protein 11 Targets TBK1/IKKi Kinases to Inhibit Antiviral Signaling

    doi: 10.1371/journal.pone.0053717

    Figure Lengend Snippet: RNF11 requires TAX1BP1 to inhibit antiviral signaling. (A) 293T cells were transfected with IFN-β luciferase reporter (200 ng), pRL-tk (20 ng), RNF11 (1 µg) and either control scrambled or TAX1BP1 siRNA (60 pmol). After 24 h, cells were transfected with poly(I:C) (15 µg) and dual luciferase assays were performed 16 h later. The data is presented as percent inhibition by RNF11 of poly(I:C)-induced IFN-β promoter induction with either control scrambled or TAX1BP1 siRNA (left panel). Knockdown of TAX1BP1 was confirmed by immunoblotting using anti-TAX1BP1 and anti-Actin (right panel). (B) Tax1bp1 +/– and Tax1bp1 –/– MEFS were transfected with poly(I:C) (20 μg), and co-IPs were performed with anti-RNF11 followed by immunoblotting with anti-IKKi (left panel) or anti-TBK1 (right panel). Immunoblots were performed with lysates using anti-IKKi, anti-RNF11, anti-TBK1 and anti-Actin. *, p

    Article Snippet: Antibodies and siRNAs Flag M2 and RNF11 antibodies were purchased from Sigma.

    Techniques: Transfection, Luciferase, Inhibition, Western Blot

    Sortilin forms homodimers on the cell surface of HEK293 cells. A , schematic of FLAG-sortilin and His 6 -sortilin. FLAG tag and His 6 tag were inserted following propeptide and 3 amino acids (Ser 78 -Ala 79 -Pro 80 ) in sortilin. SP , signal peptide; PP , propeptide. B , overexpression of FLAG-sortilin and His 6 -sortilin in HEK293 cells was validated by Western blotting. C and D , detection of binding of FLAG-sortilin and His 6 -sortilin on the cell surface of HEK293 in TR-FRET assay ( C ) and HTRF assay ( D ). Change of FRET signal by expression of His 6 -sortilin is indicated by percent change (mean ± S.D., three independent experiments). Error bars represent S.D. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Dimerization of sortilin regulates its trafficking to extracellular vesicles

    doi: 10.1074/jbc.RA117.000732

    Figure Lengend Snippet: Sortilin forms homodimers on the cell surface of HEK293 cells. A , schematic of FLAG-sortilin and His 6 -sortilin. FLAG tag and His 6 tag were inserted following propeptide and 3 amino acids (Ser 78 -Ala 79 -Pro 80 ) in sortilin. SP , signal peptide; PP , propeptide. B , overexpression of FLAG-sortilin and His 6 -sortilin in HEK293 cells was validated by Western blotting. C and D , detection of binding of FLAG-sortilin and His 6 -sortilin on the cell surface of HEK293 in TR-FRET assay ( C ) and HTRF assay ( D ). Change of FRET signal by expression of His 6 -sortilin is indicated by percent change (mean ± S.D., three independent experiments). Error bars represent S.D. *, p

    Article Snippet: Soluble sortilin with FLAG tag was eluted with 100 μg/ml FLAG peptide (Sigma, F3290, lot number SLBR6767V).

    Techniques: FLAG-tag, Over Expression, Western Blot, Binding Assay, HTRF Assay, Expressing

    Substituting the transmembrane domain of sortilin with the corresponding domain of CD43 does not decrease the dimeric form of sortilin. A , schematic of FLAG-sortilin wildtype (WT) and FLAG-sortilin CD43-TMD. The transmembrane domain of sortilin was replaced with that of CD43. SP , signal peptide; PP , propeptide. B , FLAG-sortilin WT and FLAG-sortilin CD43-TMD were transiently overexpressed in HEK293 cells, and non-reducing Western blotting was carried out using cell lysate with anti-FLAG antibody ( n = 3). Monomers, homodimers, and multimers are abbreviated as MO , D , and MU , respectively. C and D , His 6 -sortilin WT or His 6 -sortilin CD43-TMD was transiently overexpressed in HEK293 cells stably overexpressing FLAG-sortilin, and immunoprecipitation was performed using anti-FLAG M2 antibody. Western blotting was carried out using whole-cell lysates ( C ) and immunoprecipitants ( D ). His 6 -sortilin CD43-TMD coprecipitated with FLAG-sortilin as well as His 6 -sortilin WT. Arrows , sortilin wildtype or sortilin CD43-TMD ( n = 3). E , in FLAG-sortilin HEK293 cells or HEK293 cells, His 6 -sortilin CD43-TMD was overexpressed. The cells were subjected to TR-FRET assay. Change of FRET signal by expression of His 6 -sortilin WT or CD43-TMD is indicated by percent change (mean ± S.D., n = 4, one independent experiment). Error bars represent S.D. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Dimerization of sortilin regulates its trafficking to extracellular vesicles

    doi: 10.1074/jbc.RA117.000732

    Figure Lengend Snippet: Substituting the transmembrane domain of sortilin with the corresponding domain of CD43 does not decrease the dimeric form of sortilin. A , schematic of FLAG-sortilin wildtype (WT) and FLAG-sortilin CD43-TMD. The transmembrane domain of sortilin was replaced with that of CD43. SP , signal peptide; PP , propeptide. B , FLAG-sortilin WT and FLAG-sortilin CD43-TMD were transiently overexpressed in HEK293 cells, and non-reducing Western blotting was carried out using cell lysate with anti-FLAG antibody ( n = 3). Monomers, homodimers, and multimers are abbreviated as MO , D , and MU , respectively. C and D , His 6 -sortilin WT or His 6 -sortilin CD43-TMD was transiently overexpressed in HEK293 cells stably overexpressing FLAG-sortilin, and immunoprecipitation was performed using anti-FLAG M2 antibody. Western blotting was carried out using whole-cell lysates ( C ) and immunoprecipitants ( D ). His 6 -sortilin CD43-TMD coprecipitated with FLAG-sortilin as well as His 6 -sortilin WT. Arrows , sortilin wildtype or sortilin CD43-TMD ( n = 3). E , in FLAG-sortilin HEK293 cells or HEK293 cells, His 6 -sortilin CD43-TMD was overexpressed. The cells were subjected to TR-FRET assay. Change of FRET signal by expression of His 6 -sortilin WT or CD43-TMD is indicated by percent change (mean ± S.D., n = 4, one independent experiment). Error bars represent S.D. *, p

    Article Snippet: Soluble sortilin with FLAG tag was eluted with 100 μg/ml FLAG peptide (Sigma, F3290, lot number SLBR6767V).

    Techniques: Western Blot, Stable Transfection, Immunoprecipitation, Expressing

    Mutation of Cys 783 abolishes dimerization of sortilin. A , schematic of His 6 -sortilin 10CC+TMD, FLAG-sortilin WT and C783A, and His 6 -sortilin ICD+TMD WT and C783A. Cysteine 783 was replaced by alanine. SP , signal peptide; PP , propeptide. B , expression vector of His 6 -sortilin 10CC+TMD was transfected in HEK293 cells. Dimerization of His 6 -sortilin 10CC+TMD was detected in non-reducing Western blotting with anti-His 6 antibody ( n = 3). C , sortilin ICD+TMD C783A did not form homodimers in HEK293 cells in the non-reducing Western blotting ( n = 3). D and E , C783A decreased sortilin homodimers of low molecular weight in the cells ( D ) and extracellular vesicles ( E ) of HEK293 cells in non-reducing Western blotting ( n = 3). F and G , 24-h incubation with 2-FPA, an inhibitor of palmitoylation, increased sortilin homodimers of low molecular weight in HEK293 cells stably overexpressing FLAG-sortilin ( F ) and their extracellular vesicles ( G ) ( n = 3). Monomers and homodimers of high and low molecular weight are abbreviated as MO , D(HMW) , and D(LMW) , respectively. IB , immunoblotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Dimerization of sortilin regulates its trafficking to extracellular vesicles

    doi: 10.1074/jbc.RA117.000732

    Figure Lengend Snippet: Mutation of Cys 783 abolishes dimerization of sortilin. A , schematic of His 6 -sortilin 10CC+TMD, FLAG-sortilin WT and C783A, and His 6 -sortilin ICD+TMD WT and C783A. Cysteine 783 was replaced by alanine. SP , signal peptide; PP , propeptide. B , expression vector of His 6 -sortilin 10CC+TMD was transfected in HEK293 cells. Dimerization of His 6 -sortilin 10CC+TMD was detected in non-reducing Western blotting with anti-His 6 antibody ( n = 3). C , sortilin ICD+TMD C783A did not form homodimers in HEK293 cells in the non-reducing Western blotting ( n = 3). D and E , C783A decreased sortilin homodimers of low molecular weight in the cells ( D ) and extracellular vesicles ( E ) of HEK293 cells in non-reducing Western blotting ( n = 3). F and G , 24-h incubation with 2-FPA, an inhibitor of palmitoylation, increased sortilin homodimers of low molecular weight in HEK293 cells stably overexpressing FLAG-sortilin ( F ) and their extracellular vesicles ( G ) ( n = 3). Monomers and homodimers of high and low molecular weight are abbreviated as MO , D(HMW) , and D(LMW) , respectively. IB , immunoblotting.

    Article Snippet: Soluble sortilin with FLAG tag was eluted with 100 μg/ml FLAG peptide (Sigma, F3290, lot number SLBR6767V).

    Techniques: Mutagenesis, Expressing, Plasmid Preparation, Transfection, Western Blot, Molecular Weight, Incubation, Stable Transfection

    The transmembrane domain of sortilin forms homodimers via noncovalent interaction. A–D , His 6 -sortilin Full, ECD+TMD, and ICD+TMD were transiently overexpressed in HEK293 cells with stably overexpressed FLAG-sortilin Full ( A and B ) and ECD+TMD ( C and D ), respectively. Immunoprecipitation with anti-FLAG M2 antibody was performed using the cell lysates. Western blotting was carried out using whole-cell lysates ( A and C ) and immunoprecipitants ( B and D ). His 6 -sortilin Full, ECD+TMD, and ICD+TMD were coprecipitated with FLAG-sortilin Full or ECD+TMD ( B and D ) ( n = 3). IB , immunoblotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Dimerization of sortilin regulates its trafficking to extracellular vesicles

    doi: 10.1074/jbc.RA117.000732

    Figure Lengend Snippet: The transmembrane domain of sortilin forms homodimers via noncovalent interaction. A–D , His 6 -sortilin Full, ECD+TMD, and ICD+TMD were transiently overexpressed in HEK293 cells with stably overexpressed FLAG-sortilin Full ( A and B ) and ECD+TMD ( C and D ), respectively. Immunoprecipitation with anti-FLAG M2 antibody was performed using the cell lysates. Western blotting was carried out using whole-cell lysates ( A and C ) and immunoprecipitants ( B and D ). His 6 -sortilin Full, ECD+TMD, and ICD+TMD were coprecipitated with FLAG-sortilin Full or ECD+TMD ( B and D ) ( n = 3). IB , immunoblotting.

    Article Snippet: Soluble sortilin with FLAG tag was eluted with 100 μg/ml FLAG peptide (Sigma, F3290, lot number SLBR6767V).

    Techniques: Stable Transfection, Immunoprecipitation, Western Blot

    Sortilin forms homodimers in the extracellular and intracellular domains with intermolecular disulfide bonds in HEK293 cells. A , schematic of FLAG-sortilin Full, ECD+TMD, and ICD+TMD. SP , signal peptide; PP , propeptide. B and C , protein expression of FLAG-sortilin Full, ECD+TMD, and ICD+TMD was validated in reducing ( B ) and non-reducing ( C ) Western blotting using anti-FLAG antibody. FLAG-sortilin Full and ECD+TMD form homodimers and multimers. Empty vector was used as a control. D , HEK293 cells transiently overexpressing FLAG-sortilin Full or ECD+TMD were treated with a cross-linker, BS3, and the cell lysates were used for reducing Western blotting with anti-FLAG antibody, showing dimerization of sortilin Full and ECD+TMD ( n = 3). E , HEK293 cells stably overexpressing FLAG-sortilin ICD+TMD (FLAG-sortilin ICD+TMD HEK293 cells) were incubated with DMSO ( Control ), 20 μmol/liter MG-132 ( MG ) or 10 μmol/liter chloroquine ( Chlo ) for 7 h, and then reducing Western blotting was performed using anti-sortilin antibody. MG-132 increased the protein expression of FLAG-sortilin ICD+TMD, but chloroquine did not ( n = 3). F , FLAG-sortilin ICD+TMD HEK293 cells were incubated with DMSO or MG-132 (2–20 μmol/liter) for 7 or 24 h. MG-132 increased FLAG-sortilin ICD+TMD in a time- and concentration-dependent manner ( n = 3). G , following 16-h incubation of HEK293 cells ( Control ) or FLAG-sortilin ICD+TMD HEK293 cells ( ICD + TMD ) with MG-132 (5 μmol/liter) and immunoprecipitation with anti-FLAG antibody, non-reducing Western blotting showed dimerization of sortilin ICD+TMD using anti-sortilin antibody ( n = 3). Monomers, homodimers, and multimers are abbreviated as MO , D , and MU , respectively. IB , immunoblotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Dimerization of sortilin regulates its trafficking to extracellular vesicles

    doi: 10.1074/jbc.RA117.000732

    Figure Lengend Snippet: Sortilin forms homodimers in the extracellular and intracellular domains with intermolecular disulfide bonds in HEK293 cells. A , schematic of FLAG-sortilin Full, ECD+TMD, and ICD+TMD. SP , signal peptide; PP , propeptide. B and C , protein expression of FLAG-sortilin Full, ECD+TMD, and ICD+TMD was validated in reducing ( B ) and non-reducing ( C ) Western blotting using anti-FLAG antibody. FLAG-sortilin Full and ECD+TMD form homodimers and multimers. Empty vector was used as a control. D , HEK293 cells transiently overexpressing FLAG-sortilin Full or ECD+TMD were treated with a cross-linker, BS3, and the cell lysates were used for reducing Western blotting with anti-FLAG antibody, showing dimerization of sortilin Full and ECD+TMD ( n = 3). E , HEK293 cells stably overexpressing FLAG-sortilin ICD+TMD (FLAG-sortilin ICD+TMD HEK293 cells) were incubated with DMSO ( Control ), 20 μmol/liter MG-132 ( MG ) or 10 μmol/liter chloroquine ( Chlo ) for 7 h, and then reducing Western blotting was performed using anti-sortilin antibody. MG-132 increased the protein expression of FLAG-sortilin ICD+TMD, but chloroquine did not ( n = 3). F , FLAG-sortilin ICD+TMD HEK293 cells were incubated with DMSO or MG-132 (2–20 μmol/liter) for 7 or 24 h. MG-132 increased FLAG-sortilin ICD+TMD in a time- and concentration-dependent manner ( n = 3). G , following 16-h incubation of HEK293 cells ( Control ) or FLAG-sortilin ICD+TMD HEK293 cells ( ICD + TMD ) with MG-132 (5 μmol/liter) and immunoprecipitation with anti-FLAG antibody, non-reducing Western blotting showed dimerization of sortilin ICD+TMD using anti-sortilin antibody ( n = 3). Monomers, homodimers, and multimers are abbreviated as MO , D , and MU , respectively. IB , immunoblotting.

    Article Snippet: Soluble sortilin with FLAG tag was eluted with 100 μg/ml FLAG peptide (Sigma, F3290, lot number SLBR6767V).

    Techniques: Expressing, Western Blot, Plasmid Preparation, Stable Transfection, Incubation, Concentration Assay, Immunoprecipitation

    Soluble sortilin forms homodimers. A and B , orientation of sortilin on the EV membrane was determined using EVs secreted from FLAG-sortilin HEK293 cells ( A ) and sortilin-3XFLAG HEK293 cells ( B ). EVs or their lysates were subjected to immunoprecipitation with anti-FLAG M2 antibody, and FLAG-sortilin ( A ) or sortilin-3XFLAG ( B ) was detected by Western blotting with anti-FLAG antibody, showing that the extracellular domain of sortilin is located outside of EVs ( n = 3). C and D , soluble sortilin secreted by HEK293 cells overexpressing FLAG-sortilin Full and FLAG-sortilin ECD+TMD was detected in non-reducing ( C ) and reducing Western blotting ( D ), showing that they were homodimers and monomers, respectively ( n = 3). E , soluble sortilin secreted by HEK293 cells overexpressing FLAG-sortilin Full and FLAG-sortilin ECD+TMD was purified and detected in non-reducing Western blotting. IB , immunoblotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Dimerization of sortilin regulates its trafficking to extracellular vesicles

    doi: 10.1074/jbc.RA117.000732

    Figure Lengend Snippet: Soluble sortilin forms homodimers. A and B , orientation of sortilin on the EV membrane was determined using EVs secreted from FLAG-sortilin HEK293 cells ( A ) and sortilin-3XFLAG HEK293 cells ( B ). EVs or their lysates were subjected to immunoprecipitation with anti-FLAG M2 antibody, and FLAG-sortilin ( A ) or sortilin-3XFLAG ( B ) was detected by Western blotting with anti-FLAG antibody, showing that the extracellular domain of sortilin is located outside of EVs ( n = 3). C and D , soluble sortilin secreted by HEK293 cells overexpressing FLAG-sortilin Full and FLAG-sortilin ECD+TMD was detected in non-reducing ( C ) and reducing Western blotting ( D ), showing that they were homodimers and monomers, respectively ( n = 3). E , soluble sortilin secreted by HEK293 cells overexpressing FLAG-sortilin Full and FLAG-sortilin ECD+TMD was purified and detected in non-reducing Western blotting. IB , immunoblotting.

    Article Snippet: Soluble sortilin with FLAG tag was eluted with 100 μg/ml FLAG peptide (Sigma, F3290, lot number SLBR6767V).

    Techniques: Immunoprecipitation, Western Blot, Purification

    Sortilin S316E and sortilin wp increase dimerization in HEK293 cells, and the addition of propeptide decreases dimerization in the extracellular vesicles of FLAG-sortilin HEK293 cells. A , schematic of FLAG-sortilin WT, S316E, and wp. Serine 316 was replaced by glutamic acid in FLAG-sortilin S316E. Propeptide was removed in FLAG-sortilin wp. SP , signal peptide; PP , propeptide. B , S316E increased dimerization of sortilin in HEK293 cells ( n = 3). C , removal of propeptide increased dimerization of sortilin in HEK293 cells ( n = 3). D and E , addition of propeptide (100 nmol/liter) decreased dimerization of sortilin in the extracellular vesicles of FLAG-sortilin HEK293 cells ( E ), whereas a decrease in the cells was not observed ( D ) ( n = 2). Monomers and homodimers of high and low molecular weight are abbreviated as MO , D(HMW) , and D(LMW) , respectively. Vec , vector; IB , immunoblotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Dimerization of sortilin regulates its trafficking to extracellular vesicles

    doi: 10.1074/jbc.RA117.000732

    Figure Lengend Snippet: Sortilin S316E and sortilin wp increase dimerization in HEK293 cells, and the addition of propeptide decreases dimerization in the extracellular vesicles of FLAG-sortilin HEK293 cells. A , schematic of FLAG-sortilin WT, S316E, and wp. Serine 316 was replaced by glutamic acid in FLAG-sortilin S316E. Propeptide was removed in FLAG-sortilin wp. SP , signal peptide; PP , propeptide. B , S316E increased dimerization of sortilin in HEK293 cells ( n = 3). C , removal of propeptide increased dimerization of sortilin in HEK293 cells ( n = 3). D and E , addition of propeptide (100 nmol/liter) decreased dimerization of sortilin in the extracellular vesicles of FLAG-sortilin HEK293 cells ( E ), whereas a decrease in the cells was not observed ( D ) ( n = 2). Monomers and homodimers of high and low molecular weight are abbreviated as MO , D(HMW) , and D(LMW) , respectively. Vec , vector; IB , immunoblotting.

    Article Snippet: Soluble sortilin with FLAG tag was eluted with 100 μg/ml FLAG peptide (Sigma, F3290, lot number SLBR6767V).

    Techniques: Molecular Weight, Plasmid Preparation

    Smed-β-catenin-2 interacts with the cadherin complex. A , HEK293T cells were transfected with the indicated plasmids, and lysates were immunoprecipitated ( IP ) with FLAG-M2 beads. Western blotting ( IB ) with anti-GFP revealed co-IP of Smed-β-catenin-2

    Journal: The Journal of Biological Chemistry

    Article Title: Complete Functional Segregation of Planarian ?-Catenin-1 and -2 in Mediating Wnt Signaling and Cell Adhesion *

    doi: 10.1074/jbc.M110.113662

    Figure Lengend Snippet: Smed-β-catenin-2 interacts with the cadherin complex. A , HEK293T cells were transfected with the indicated plasmids, and lysates were immunoprecipitated ( IP ) with FLAG-M2 beads. Western blotting ( IB ) with anti-GFP revealed co-IP of Smed-β-catenin-2

    Article Snippet: The remaining supernatant was incubated with FLAG-M2 beads (Sigma) or other antibodies (anti-Myc or anti-hemagglutinin) at 4 °C for 4 h and followed by incubation with protein A/G-Sepharose (Santa Cruz Biotechnology, Inc.) overnight.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay

    SOCS-6 associates with elongins B and C. (A) Endogenous SOCS-6 binds to endogenous elongins B and C. M1 cells were lysed and subjected to immunoprecipitation with either the 1C3 anti-SOCS-6 antibody, a control isotype matched (IgG2B) antibody, or a monoclonal antibody that recognizes both elongins B and C. This was followed by Western blotting with the polyclonal antibody that recognizes both elongins B and C (top). To confirm that SOCS-6 was immunoprecipitated, 1/10 of each immunoprecipitate was subjected to Western blotting with the 3A7 anti-SOCS-6 antibody (bottom). (B) SOCS-6 binds to elongins B and C through its SOCS box. 293T cells were transfected with cDNAs encoding FLAG-S6, N, N+SH2, or SH2+SB proteins or with the empty vector (V). Lysates were prepared and subjected to immunoprecipitation using M2 anti-FLAG resin followed by Western blotting with a polyclonal antibody that recognizes both elongins B and C (top). The filter was then stripped and reprobed with anti-FLAG antibodies (bottom). Molecular masses (kilodaltons) are on the left. IPPT, immunoprecipitate; S6, SOCS-6; El, elongin; C, control antibody; IgH, immunoglobulin heavy chain.

    Journal: Molecular and Cellular Biology

    Article Title: SOCS-6 Binds to Insulin Receptor Substrate 4, and Mice Lacking the SOCS-6 Gene Exhibit Mild Growth Retardation

    doi: 10.1128/MCB.22.13.4567-4578.2002

    Figure Lengend Snippet: SOCS-6 associates with elongins B and C. (A) Endogenous SOCS-6 binds to endogenous elongins B and C. M1 cells were lysed and subjected to immunoprecipitation with either the 1C3 anti-SOCS-6 antibody, a control isotype matched (IgG2B) antibody, or a monoclonal antibody that recognizes both elongins B and C. This was followed by Western blotting with the polyclonal antibody that recognizes both elongins B and C (top). To confirm that SOCS-6 was immunoprecipitated, 1/10 of each immunoprecipitate was subjected to Western blotting with the 3A7 anti-SOCS-6 antibody (bottom). (B) SOCS-6 binds to elongins B and C through its SOCS box. 293T cells were transfected with cDNAs encoding FLAG-S6, N, N+SH2, or SH2+SB proteins or with the empty vector (V). Lysates were prepared and subjected to immunoprecipitation using M2 anti-FLAG resin followed by Western blotting with a polyclonal antibody that recognizes both elongins B and C (top). The filter was then stripped and reprobed with anti-FLAG antibodies (bottom). Molecular masses (kilodaltons) are on the left. IPPT, immunoprecipitate; S6, SOCS-6; El, elongin; C, control antibody; IgH, immunoglobulin heavy chain.

    Article Snippet: Antiphosphotyrosine (clone 4G10), anti-p85, and anti-IRS-4 antibodies were from Upstate Biotechnology, Lake Placid, N.Y.; M2 anti-FLAG resin was from Sigma, Castle Hill, New South Wales, Australia; sheep anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP) antibodies were from Amersham Pharmacia, Sydney, New South Wales, Australia; goat anti-rabbit IgG-HRP antibodies were from Bio-Rad, Hercules, Calif.; goat anti-rat IgM/IgG-HRP antibodies were from Southern Biotechnology, Birmingham, Ala.

    Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation

    APMAP interacts physically and co-localizes with γ-secretase, APP-FL and APP-CTFs. ( A ) Velocity co-sedimentation and co-immunoprecipitation of APMAP with the γ-secretase complex, APP-FL and APP-CTFs. Total membrane protein extracts from HEK-APPSwe cells transiently overexpressing hAPMAP1 or hAPMAP1-Flag were sedimented on an 18–28% glycerol gradient containing 0.1% CHAPSO. Each fraction was collected and analyzed by western blot for APMAP1, APP-FL, APP-CTFs and mature and immature γ-secretase (top panels). Next, proteins interacting with hAPMAP1-Flag (Flag) were affinity-precipitated in the fractions labeled in red with M2 anti-Flag affinity resin (lower panel). Untagged APMAP (hAPMAP1, also labeled ‘-’ in the figure) served as a control for the specific co-precipitation. ( B ) Immunohistochemical co-localization of APMAP (green) with the γ-secretase subunit Nicastrin (red, upper panel) or APP (red, lower panel) in 14 days in vitro mouse primary cortical neurons. Scale bar: 10 µm. Both confocal images (left panels) and Z-stack projections (right panels) are shown with a microscope objective magnification of 40×. For comparison, un-merged images for APMAP, NCT, APP-CTFs and DAPI are shown in Supplementary Material, Fig. S10 . mNCT and iNCT, mature and immature Nicastrin.

    Journal: Human Molecular Genetics

    Article Title: The adipocyte differentiation protein APMAP is an endogenous suppressor of Aβ production in the brain

    doi: 10.1093/hmg/ddu449

    Figure Lengend Snippet: APMAP interacts physically and co-localizes with γ-secretase, APP-FL and APP-CTFs. ( A ) Velocity co-sedimentation and co-immunoprecipitation of APMAP with the γ-secretase complex, APP-FL and APP-CTFs. Total membrane protein extracts from HEK-APPSwe cells transiently overexpressing hAPMAP1 or hAPMAP1-Flag were sedimented on an 18–28% glycerol gradient containing 0.1% CHAPSO. Each fraction was collected and analyzed by western blot for APMAP1, APP-FL, APP-CTFs and mature and immature γ-secretase (top panels). Next, proteins interacting with hAPMAP1-Flag (Flag) were affinity-precipitated in the fractions labeled in red with M2 anti-Flag affinity resin (lower panel). Untagged APMAP (hAPMAP1, also labeled ‘-’ in the figure) served as a control for the specific co-precipitation. ( B ) Immunohistochemical co-localization of APMAP (green) with the γ-secretase subunit Nicastrin (red, upper panel) or APP (red, lower panel) in 14 days in vitro mouse primary cortical neurons. Scale bar: 10 µm. Both confocal images (left panels) and Z-stack projections (right panels) are shown with a microscope objective magnification of 40×. For comparison, un-merged images for APMAP, NCT, APP-CTFs and DAPI are shown in Supplementary Material, Fig. S10 . mNCT and iNCT, mature and immature Nicastrin.

    Article Snippet: The lysate was further diluted six times in 0.1% digitonin-TBS buffer (50 mm Tris–HCl pH 7.4, 150 mm NaCl) and bound to M2 anti-Flag affinity resin (Sigma-Aldrich) overnight.

    Techniques: Sedimentation, Immunoprecipitation, Western Blot, Labeling, Immunohistochemistry, In Vitro, Microscopy

    MMG8 associates with γTuCs and is required for γTuC attachment to the Golgi. (A,B) Anti-MMG8 (A) and anti-GCP3 (B) immunoprecipitations (IPs) were performed using HeLa extracts. The immunoprecipitates and inputs were immunoblotted. (C) FLAG–MMG8 and FLAG–MMG8 (389–1116) transiently expressed in HEK293T cells were immunoprecipitated in RIPA buffer. The beads were then used in a pull-down assay together with RPE1 lysates of cells that were transfected with control or AKAP450 siRNA. The pull-downs were examined on immunoblots. (D) RPE1 cells were double-stained for γ-tubulin and MMG8. (E) Cells transfected with siRNAs were immunostained for γ-tubulin and mannosidase II (Man II). Arrows indicate centrosomes. Images shown in D,E are representative ( > 90%) of 200 cells analyzed for each sample. Scale bars: 5 µm. (F) Golgi membranes were isolated from siRNA-transfected cells. Both the Golgi fractions and the whole cell lysates (WCLs) were analyzed on the immunoblots. The graph shows the protein quantification of the isolated Golgi membranes as the mean±s.d. from three independent experiments; ** P

    Journal: Journal of Cell Science

    Article Title: A newly identified myomegalin isoform functions in Golgi microtubule organization and ER–Golgi transport

    doi: 10.1242/jcs.155408

    Figure Lengend Snippet: MMG8 associates with γTuCs and is required for γTuC attachment to the Golgi. (A,B) Anti-MMG8 (A) and anti-GCP3 (B) immunoprecipitations (IPs) were performed using HeLa extracts. The immunoprecipitates and inputs were immunoblotted. (C) FLAG–MMG8 and FLAG–MMG8 (389–1116) transiently expressed in HEK293T cells were immunoprecipitated in RIPA buffer. The beads were then used in a pull-down assay together with RPE1 lysates of cells that were transfected with control or AKAP450 siRNA. The pull-downs were examined on immunoblots. (D) RPE1 cells were double-stained for γ-tubulin and MMG8. (E) Cells transfected with siRNAs were immunostained for γ-tubulin and mannosidase II (Man II). Arrows indicate centrosomes. Images shown in D,E are representative ( > 90%) of 200 cells analyzed for each sample. Scale bars: 5 µm. (F) Golgi membranes were isolated from siRNA-transfected cells. Both the Golgi fractions and the whole cell lysates (WCLs) were analyzed on the immunoblots. The graph shows the protein quantification of the isolated Golgi membranes as the mean±s.d. from three independent experiments; ** P

    Article Snippet: In the pull-down assay of γTuCs, FLAG–MMG8 transiently expressed in HEK293T cells was immunoprecipitated using anti-FLAG-coupled beads (M2, Sigma Aldrich) in RIPA buffer.

    Techniques: Immunoprecipitation, Pull Down Assay, Transfection, Western Blot, Staining, Isolation

    MMG8 targets to the Golgi by interacting with AKAP450. (A) Anti-MMG8 and anti-AKAP450 immunoprecipitations (IPs) were performed using HeLa extracts. The immunoprecipitated proteins and inputs were analyzed by immunoblotting and quantifying the samples. The graph shows the amount of the precipitated proteins relative to that of the respective inputs. Data show the mean±s.d. (three independent experiments). (B) Proteins coimmunoprecipitated with MMG8 were stained with Sypro Ruby or detected on immunoblots (IBs). The Sypro Ruby intensities of MMG8 and AKAP450 were quantified. Data from three independent experiments showed that the molar ratio of AKAP450 to MMG8 was 0.98±0.1∶1. (C) HEK293T cells expressing constructs encoding FLAG–MMG8 were subjected to anti-FLAG immunoprecipitation. The immunoprecipitates and inputs were immunoblotted for the MMG8 proteins (anti-FLAG) and AKAP450. (D) RPE1 cells were double-stained for AKAP450 and MMG8. The boxed area is enlarged on the right. (E) MMG8 fragments were ectopically expressed (GFP) in HeLa cells. The cells were examined for the transfected proteins and a Golgi marker. The boxed areas are enlarged on the right. (F) HeLa cells in which MMG8 or AKAP450 was depleted using RNAi were treated with MG132 for 12 h. The cells were then immunostained. The phenotypes shown in E,F were observed in > 90% of 100 cells analyzed for each sample. Scale bars: 5 µm.

    Journal: Journal of Cell Science

    Article Title: A newly identified myomegalin isoform functions in Golgi microtubule organization and ER–Golgi transport

    doi: 10.1242/jcs.155408

    Figure Lengend Snippet: MMG8 targets to the Golgi by interacting with AKAP450. (A) Anti-MMG8 and anti-AKAP450 immunoprecipitations (IPs) were performed using HeLa extracts. The immunoprecipitated proteins and inputs were analyzed by immunoblotting and quantifying the samples. The graph shows the amount of the precipitated proteins relative to that of the respective inputs. Data show the mean±s.d. (three independent experiments). (B) Proteins coimmunoprecipitated with MMG8 were stained with Sypro Ruby or detected on immunoblots (IBs). The Sypro Ruby intensities of MMG8 and AKAP450 were quantified. Data from three independent experiments showed that the molar ratio of AKAP450 to MMG8 was 0.98±0.1∶1. (C) HEK293T cells expressing constructs encoding FLAG–MMG8 were subjected to anti-FLAG immunoprecipitation. The immunoprecipitates and inputs were immunoblotted for the MMG8 proteins (anti-FLAG) and AKAP450. (D) RPE1 cells were double-stained for AKAP450 and MMG8. The boxed area is enlarged on the right. (E) MMG8 fragments were ectopically expressed (GFP) in HeLa cells. The cells were examined for the transfected proteins and a Golgi marker. The boxed areas are enlarged on the right. (F) HeLa cells in which MMG8 or AKAP450 was depleted using RNAi were treated with MG132 for 12 h. The cells were then immunostained. The phenotypes shown in E,F were observed in > 90% of 100 cells analyzed for each sample. Scale bars: 5 µm.

    Article Snippet: In the pull-down assay of γTuCs, FLAG–MMG8 transiently expressed in HEK293T cells was immunoprecipitated using anti-FLAG-coupled beads (M2, Sigma Aldrich) in RIPA buffer.

    Techniques: Immunoprecipitation, Staining, Western Blot, Expressing, Construct, Transfection, Marker

    PHD-induced hydroxylation of wild type and mutant HIF-1α-(531–652) A , in vitro translated, 35 S-labeled wild type or mutant GAL4-HIF-1α-(531–652) was incubated with 0.5 μg of His 6 -PHD1, His 6 -PHD2, or His 6 -PHD3 for the indicated times. Hydroxylated reaction products were then isolated by first incubating with His 6 -FLAG-VBC and then by immunoprecipitating with anti-FLAG antibodies coupled to agarose. The immunoprecipitated reaction products were subjected to SDS-PAGE and autoradiography. B , same as in A except that His 6 -PHD2 and either HA-HIF-1α or GAL4-HIF-1α-(531–652) were employed. Input ( In ) designates 20% of the substrate analyzed at a given time point. One of three representative results is shown.

    Journal: The Journal of biological chemistry

    Article Title: Sequence Determinants in Hypoxia-inducible Factor-1? for Hydroxylation by the Prolyl Hydroxylases PHD1, PHD2, and PHD3

    doi: 10.1074/jbc.M206955200

    Figure Lengend Snippet: PHD-induced hydroxylation of wild type and mutant HIF-1α-(531–652) A , in vitro translated, 35 S-labeled wild type or mutant GAL4-HIF-1α-(531–652) was incubated with 0.5 μg of His 6 -PHD1, His 6 -PHD2, or His 6 -PHD3 for the indicated times. Hydroxylated reaction products were then isolated by first incubating with His 6 -FLAG-VBC and then by immunoprecipitating with anti-FLAG antibodies coupled to agarose. The immunoprecipitated reaction products were subjected to SDS-PAGE and autoradiography. B , same as in A except that His 6 -PHD2 and either HA-HIF-1α or GAL4-HIF-1α-(531–652) were employed. Input ( In ) designates 20% of the substrate analyzed at a given time point. One of three representative results is shown.

    Article Snippet: Hydroxylated reaction product was then isolated by first incubating with 1 μg of His6 -FLAG-VBC in 500 μl of buffer A for 1 h at 4 °C with rocking, and then immunoprecipitating the His6 -FLAG-VBC complex with 10 μl of anti-FLAG-agarose (M2-agarose, Sigma) for 1 h at 4 °C with rocking.

    Techniques: Mutagenesis, In Vitro, Labeling, Incubation, Isolation, Immunoprecipitation, SDS Page, Autoradiography