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  • 99
    Millipore streptavidin agarose magnetic beads
    Comparison of different ε and non-ε isoforms for the ability to bind and activate the H + -ATPase. A. Densitometric analysis of a relative amount of GF14 isoforms bound to the H + -ATPase (left panel) and to its recombinant C-terminal domain (right panel) estimated from overlay assays. Ten µg of plasma membrane preparation from maize roots or 1 µg of the recombinant GST-fused MHA2 C-terminal domain were subjected to the SDS-PAGE, then blotted onto nitrocellulose membrane and incubated with 0.1 μM 32 P-labeled-GF14 isoforms in the presence or in the absence of 10 µM FC. Densitometric analysis was performed on four independent overlay experiments and data are the means ± S.E. B. Peptide binding assay: 0.05 nmol of bL15Vp biotinyl-peptide reproducing the last 15 amino acids of MHA2 H + -ATPase, phosphorylated on the threonine residue at position −1 from the C-terminus, were immobilized onto <t>streptavidin–agarose</t> magnetic beads and incubated with 0.1 nmol 32 P-labeled-GF14 isoforms in the presence or in the absence of 10 µM FC. After washing, the amount of peptide-associated 14-3-3 was estimated by measuring the beads bound radioactivity. Data are the means ± S.E. of three independent experiments. C. Comparison of GF14 isoforms ability to stimulate the H + -ATPase: phosphohydrolytic activity of H + -ATPase was determined by incubating 10 µg of ER yeast vesicles, containing AHA1, with GF14 isoforms at different concentrations (ranging from 0 to 4 μM) in the presence of 10 µM FC. Data are the means ± S.E. of four independent experiments.
    Streptavidin Agarose Magnetic Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin coated agarose beads
    p120 regulates E-cadherin turnover at the cell membrane. (a) E-cadherin synthesis and processing in p120 knockdown cells. E-cadherin turnover was examined by pulse-chase analysis of parental and h p120 siRNA-expressing A431 cells. α- and β-Catenin processing from the same experiment are shown below. Chase times are indicated across the top. At chase time 0 (15 min after initiation of the pulse labeling), E-cadherin synthesis was identical in the presence and absence of p120 (a, compare E-cadherin bands). The processing of the pro- (pro-E-cad) and mature (E-cad.) forms were identical for at least 1 h. Soon thereafter, E-cadherin degradation was significantly accelerated in the absence of p120. (b) Analysis of total E-cadherin surface levels in parental and p120 knockdown A431 cells. The parental and p120 knockdown A431 cells were biotinylated for 20 min at 4°C to label surface cadherins. To specifically measure the surface levels, E-cadherin was first immunoprecipitated directly with E-cadherin mAb HECD-1. The sample was eluted with 0.5% SDS and then reprecipitated with <t>streptavidin-coated</t> beads to isolate the surface-labeled pool. E-cadherin levels at the surface in p120 knockdown cells (lane 1) are at least 100-fold diminished relative to the parental cells (lane 2). The result in lane 2 shows that the surface E-cadherin can be efficiently labeled (and detected) by this method. (c) Tracking the arrival of newly synthesized E-cadherin to the cell surface. The methods in a and b were combined to determine whether newly synthesized E-cadherin could transit to the cell surface in the absence of p120. The results in c were quantified by densitometry and represented graphically in d. Parental and p120 knockdown (h siRNA) cells were labeled with [ 35 S]methionine for 15 min, chased at 37°C for the times indicated across top, and placed on ice (4°C) to suspend trafficking. Cell surface proteins were immediately biotinylated at 4°C for 20 min as in b. Surface E-cadherin was then isolated as in b, and the nascent [ 35 S]methionine E-cadherin pool was visualized by SDS-PAGE and radiography. Nascent E-cadherin appeared at the surface at 30 min and peaked at 1 h. The absence of p120 had no effect on this result. Therefore, p120 is not required for E-cadherin synthesis or trafficking, but is essential to regulate E-cadherin turnover soon after its arrival at the cell surface.
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    94
    Merck KGaA streptavidin agarose
    p120 regulates E-cadherin turnover at the cell membrane. (a) E-cadherin synthesis and processing in p120 knockdown cells. E-cadherin turnover was examined by pulse-chase analysis of parental and h p120 siRNA-expressing A431 cells. α- and β-Catenin processing from the same experiment are shown below. Chase times are indicated across the top. At chase time 0 (15 min after initiation of the pulse labeling), E-cadherin synthesis was identical in the presence and absence of p120 (a, compare E-cadherin bands). The processing of the pro- (pro-E-cad) and mature (E-cad.) forms were identical for at least 1 h. Soon thereafter, E-cadherin degradation was significantly accelerated in the absence of p120. (b) Analysis of total E-cadherin surface levels in parental and p120 knockdown A431 cells. The parental and p120 knockdown A431 cells were biotinylated for 20 min at 4°C to label surface cadherins. To specifically measure the surface levels, E-cadherin was first immunoprecipitated directly with E-cadherin mAb HECD-1. The sample was eluted with 0.5% SDS and then reprecipitated with <t>streptavidin-coated</t> beads to isolate the surface-labeled pool. E-cadherin levels at the surface in p120 knockdown cells (lane 1) are at least 100-fold diminished relative to the parental cells (lane 2). The result in lane 2 shows that the surface E-cadherin can be efficiently labeled (and detected) by this method. (c) Tracking the arrival of newly synthesized E-cadherin to the cell surface. The methods in a and b were combined to determine whether newly synthesized E-cadherin could transit to the cell surface in the absence of p120. The results in c were quantified by densitometry and represented graphically in d. Parental and p120 knockdown (h siRNA) cells were labeled with [ 35 S]methionine for 15 min, chased at 37°C for the times indicated across top, and placed on ice (4°C) to suspend trafficking. Cell surface proteins were immediately biotinylated at 4°C for 20 min as in b. Surface E-cadherin was then isolated as in b, and the nascent [ 35 S]methionine E-cadherin pool was visualized by SDS-PAGE and radiography. Nascent E-cadherin appeared at the surface at 30 min and peaked at 1 h. The absence of p120 had no effect on this result. Therefore, p120 is not required for E-cadherin synthesis or trafficking, but is essential to regulate E-cadherin turnover soon after its arrival at the cell surface.
    Streptavidin Agarose, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 94/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore streptavidin agarose
    Schematic of poliovirus genomic RNAs containing S1 and D8 aptamer tags within the highly structured 5′NCR, for positive-strand isolation. Insertion of either the S1 <t>streptavidin-binding</t> or D8 Sephadex-binding aptamer sequence within three regions of the poliovirus 5′NCR resulted in the production of 12 poliovirus cDNA constructs. Tag insertions in place of S-L III (blue), in place of S-L VI (green), or at nucleotide position 702 (purple) are depicted on the left, middle, and right, respectively. Constructs for negative-strand isolation (aptamers inserted in a reverse orientation) are not shown.
    Streptavidin Agarose, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin agarose beads
    PRMT5 peptide pull-downs confirm an in vitro interaction with NHERF2. A , GST-fused PDZ domains of GRIP1 (residues 672–754), MPP7 (residues 139–220), PDZ-LIM55 (residues 2–85), NHERF1 FL (residues 1–355), NHERF2 FL (residues 1–337), SCRIB (residues 714–801), PDZ-LIM2 (residues 1–84), and GST were incubated with biotinylated PRMT5 C terminus unphosphorylated peptide. Bound proteins were detected with α-GST antibody (short and long exposure are shown). Peptide loading was assessed with HRP-conjugated <t>streptavidin</t> ( SA-HRP ). The Coomassie stain demonstrates roughly equal input of the GST fusion proteins. B , schematic representation of the constructs used for peptide pull-down in C. C , purified recombinant GST, GST-tagged human NHERF2 full-length (NHERF2-PDZ 1–2), PDZ1 (amino acids 1–152), PDZ2 (amino acids 107–337), and 14-3-3ϵ were incubated with biotinylated PRMT5 C terminus unphosphorylated and Thr-634-phosphorylated peptides bound to streptavidin-agarose beads and detected by α-GST. Left lane , inputs of the GST fusion proteins. D , 293T cells were transfected with constructs expressing GFP-14-3-3ϵ and Myc-PRMT5 wild type or T634A mutant. Cell lysates were then incubated with normal mouse IgG or α-Myc antibody. Immunocomplexes were captured by Protein A beads and detected by either α-Myc or α-GFP. IB , immunoblotting.
    Streptavidin Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 2026 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin agarose resin
    N63Y mutant CVB3 has a growth defect in cell culture and reduced glycan-mediated cell attachment. Single-cycle assays of viral replication in HeLa (A) and Huh7 (B) cells were performed. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. n = 3. (C) Cell attachment of 35 S-labeled WT or N63Y mutant CVB3. Virus was incubated with cells at 4°C for 40 min. Cells were washed and trypsinized, and cell-associated 35 S was quantified. (D) 35 S-labeled WT or N63Y mutant CVB3 was incubated with CHO cells (CHO-K1, pgsA745, pgsD677, and pgsB761) that vary in GAG expression. Plus and minus signs indicate the relative levels of GAGs on the cell surface. (E) Effect of heparinase treatment on CVB3 cell attachment. Huh7 cells were treated with or without heparinase I for 90 min prior to quantification of 35 S CVB3 attachment. n = 7. (F) Heparin-agarose pulldown assay. 35 S-labeled WT or N63Y mutant CVB3 was incubated with heparin-agarose resin or <t>streptavidin-agarose</t> resin (control). Resin was washed, and bound 35 S-labeled CVB3 was quantified. n = 3. *, P
    Streptavidin Agarose Resin, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin agarose column
    N63Y mutant CVB3 has a growth defect in cell culture and reduced glycan-mediated cell attachment. Single-cycle assays of viral replication in HeLa (A) and Huh7 (B) cells were performed. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. n = 3. (C) Cell attachment of 35 S-labeled WT or N63Y mutant CVB3. Virus was incubated with cells at 4°C for 40 min. Cells were washed and trypsinized, and cell-associated 35 S was quantified. (D) 35 S-labeled WT or N63Y mutant CVB3 was incubated with CHO cells (CHO-K1, pgsA745, pgsD677, and pgsB761) that vary in GAG expression. Plus and minus signs indicate the relative levels of GAGs on the cell surface. (E) Effect of heparinase treatment on CVB3 cell attachment. Huh7 cells were treated with or without heparinase I for 90 min prior to quantification of 35 S CVB3 attachment. n = 7. (F) Heparin-agarose pulldown assay. 35 S-labeled WT or N63Y mutant CVB3 was incubated with heparin-agarose resin or <t>streptavidin-agarose</t> resin (control). Resin was washed, and bound 35 S-labeled CVB3 was quantified. n = 3. *, P
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    Millipore streptavidin agarose slurry
    Specific binding of RAG-2 to histone H3 containing di- or trimethylated lysine 4. (A) Whole-cell lysates of 293T cells expressing full-length, wild-type RAG-2 were incubated with <t>streptavidin</t> bead-bound, biotinylated peptides corresponding to residues
    Streptavidin Agarose Slurry, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin agarose sample
    Specific binding of RAG-2 to histone H3 containing di- or trimethylated lysine 4. (A) Whole-cell lysates of 293T cells expressing full-length, wild-type RAG-2 were incubated with <t>streptavidin</t> bead-bound, biotinylated peptides corresponding to residues
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    Millipore streptavidin agarose dynabeads
    Specific binding of RAG-2 to histone H3 containing di- or trimethylated lysine 4. (A) Whole-cell lysates of 293T cells expressing full-length, wild-type RAG-2 were incubated with <t>streptavidin</t> bead-bound, biotinylated peptides corresponding to residues
    Streptavidin Agarose Dynabeads, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin agarose conjugate
    Specific binding of RAG-2 to histone H3 containing di- or trimethylated lysine 4. (A) Whole-cell lysates of 293T cells expressing full-length, wild-type RAG-2 were incubated with <t>streptavidin</t> bead-bound, biotinylated peptides corresponding to residues
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    Millipore streptavidine agarose beads
    Sam68 regulates alternative splicing of Sgce exon 8 in male germ cells. RT–PCR analysis of Sgce exon 8 inclusion in total RNA extracted from Sam68 wild-type or knockout testes at 8, 16 and 30 days post partum (dpp) ( A ) or from isolated wild-type or knockout spermatocytes and spermatids. ( B ) Bands corresponding to mRNAs containing or not, exon 8 are indicated on the right of the panels. ( C and D ) Chromatin immunoprecipitation (ChIP) analysis of Sam68 wild-type and knockout germ cells. Sonicated chromatin (100 µg of DNA/sample) was immunoprecipitated with 2µg of H5, H14 antibodies or control rabbit IgGs and co-precipitated DNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( E ) CLIP analysis of the binding of Sam68 to Sgce pre-mRNA. After UV crosslink, Sam68 wild-type and knockout germ cell extracts were sonicated and treated with DNase and RNase to yield RNA fragments of ∼200 to 250 nt. Wild-type and knockout germ cell extracts were immunoprecipitated with 2 µg of rabbit IgGs or anti-Sam68 antibodies and co-precipitated RNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( F ) Electrophoretic mobility shift assays (EMSAs) of the binding of purified GST-Sam68 1–277 to a labelled Sgce probe containing the sequences encoded at the intron 7/exon 8 boundary. The position of the free probe and the probe complexed with GST-Sam68 1–277 are shown on the left side. Competition with the wild-type (middle panel) or mutated (right panel) cold probes is shown. The scheme above the gels shows the wild-type and mutated sequence used in the EMSAs. The mutated bases that interfere with the Sam68 consensus are shown in violet. ( G ) The western blot analysis of RNA pulldown assay of U2AF65 binding to Sgce exon 8. Biotinylated RNAs encoding the Sgce wild-type and mutated exon 8 sequences (from −32 to +63) were bound to <t>Streptavidine</t> agarose beads and incubated with testicular nuclear extract (100 µg) in the presence of 1 µg of purified GST or GST-Sam68 1–277, as indicated.
    Streptavidine Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin agarose solution
    P cr -400 interacts with the coiled-coil of MBP-Fishhook. (A) MBP (lanes 2 and 3), MBP-Hairpin (lanes 4 and 5), or MBP-Fishhook (lanes 6 and 7) was incubated without (−) or with (+) P cr -400, and the complexes were analyzed by native PAGE and stained with Coomasie blue. P cr -400 induces a shift in the electrophoretic mobility of the trimeric MBP-Fishhook (T F ), causing it to migrate similarly to trimeric MBP-Hairpin (T H ). A, aggregate. Lane M, molecular-mass markers. (B) Increasing amounts of P cr -400 were incubated with MBP-Fishhook, and the complexes were analyzed by native PAGE. Lanes 2 and 3, MBP-Hairpin and MBP-Fishhook controls. The amount of MBP-Fishhook (T F ) shifting to a complex that mimics MBP-Hairpin (T H ) increases with the amount of P cr -400 added (lanes 4 to 12). (C) Increasing amounts of biotinylated P cr -400 were incubated with MBP-Fishhook (lanes 4 to 8). As a control, MBP was also incubated with biotinylated P cr -400 (lane 9). <t>Streptavidin-agarose</t> was then added to the reaction mixture, and the bound complexes were precipitated, heat denatured, and analyzed by SDS-PAGE.
    Streptavidin Agarose Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore streptavidin agarose resin beads
    P cr -400 interacts with the coiled-coil of MBP-Fishhook. (A) MBP (lanes 2 and 3), MBP-Hairpin (lanes 4 and 5), or MBP-Fishhook (lanes 6 and 7) was incubated without (−) or with (+) P cr -400, and the complexes were analyzed by native PAGE and stained with Coomasie blue. P cr -400 induces a shift in the electrophoretic mobility of the trimeric MBP-Fishhook (T F ), causing it to migrate similarly to trimeric MBP-Hairpin (T H ). A, aggregate. Lane M, molecular-mass markers. (B) Increasing amounts of P cr -400 were incubated with MBP-Fishhook, and the complexes were analyzed by native PAGE. Lanes 2 and 3, MBP-Hairpin and MBP-Fishhook controls. The amount of MBP-Fishhook (T F ) shifting to a complex that mimics MBP-Hairpin (T H ) increases with the amount of P cr -400 added (lanes 4 to 12). (C) Increasing amounts of biotinylated P cr -400 were incubated with MBP-Fishhook (lanes 4 to 8). As a control, MBP was also incubated with biotinylated P cr -400 (lane 9). <t>Streptavidin-agarose</t> was then added to the reaction mixture, and the bound complexes were precipitated, heat denatured, and analyzed by SDS-PAGE.
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    Millipore streptavidin agarose mini column
    Cys37 in VPAC1 is S-palmitoylated determined by acyl-biotin exchange assay and click chemistry palmitolaytion assay ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by <t>streptavidin-agarose</t> and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.
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    Cys37 in VPAC1 is S-palmitoylated determined by acyl-biotin exchange assay and click chemistry palmitolaytion assay ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by <t>streptavidin-agarose</t> and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.
    Streptavidin Agarose Beads Slurry, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    H3K27m3 and DNA methylation co‐exist at ATCOPIA93 IGB (integrative genome browser) views showing H3K9m2 levels and H3K27m3 levels in WT and met1 rosette leaves, at ATCOPIA93 EVD and ATR (ChIP‐chip public data, Deleris et al , 2012 ). Orange horizontal bars: protein‐coding genes; horizontal green bars: transposable elements. The LTRs are delineated by pink bars. Vertical blue bars: H3K9m2 signal relative to H3 (two top lanes); vertical purple bars: H3K27m3 signal relative to H3 for each probe. Analysis of H3K27m3 marks at ATCOPIA93 EVD and ATR by ChIP on rosette leaves, followed by qPCR, in wild‐type plants and in clf plants mutated for the H3K27 methyltransferase CURLY LEAF. Data were normalized to the input DNA. ATCOPIA93 CDS is a region in ATCOPIA93 GAG common to EVD and ATR . AT5g17120 is a region in the protein‐coding gene located upstream of EVD . FLC is a region located in the first intron of FLOWERING LOCUS C which shows high levels of H3K27m3 in vegetative tissues and serves as a positive control. TA3 is a transposon and serves as a negative control. Because of technical variability in the ChIP efficiency, one ChIP experiment is presented here and two other independent experiments are presented in Fig EV3 B. ChIPs were performed on a pool of rosette leaves from eight to 10 plants/genotype. Genomic distribution of H3K27m3 marks between EVD and ATR loci by ChIP‐PCR pyrosequencing. Upper panel: Depiction of the pyrosequenced region (in yellow) within the GAG biotinylated qPCR amplicon obtained after H3K27m3 ChIP‐qPCR and purification with <t>streptavidin</t> beads. The position interrogated corresponds to the discriminating SNP between EVD (C/G) and ATR (A/T). Lower panel: The % indicated represents the % of G ( EVD , dark blue bar) or T ( ATR , light blue bar) at that position. The PCR and sequencing primers were designed so that other ATCOPIA93 ‐derived sequences (divergent and presumably nonfunctional) such as AT4G04410 and AT1G43775 cannot be amplified and so that the allelic ratio between the two active ATCOPIA93 copies EVD and ATR only can be evaluated. To verify this, the qPCR GAG product is also amplified from the Input gDNA as a control where a 50–50% ratio is expected. For clarity, an average of two experiments performed on two independent Input and ChIPs samples is shown (error bars represent standard error (SE) of the mean) and individual datasets presented in Fig EV3 C. Methylation status of the DNA captured with H3K27m3 by Sau96I Chop‐qPCR. H3K27m3 ChIP‐DNA from two independent ChIPs was digested with the methylation‐sensitive restriction endonuclease Sau96I which is sensitive to the methylation of the second C at the GGGCCG site in the LTR (as in Fig EV1 ). The values plotted correspond to the ratio between the amount of amplified DNA in the Sau96I digestion and the amount of amplified DNA in the undigested control, as calculated by the formula 2 −(Ct.digestedDNA–Ct.undigestedDNA) and using primers specific for a region of EVD‐ LTR spanning this Sau96I restriction site. Dark and light symbols are used for the first and second experiments, respectively. Results show that the WT ChIP‐DNA had significantly less digestion compared with the ddm1 control; thus, there was more methylation. Source data are available online for this figure.
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    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
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    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
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    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
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    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
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    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
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    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
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    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
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    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
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    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
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    Comparison of the inhibitory rate on the proliferation of U87MG and U87-EGFRvIII cells following c-Met gene silencing. The inhibitory rate was calculated according to the OD values of the control and experimental groups obtained from the MTT assay. OD, optical density; BA, 5′-biotin-labeled aptamer 32; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + <t>streptavidin;</t> lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Data show the means ± standard deviation of three independent experiments. ** P
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    Comparison of the inhibitory rate on the proliferation of U87MG and U87-EGFRvIII cells following c-Met gene silencing. The inhibitory rate was calculated according to the OD values of the control and experimental groups obtained from the MTT assay. OD, optical density; BA, 5′-biotin-labeled aptamer 32; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + <t>streptavidin;</t> lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Data show the means ± standard deviation of three independent experiments. ** P
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    DNA binding activity of RUNX2 or RUNX2 R131G HA tagged RUNX2 or RUNX2 R131G proteins were expressed in HeLa cells. Nuclear extracts from transfected HeLa cells were incubated with biotinylated wild type (Wt) or mutant (Mu) RUNX2 binding oligonucleotides for 1h at RT. <t>Streptavidin</t> coated Sepharose <t>CL4B</t> beads were added to nuclear protein/DNA complexes and incubated at RT for 1 hr with rotation. Reacted (A) or non-reacted (B) nuclear extracts were separated in 10% SDS-polyacrylamide gels, transferred, and immunoblotted with antibodies to RUNX2. Lamin B antibody was used as internal loading control.
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    Image Search Results


    Comparison of different ε and non-ε isoforms for the ability to bind and activate the H + -ATPase. A. Densitometric analysis of a relative amount of GF14 isoforms bound to the H + -ATPase (left panel) and to its recombinant C-terminal domain (right panel) estimated from overlay assays. Ten µg of plasma membrane preparation from maize roots or 1 µg of the recombinant GST-fused MHA2 C-terminal domain were subjected to the SDS-PAGE, then blotted onto nitrocellulose membrane and incubated with 0.1 μM 32 P-labeled-GF14 isoforms in the presence or in the absence of 10 µM FC. Densitometric analysis was performed on four independent overlay experiments and data are the means ± S.E. B. Peptide binding assay: 0.05 nmol of bL15Vp biotinyl-peptide reproducing the last 15 amino acids of MHA2 H + -ATPase, phosphorylated on the threonine residue at position −1 from the C-terminus, were immobilized onto streptavidin–agarose magnetic beads and incubated with 0.1 nmol 32 P-labeled-GF14 isoforms in the presence or in the absence of 10 µM FC. After washing, the amount of peptide-associated 14-3-3 was estimated by measuring the beads bound radioactivity. Data are the means ± S.E. of three independent experiments. C. Comparison of GF14 isoforms ability to stimulate the H + -ATPase: phosphohydrolytic activity of H + -ATPase was determined by incubating 10 µg of ER yeast vesicles, containing AHA1, with GF14 isoforms at different concentrations (ranging from 0 to 4 μM) in the presence of 10 µM FC. Data are the means ± S.E. of four independent experiments.

    Journal: PLoS ONE

    Article Title: Specificity of ? and Non-? Isoforms of Arabidopsis 14-3-3 Proteins Towards the H+-ATPase and Other Targets

    doi: 10.1371/journal.pone.0090764

    Figure Lengend Snippet: Comparison of different ε and non-ε isoforms for the ability to bind and activate the H + -ATPase. A. Densitometric analysis of a relative amount of GF14 isoforms bound to the H + -ATPase (left panel) and to its recombinant C-terminal domain (right panel) estimated from overlay assays. Ten µg of plasma membrane preparation from maize roots or 1 µg of the recombinant GST-fused MHA2 C-terminal domain were subjected to the SDS-PAGE, then blotted onto nitrocellulose membrane and incubated with 0.1 μM 32 P-labeled-GF14 isoforms in the presence or in the absence of 10 µM FC. Densitometric analysis was performed on four independent overlay experiments and data are the means ± S.E. B. Peptide binding assay: 0.05 nmol of bL15Vp biotinyl-peptide reproducing the last 15 amino acids of MHA2 H + -ATPase, phosphorylated on the threonine residue at position −1 from the C-terminus, were immobilized onto streptavidin–agarose magnetic beads and incubated with 0.1 nmol 32 P-labeled-GF14 isoforms in the presence or in the absence of 10 µM FC. After washing, the amount of peptide-associated 14-3-3 was estimated by measuring the beads bound radioactivity. Data are the means ± S.E. of three independent experiments. C. Comparison of GF14 isoforms ability to stimulate the H + -ATPase: phosphohydrolytic activity of H + -ATPase was determined by incubating 10 µg of ER yeast vesicles, containing AHA1, with GF14 isoforms at different concentrations (ranging from 0 to 4 μM) in the presence of 10 µM FC. Data are the means ± S.E. of four independent experiments.

    Article Snippet: Streptavidin-agarose magnetic beads, glutathione-sepharose resin, the catalytic subunit of protein kinase A and thrombin were obtained from Sigma (St. Louis, MO, USA).

    Techniques: Recombinant, SDS Page, Incubation, Labeling, Binding Assay, Magnetic Beads, Radioactivity, Activity Assay

    Effect of C-terminal deletion on the binding properties of GF14ε and GF14ω. A. Peptide binding assay: 0.05 nmol of bL15Vp biotinyl-peptide were immobilized onto streptavidin–agarose magnetic beads and incubated with 0.1 nmol wild type 32 P-labeled-GF14ε and 32 P-labeled-GF14ω or corresponding C-terminal deletion mutants in the presence or in the absence of 10 µM FC. Data are the means ± S.E. of three independent experiments. *, p

    Journal: PLoS ONE

    Article Title: Specificity of ? and Non-? Isoforms of Arabidopsis 14-3-3 Proteins Towards the H+-ATPase and Other Targets

    doi: 10.1371/journal.pone.0090764

    Figure Lengend Snippet: Effect of C-terminal deletion on the binding properties of GF14ε and GF14ω. A. Peptide binding assay: 0.05 nmol of bL15Vp biotinyl-peptide were immobilized onto streptavidin–agarose magnetic beads and incubated with 0.1 nmol wild type 32 P-labeled-GF14ε and 32 P-labeled-GF14ω or corresponding C-terminal deletion mutants in the presence or in the absence of 10 µM FC. Data are the means ± S.E. of three independent experiments. *, p

    Article Snippet: Streptavidin-agarose magnetic beads, glutathione-sepharose resin, the catalytic subunit of protein kinase A and thrombin were obtained from Sigma (St. Louis, MO, USA).

    Techniques: Binding Assay, Magnetic Beads, Incubation, Labeling

    Verification of SSO incorporation into its homologous DNA target ( A ) A schematic illustration of the experimental procedure. Biotinylated recombination products were purified using magnetic streptavidin beads. The presence of (corrected) pmKan was confirmed by the detection of a 496 bp PCR product. ( B ) pmKan and ddH 2 O were used as templates for the negative and positive PCR controls (lanes 2 and 3 respectively). DY380/pmKan cells were incubated at 42°C for 15 min to induce λ-Red protein expression prior to electroporation with biotinylated-SSO (lane 6) or unmodified SSO (lane 4). As a control, DY380/pmKan cells that had been incubated at 32°C for 15 min (i.e. no λ-Red induction) were similarly electroporated with biotinylated-SSO (lane 5). Plasmid DNA were extracted from the electroporated cells after a 15 min recovery period. Three independent experiments were performed; a representative experiment is shown.

    Journal: Nucleic Acids Research

    Article Title: The involvement of replication in single stranded oligonucleotide-mediated gene repair

    doi: 10.1093/nar/gkl852

    Figure Lengend Snippet: Verification of SSO incorporation into its homologous DNA target ( A ) A schematic illustration of the experimental procedure. Biotinylated recombination products were purified using magnetic streptavidin beads. The presence of (corrected) pmKan was confirmed by the detection of a 496 bp PCR product. ( B ) pmKan and ddH 2 O were used as templates for the negative and positive PCR controls (lanes 2 and 3 respectively). DY380/pmKan cells were incubated at 42°C for 15 min to induce λ-Red protein expression prior to electroporation with biotinylated-SSO (lane 6) or unmodified SSO (lane 4). As a control, DY380/pmKan cells that had been incubated at 32°C for 15 min (i.e. no λ-Red induction) were similarly electroporated with biotinylated-SSO (lane 5). Plasmid DNA were extracted from the electroporated cells after a 15 min recovery period. Three independent experiments were performed; a representative experiment is shown.

    Article Snippet: After isolation of the plasmid DNA, biotinylated DNA was captured on streptavidin-conjugated magnetic agarose beads (Novagen), and was purified as described by Edwards et al . ( ).

    Techniques: Purification, Polymerase Chain Reaction, Incubation, Expressing, Electroporation, Plasmid Preparation

    The binding of Cwc25 to the spliceosome requires Prp2 and Yju2. The spliceosome formed with biotinylated ACAC pre-mRNA was isolated by precipitation with streptavidin-Sepharose, and the components were analyzed by Western blotting. (A) Splicing was carried

    Journal: Molecular and Cellular Biology

    Article Title: Cwc25 Is a Novel Splicing Factor Required after Prp2 and Yju2 To Facilitate the First Catalytic Reaction ▿

    doi: 10.1128/MCB.00773-09

    Figure Lengend Snippet: The binding of Cwc25 to the spliceosome requires Prp2 and Yju2. The spliceosome formed with biotinylated ACAC pre-mRNA was isolated by precipitation with streptavidin-Sepharose, and the components were analyzed by Western blotting. (A) Splicing was carried

    Article Snippet: Protein A-Sepharose was obtained from Amersham Inc., and streptavidin-Sepharose was obtained from Sigma-Aldrich.

    Techniques: Binding Assay, Isolation, Western Blot

    Western blot analysis of a biotin internalization assay of RSV F proteins. RSV-infected and noninfected HEp-2 cells were biotin labeled using a membrane-impermeable biotinylation reagent. The cells were shifted with or without antibodies to 37°C for 90 min to allow endocytosis. Noninternalized biotinylated surface proteins were removed by cleavage with glutathione, while internalized proteins were protected from biotin removal. After cell lysis, biotinylated proteins were immunoprecipitated using Streptavidin Mag Sepharose, separated by SDS-PAGE under nonreducing conditions, and detected with RSV F-specific antibodies. (A) Sample of noninfected cells. (B) Internalized RSV F proteins after incubation at 37°C. (C) Internalized RSV F proteins after incubation with RSV F-specific antibodies. (D) Amount of biotinylated surface RSV F proteins after cleavage with glutathione. (E) Total amount of biotinylated surface RSV F proteins before 37°C incubation step. A representative blot of a duplicate experiment is shown.

    Journal: Journal of Virology

    Article Title: Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

    doi: 10.1128/JVI.00184-17

    Figure Lengend Snippet: Western blot analysis of a biotin internalization assay of RSV F proteins. RSV-infected and noninfected HEp-2 cells were biotin labeled using a membrane-impermeable biotinylation reagent. The cells were shifted with or without antibodies to 37°C for 90 min to allow endocytosis. Noninternalized biotinylated surface proteins were removed by cleavage with glutathione, while internalized proteins were protected from biotin removal. After cell lysis, biotinylated proteins were immunoprecipitated using Streptavidin Mag Sepharose, separated by SDS-PAGE under nonreducing conditions, and detected with RSV F-specific antibodies. (A) Sample of noninfected cells. (B) Internalized RSV F proteins after incubation at 37°C. (C) Internalized RSV F proteins after incubation with RSV F-specific antibodies. (D) Amount of biotinylated surface RSV F proteins after cleavage with glutathione. (E) Total amount of biotinylated surface RSV F proteins before 37°C incubation step. A representative blot of a duplicate experiment is shown.

    Article Snippet: After lysis of the cells with radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore), biotinylated proteins were immunoprecipitated using Streptavidin Mag Sepharose (Sigma) and then SDS-PAGE under nonreducing conditions.

    Techniques: Western Blot, Infection, Labeling, Lysis, Immunoprecipitation, SDS Page, Incubation

    p120 regulates E-cadherin turnover at the cell membrane. (a) E-cadherin synthesis and processing in p120 knockdown cells. E-cadherin turnover was examined by pulse-chase analysis of parental and h p120 siRNA-expressing A431 cells. α- and β-Catenin processing from the same experiment are shown below. Chase times are indicated across the top. At chase time 0 (15 min after initiation of the pulse labeling), E-cadherin synthesis was identical in the presence and absence of p120 (a, compare E-cadherin bands). The processing of the pro- (pro-E-cad) and mature (E-cad.) forms were identical for at least 1 h. Soon thereafter, E-cadherin degradation was significantly accelerated in the absence of p120. (b) Analysis of total E-cadherin surface levels in parental and p120 knockdown A431 cells. The parental and p120 knockdown A431 cells were biotinylated for 20 min at 4°C to label surface cadherins. To specifically measure the surface levels, E-cadherin was first immunoprecipitated directly with E-cadherin mAb HECD-1. The sample was eluted with 0.5% SDS and then reprecipitated with streptavidin-coated beads to isolate the surface-labeled pool. E-cadherin levels at the surface in p120 knockdown cells (lane 1) are at least 100-fold diminished relative to the parental cells (lane 2). The result in lane 2 shows that the surface E-cadherin can be efficiently labeled (and detected) by this method. (c) Tracking the arrival of newly synthesized E-cadherin to the cell surface. The methods in a and b were combined to determine whether newly synthesized E-cadherin could transit to the cell surface in the absence of p120. The results in c were quantified by densitometry and represented graphically in d. Parental and p120 knockdown (h siRNA) cells were labeled with [ 35 S]methionine for 15 min, chased at 37°C for the times indicated across top, and placed on ice (4°C) to suspend trafficking. Cell surface proteins were immediately biotinylated at 4°C for 20 min as in b. Surface E-cadherin was then isolated as in b, and the nascent [ 35 S]methionine E-cadherin pool was visualized by SDS-PAGE and radiography. Nascent E-cadherin appeared at the surface at 30 min and peaked at 1 h. The absence of p120 had no effect on this result. Therefore, p120 is not required for E-cadherin synthesis or trafficking, but is essential to regulate E-cadherin turnover soon after its arrival at the cell surface.

    Journal: The Journal of Cell Biology

    Article Title: A core function for p120-catenin in cadherin turnover

    doi: 10.1083/jcb.200307111

    Figure Lengend Snippet: p120 regulates E-cadherin turnover at the cell membrane. (a) E-cadherin synthesis and processing in p120 knockdown cells. E-cadherin turnover was examined by pulse-chase analysis of parental and h p120 siRNA-expressing A431 cells. α- and β-Catenin processing from the same experiment are shown below. Chase times are indicated across the top. At chase time 0 (15 min after initiation of the pulse labeling), E-cadherin synthesis was identical in the presence and absence of p120 (a, compare E-cadherin bands). The processing of the pro- (pro-E-cad) and mature (E-cad.) forms were identical for at least 1 h. Soon thereafter, E-cadherin degradation was significantly accelerated in the absence of p120. (b) Analysis of total E-cadherin surface levels in parental and p120 knockdown A431 cells. The parental and p120 knockdown A431 cells were biotinylated for 20 min at 4°C to label surface cadherins. To specifically measure the surface levels, E-cadherin was first immunoprecipitated directly with E-cadherin mAb HECD-1. The sample was eluted with 0.5% SDS and then reprecipitated with streptavidin-coated beads to isolate the surface-labeled pool. E-cadherin levels at the surface in p120 knockdown cells (lane 1) are at least 100-fold diminished relative to the parental cells (lane 2). The result in lane 2 shows that the surface E-cadherin can be efficiently labeled (and detected) by this method. (c) Tracking the arrival of newly synthesized E-cadherin to the cell surface. The methods in a and b were combined to determine whether newly synthesized E-cadherin could transit to the cell surface in the absence of p120. The results in c were quantified by densitometry and represented graphically in d. Parental and p120 knockdown (h siRNA) cells were labeled with [ 35 S]methionine for 15 min, chased at 37°C for the times indicated across top, and placed on ice (4°C) to suspend trafficking. Cell surface proteins were immediately biotinylated at 4°C for 20 min as in b. Surface E-cadherin was then isolated as in b, and the nascent [ 35 S]methionine E-cadherin pool was visualized by SDS-PAGE and radiography. Nascent E-cadherin appeared at the surface at 30 min and peaked at 1 h. The absence of p120 had no effect on this result. Therefore, p120 is not required for E-cadherin synthesis or trafficking, but is essential to regulate E-cadherin turnover soon after its arrival at the cell surface.

    Article Snippet: At the end of the chase, cell surface proteins were labeled with 1 mg/ml EZ-Link sulfo-NHS-SS-biotin (Pierce Chemical Co.) at 4°C for 30 min. E-cadherin was immunoprecipitated from NP-40 cell lysates, and surface cadherin was detected by dividing the E-cadherin immunoprecipitations in half, eluting E-cadherin from the beads with 0.5% SDS, reconstituting elutions in 50 mM Tris-HCl (pH 7.5), and pulling down biotinylated E-cadherin with 10 μl per sample of packed streptavidin-coated agarose beads (Sigma-Aldrich) for 1 h at 4°C.

    Techniques: Pulse Chase, Expressing, Labeling, Immunoprecipitation, Synthesized, Isolation, SDS Page

    Schematic of poliovirus genomic RNAs containing S1 and D8 aptamer tags within the highly structured 5′NCR, for positive-strand isolation. Insertion of either the S1 streptavidin-binding or D8 Sephadex-binding aptamer sequence within three regions of the poliovirus 5′NCR resulted in the production of 12 poliovirus cDNA constructs. Tag insertions in place of S-L III (blue), in place of S-L VI (green), or at nucleotide position 702 (purple) are depicted on the left, middle, and right, respectively. Constructs for negative-strand isolation (aptamers inserted in a reverse orientation) are not shown.

    Journal: Viruses

    Article Title: Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

    doi: 10.3390/v8020039

    Figure Lengend Snippet: Schematic of poliovirus genomic RNAs containing S1 and D8 aptamer tags within the highly structured 5′NCR, for positive-strand isolation. Insertion of either the S1 streptavidin-binding or D8 Sephadex-binding aptamer sequence within three regions of the poliovirus 5′NCR resulted in the production of 12 poliovirus cDNA constructs. Tag insertions in place of S-L III (blue), in place of S-L VI (green), or at nucleotide position 702 (purple) are depicted on the left, middle, and right, respectively. Constructs for negative-strand isolation (aptamers inserted in a reverse orientation) are not shown.

    Article Snippet: In vitro transcribed RNA (11 μg) was renatured in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating at 56 °C for 5 min, 37 °C for 10 min, and incubating at room temperature for 15 min. Streptavidin-agarose (SigmaAldrich,) was prepared by washing 10 times in lysis buffer (50 mM HEPES pH 7.4, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT, 0.1% Triton X-100, 10% glycerol), Complete protease inhibitors (Roche) and resuspended to 50% slurry in lysis buffer.

    Techniques: Isolation, Binding Assay, Sequencing, Construct

    Phenotypic analyses of mCherry-tagged PbI MC1c parasite lines. A: Western blot using anti-mCherry antibodies of blood stage parasites from parasite line IMC1c/mCherry-WT (WT) and IMC1c/mCherry-Mutant 1 (M1). Positions of molecular weight markers (PageRuler™ Prestained Protein Ladder +) are shown on the right hand side. B: Confocal fluorescence and brightfield images of ookinetes (top), sporulating oocysts (middle) and sporozoites (bottom) of parasite lines IMC1a/mCherry-WT and Mutant 1. White square marks an area of the oocyst showing transversally cross-sectioned sporozoites (inset), clearly showing the cortical fluorescence. C: Western blot of blood stage parasite lysates of parasite lines IMC1c/mCherry-WT and Mutant 1 after acyl-biotin exchange in the absence (−HAM) or presence (+HAM) of hydroxylamine, before and after pull-down with streptavidin-agarose beads.

    Journal: Molecular and Biochemical Parasitology

    Article Title: Palmitoylation of Plasmodium alveolins promotes cytoskeletal function

    doi: 10.1016/j.molbiopara.2017.02.003

    Figure Lengend Snippet: Phenotypic analyses of mCherry-tagged PbI MC1c parasite lines. A: Western blot using anti-mCherry antibodies of blood stage parasites from parasite line IMC1c/mCherry-WT (WT) and IMC1c/mCherry-Mutant 1 (M1). Positions of molecular weight markers (PageRuler™ Prestained Protein Ladder +) are shown on the right hand side. B: Confocal fluorescence and brightfield images of ookinetes (top), sporulating oocysts (middle) and sporozoites (bottom) of parasite lines IMC1a/mCherry-WT and Mutant 1. White square marks an area of the oocyst showing transversally cross-sectioned sporozoites (inset), clearly showing the cortical fluorescence. C: Western blot of blood stage parasite lysates of parasite lines IMC1c/mCherry-WT and Mutant 1 after acyl-biotin exchange in the absence (−HAM) or presence (+HAM) of hydroxylamine, before and after pull-down with streptavidin-agarose beads.

    Article Snippet: Streptavidin-agarose suspension (Sigma) was added and the mixture incubated for 1 h at room temperature with nutation to allow binding to and subsequent pulldown of the biotinylated protein fraction.

    Techniques: Western Blot, Mutagenesis, Molecular Weight, Fluorescence

    IQGAP1 knockdown inhibits TGF-β1 downregulation of TβRII, TβRII ubiquitination, and the plasma membrane targeting of SMURF1. ( A ) Top: HSCs with their cell-surface proteins prelabeled with biotin were incubated with TGF-1 for indicated times and cells were harvested for streptavidin pull-down and TβRII WB to determine internalized TβRII. Bottom: TβRII degradation curves generated by densitometric analysis are shown. IQGAP1 knockdown inhibited TGF-β1 downregulation of cell surface TβRII. Chlo, chloroquine; Tν, half-life of TβRII. Data are representative of multiple independent experiments. Asterisks designate a point where TβRII was down to 50%. ( B ) HSCs expressing TβRI-FLAG were transduced with lentiviruses encoding either NT shRNA or IQGAP1 shRNA, and TβRI protein levels were detected by Flag WB. IQGAP1 knockdown increased TβRI-Flag in HSCs. n = 3 experiments with similar results. ( C ) TβRII-HA was precipitated from HSCs by IP using anti-HA; TβRII ubiquitination was detected by WB. IQGAP1 knockdown markedly inhibited TβRII ubiquitination. ( D ) Double IF for IQGAP1 (red) and SMURF1 (green) revealed that IQGAP1 and SMURF1 colocalized at the periphery plasma membrane in control cells (arrows, upper panels), and that IQGAP1 knockdown reduced the localization of SMURF1 at the plasma membrane (lower panels). Scale bar: 20 μm. ( E ) TβRII and SMURF1 colocalized at the peripheral plasma membrane (arrowheads, upper panels), and IQGAP1 knockdown reduced TβRII/SMURF1 colocalization at the plasma membrane (lower panels). Scale bar: 20 μm. ( F ) IQGAP1 knockdown reduced SMURF1 protein levels in HSCs by WB. β-actin WB was used as a loading control. n = 3 independent experiments with identical results.

    Journal: The Journal of Clinical Investigation

    Article Title: IQGAP1 suppresses T?RII-mediated myofibroblastic activation and metastatic growth in liver

    doi: 10.1172/JCI63836

    Figure Lengend Snippet: IQGAP1 knockdown inhibits TGF-β1 downregulation of TβRII, TβRII ubiquitination, and the plasma membrane targeting of SMURF1. ( A ) Top: HSCs with their cell-surface proteins prelabeled with biotin were incubated with TGF-1 for indicated times and cells were harvested for streptavidin pull-down and TβRII WB to determine internalized TβRII. Bottom: TβRII degradation curves generated by densitometric analysis are shown. IQGAP1 knockdown inhibited TGF-β1 downregulation of cell surface TβRII. Chlo, chloroquine; Tν, half-life of TβRII. Data are representative of multiple independent experiments. Asterisks designate a point where TβRII was down to 50%. ( B ) HSCs expressing TβRI-FLAG were transduced with lentiviruses encoding either NT shRNA or IQGAP1 shRNA, and TβRI protein levels were detected by Flag WB. IQGAP1 knockdown increased TβRI-Flag in HSCs. n = 3 experiments with similar results. ( C ) TβRII-HA was precipitated from HSCs by IP using anti-HA; TβRII ubiquitination was detected by WB. IQGAP1 knockdown markedly inhibited TβRII ubiquitination. ( D ) Double IF for IQGAP1 (red) and SMURF1 (green) revealed that IQGAP1 and SMURF1 colocalized at the periphery plasma membrane in control cells (arrows, upper panels), and that IQGAP1 knockdown reduced the localization of SMURF1 at the plasma membrane (lower panels). Scale bar: 20 μm. ( E ) TβRII and SMURF1 colocalized at the peripheral plasma membrane (arrowheads, upper panels), and IQGAP1 knockdown reduced TβRII/SMURF1 colocalization at the plasma membrane (lower panels). Scale bar: 20 μm. ( F ) IQGAP1 knockdown reduced SMURF1 protein levels in HSCs by WB. β-actin WB was used as a loading control. n = 3 independent experiments with identical results.

    Article Snippet: Streptavidin-agarose pull-down (S1638; Sigma-Aldrich) followed by WB for TβRII was used to quantitate TβRII that was internalized and spared from degradation.

    Techniques: Incubation, Western Blot, Generated, Expressing, Transduction, shRNA

    HER3 can associate with the cyclin D1 promoter. A. Nuclear EGFR and HER2 associate with the cyclin D1 promoter. ChIP using an anti-EGFR, anti-HER2, or normal rabbit IgG antibody was performed with SKBr3 cells and isolated DNA was subsequently used for qPCR with primers flanking the 122 bp cyclin D1 promoter region (n = 3). qPCR specificity for the 122 bp cyclin D1 promoter was confirmed by agarose gel electrophoresis of semi-qPCR products. DAPA analysis was performed using a biotinylated 122 bp cyclin D1 promoter probe incubated with 400 ug of nuclear lysate harvested from SKBr3 cells. Bound proteins were isolated with streptavidin agarose beads and subsequently fractionated on SDS-PAGE followed by immunoblotting for EGFR or HER2. Nuclear lysate incubated with beads only lacked association. B. Nuclear HER3 can associate with the cyclin D1 promoter. ChIP using a N-TERM anti-HER3 or a human IgG antibody was performed with H226 R , SKBr3, and BT549 cells. qPCR was performed as in 5A. C. HER3 can associate with a cyclin D1 promoter probe. DAPA analysis was performed using nuclear lysate harvested from H226 R , SKBr3, MCF-7, and BT549 cells as in 5A. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. D. HER3 association with the cyclin D1 promoter probe is specific. DAPA analysis was performed as in 5A using nuclear lysate harvested from H226 R , SKBr3, MCF-7, and BT549 cells transfected with either non-targeting (NT) or HER3 siRNA for 48 hr. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. All data points for ChIP are represented as mean +/− s.e.m and normalized to the IgG control. P

    Journal: PLoS ONE

    Article Title: Mapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3

    doi: 10.1371/journal.pone.0071518

    Figure Lengend Snippet: HER3 can associate with the cyclin D1 promoter. A. Nuclear EGFR and HER2 associate with the cyclin D1 promoter. ChIP using an anti-EGFR, anti-HER2, or normal rabbit IgG antibody was performed with SKBr3 cells and isolated DNA was subsequently used for qPCR with primers flanking the 122 bp cyclin D1 promoter region (n = 3). qPCR specificity for the 122 bp cyclin D1 promoter was confirmed by agarose gel electrophoresis of semi-qPCR products. DAPA analysis was performed using a biotinylated 122 bp cyclin D1 promoter probe incubated with 400 ug of nuclear lysate harvested from SKBr3 cells. Bound proteins were isolated with streptavidin agarose beads and subsequently fractionated on SDS-PAGE followed by immunoblotting for EGFR or HER2. Nuclear lysate incubated with beads only lacked association. B. Nuclear HER3 can associate with the cyclin D1 promoter. ChIP using a N-TERM anti-HER3 or a human IgG antibody was performed with H226 R , SKBr3, and BT549 cells. qPCR was performed as in 5A. C. HER3 can associate with a cyclin D1 promoter probe. DAPA analysis was performed using nuclear lysate harvested from H226 R , SKBr3, MCF-7, and BT549 cells as in 5A. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. D. HER3 association with the cyclin D1 promoter probe is specific. DAPA analysis was performed as in 5A using nuclear lysate harvested from H226 R , SKBr3, MCF-7, and BT549 cells transfected with either non-targeting (NT) or HER3 siRNA for 48 hr. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. All data points for ChIP are represented as mean +/− s.e.m and normalized to the IgG control. P

    Article Snippet: Following annealing of Sense and Anti-sense oligonucleotdies for 1 hr at 95°C, 4 ug of annealed biotinylated probe was incubated with 500 ug of nuclear cell lysate, and 40 ul of streptavidin-agarose bead suspension (Sigma) in PBS diluted with 1 mM Na3 VO4 , 1 mM PMSF, 1 mM BGP, 10 ug/ml of leupeptin and aprotinin for 2–3 hr rocking at room temperature.

    Techniques: Chromatin Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Incubation, SDS Page, Transfection

    Palmitoylation-deficient mutants of Furin and PC7 are not active in cleaving anthrax toxin because they do not preferentially reside in microdomains. ( A ) RPE-1 cells were transfected with WT or palmitoylation-deficient mutants of Furin (shown) or PC7 (in SI Appendix , Fig. S5 A ). The cells were biotinylated for 30 min and then quenched, washed, and lysed. The surface fraction was pulled down using streptavidin beads. SDS/PAGE was performed on TCE and pull-down (PD) fractions with the percent of TCE shown. Statistics : unpaired two-tailed t test. ( B ) As in A , RPE-1 cells were transfected with V5-tagged Furin (shown) or PC7 (shown in SI Appendix , Fig. S5 B ) after being silenced for ZDHHC5 or not (Control). Then, the surface proteins were labeled with biotin and isolated. Statistics : unpaired two-tailed t test. ( C ) Analytical flow cytometry was performed as described in Fig. 1 but with Furin overexpression (using an antibody against V5 to select for transfected cells) or not in cells silenced for Control (Ctrl) or ZDHHC5 (Z5). The shift in MEK cleavage between no toxin (−TOX, black) and toxin (+TOX, red) is noted by the bar on top of the graph. Quantification of recomplementation flow cytometry peaks as in C with overexpression of both Furin and PC7, WT or palmitoylation-deficient mutants (ΔPalm), in ZDHHC5- ( D ), Control- ( E ), and F/PC7- ( F ) silenced cells. Statistics : paired two-tailed t test on the original data. ( G ) OptiPrep ultracentrifugation gradients were used to probe presence of Furin, WT or CS mutant in DRMs. Representative blot with DRM control (caveolin 1) and non-DRM control (GAPDH) for Furin (shown) or PC7 (shown in SI Appendix , Fig. S5 G ). Statistics : ratio paired two-tailed t test on the original data. * P

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

    Article Title: Anthrax toxin requires ZDHHC5-mediated palmitoylation of its surface-processing host enzymes

    doi: 10.1073/pnas.1812588116

    Figure Lengend Snippet: Palmitoylation-deficient mutants of Furin and PC7 are not active in cleaving anthrax toxin because they do not preferentially reside in microdomains. ( A ) RPE-1 cells were transfected with WT or palmitoylation-deficient mutants of Furin (shown) or PC7 (in SI Appendix , Fig. S5 A ). The cells were biotinylated for 30 min and then quenched, washed, and lysed. The surface fraction was pulled down using streptavidin beads. SDS/PAGE was performed on TCE and pull-down (PD) fractions with the percent of TCE shown. Statistics : unpaired two-tailed t test. ( B ) As in A , RPE-1 cells were transfected with V5-tagged Furin (shown) or PC7 (shown in SI Appendix , Fig. S5 B ) after being silenced for ZDHHC5 or not (Control). Then, the surface proteins were labeled with biotin and isolated. Statistics : unpaired two-tailed t test. ( C ) Analytical flow cytometry was performed as described in Fig. 1 but with Furin overexpression (using an antibody against V5 to select for transfected cells) or not in cells silenced for Control (Ctrl) or ZDHHC5 (Z5). The shift in MEK cleavage between no toxin (−TOX, black) and toxin (+TOX, red) is noted by the bar on top of the graph. Quantification of recomplementation flow cytometry peaks as in C with overexpression of both Furin and PC7, WT or palmitoylation-deficient mutants (ΔPalm), in ZDHHC5- ( D ), Control- ( E ), and F/PC7- ( F ) silenced cells. Statistics : paired two-tailed t test on the original data. ( G ) OptiPrep ultracentrifugation gradients were used to probe presence of Furin, WT or CS mutant in DRMs. Representative blot with DRM control (caveolin 1) and non-DRM control (GAPDH) for Furin (shown) or PC7 (shown in SI Appendix , Fig. S5 G ). Statistics : ratio paired two-tailed t test on the original data. * P

    Article Snippet: The rest of the lysate was incubated with prewashed streptavidin-coupled beads (Sigma S1638) overnight at 4 °C.

    Techniques: Transfection, SDS Page, Two Tailed Test, Labeling, Isolation, Flow Cytometry, Cytometry, Over Expression, Mutagenesis

    MARF1 interacts with the DCP1:DCP2 mRNA decapping complex. ( A ) Schematic diagram of full-length MARF1. ( B ) Dot plot depicting high-confidence protein interactions identified by affinity purification of FLAG-MARF1 in HEK293 cells. SAINT analysis of two independent experiments was performed and a subset of high-confident preys is presented in this dot plot. Node color represents the average spectral counts, and the node edge color corresponds to the SAINTexpress Bayesian FDR value (BFDR). ( C ) Western blot analysis of lysates derived from HEK293 cells expressing either FLAG-BirA* or FLAG-BirA*-MARF1 and probed with anti-FLAG antibody. ( D ) Immunoprecipitation (IP) of FLAG-BirA* and FLAG-BirA*-MARF1 from benzonase–treated HEK293 cell extracts using anti-FLAG antibody. Immunoprecipitated complexes were separated by SDS-PAGE and probed with antibodies against the indicated proteins. ( E ) Streptavidin pulldowns of biotinylated proteins from benzonase-treated lysates outlined in (C). Precipitated proteins were subjected to SDS-PAGE and probed with antibodies against the indicated endogenous proteins. Inputs represent 2% of total lysates.

    Journal: Nucleic Acids Research

    Article Title: Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs

    doi: 10.1093/nar/gky1011

    Figure Lengend Snippet: MARF1 interacts with the DCP1:DCP2 mRNA decapping complex. ( A ) Schematic diagram of full-length MARF1. ( B ) Dot plot depicting high-confidence protein interactions identified by affinity purification of FLAG-MARF1 in HEK293 cells. SAINT analysis of two independent experiments was performed and a subset of high-confident preys is presented in this dot plot. Node color represents the average spectral counts, and the node edge color corresponds to the SAINTexpress Bayesian FDR value (BFDR). ( C ) Western blot analysis of lysates derived from HEK293 cells expressing either FLAG-BirA* or FLAG-BirA*-MARF1 and probed with anti-FLAG antibody. ( D ) Immunoprecipitation (IP) of FLAG-BirA* and FLAG-BirA*-MARF1 from benzonase–treated HEK293 cell extracts using anti-FLAG antibody. Immunoprecipitated complexes were separated by SDS-PAGE and probed with antibodies against the indicated proteins. ( E ) Streptavidin pulldowns of biotinylated proteins from benzonase-treated lysates outlined in (C). Precipitated proteins were subjected to SDS-PAGE and probed with antibodies against the indicated endogenous proteins. Inputs represent 2% of total lysates.

    Article Snippet: Lysates were sonicated and incubated with streptavidin-coupled Agarose (Millipore) for 3 h at 4°C to capture biotinylated proteins.

    Techniques: Affinity Purification, Western Blot, Derivative Assay, Expressing, Immunoprecipitation, SDS Page

    PRMT5 peptide pull-downs confirm an in vitro interaction with NHERF2. A , GST-fused PDZ domains of GRIP1 (residues 672–754), MPP7 (residues 139–220), PDZ-LIM55 (residues 2–85), NHERF1 FL (residues 1–355), NHERF2 FL (residues 1–337), SCRIB (residues 714–801), PDZ-LIM2 (residues 1–84), and GST were incubated with biotinylated PRMT5 C terminus unphosphorylated peptide. Bound proteins were detected with α-GST antibody (short and long exposure are shown). Peptide loading was assessed with HRP-conjugated streptavidin ( SA-HRP ). The Coomassie stain demonstrates roughly equal input of the GST fusion proteins. B , schematic representation of the constructs used for peptide pull-down in C. C , purified recombinant GST, GST-tagged human NHERF2 full-length (NHERF2-PDZ 1–2), PDZ1 (amino acids 1–152), PDZ2 (amino acids 107–337), and 14-3-3ϵ were incubated with biotinylated PRMT5 C terminus unphosphorylated and Thr-634-phosphorylated peptides bound to streptavidin-agarose beads and detected by α-GST. Left lane , inputs of the GST fusion proteins. D , 293T cells were transfected with constructs expressing GFP-14-3-3ϵ and Myc-PRMT5 wild type or T634A mutant. Cell lysates were then incubated with normal mouse IgG or α-Myc antibody. Immunocomplexes were captured by Protein A beads and detected by either α-Myc or α-GFP. IB , immunoblotting.

    Journal: The Journal of Biological Chemistry

    Article Title: PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch *

    doi: 10.1074/jbc.M116.760330

    Figure Lengend Snippet: PRMT5 peptide pull-downs confirm an in vitro interaction with NHERF2. A , GST-fused PDZ domains of GRIP1 (residues 672–754), MPP7 (residues 139–220), PDZ-LIM55 (residues 2–85), NHERF1 FL (residues 1–355), NHERF2 FL (residues 1–337), SCRIB (residues 714–801), PDZ-LIM2 (residues 1–84), and GST were incubated with biotinylated PRMT5 C terminus unphosphorylated peptide. Bound proteins were detected with α-GST antibody (short and long exposure are shown). Peptide loading was assessed with HRP-conjugated streptavidin ( SA-HRP ). The Coomassie stain demonstrates roughly equal input of the GST fusion proteins. B , schematic representation of the constructs used for peptide pull-down in C. C , purified recombinant GST, GST-tagged human NHERF2 full-length (NHERF2-PDZ 1–2), PDZ1 (amino acids 1–152), PDZ2 (amino acids 107–337), and 14-3-3ϵ were incubated with biotinylated PRMT5 C terminus unphosphorylated and Thr-634-phosphorylated peptides bound to streptavidin-agarose beads and detected by α-GST. Left lane , inputs of the GST fusion proteins. D , 293T cells were transfected with constructs expressing GFP-14-3-3ϵ and Myc-PRMT5 wild type or T634A mutant. Cell lysates were then incubated with normal mouse IgG or α-Myc antibody. Immunocomplexes were captured by Protein A beads and detected by either α-Myc or α-GFP. IB , immunoblotting.

    Article Snippet: 15 μg of biotin-labeled peptides were immobilized on streptavidin-agarose beads (Sigma) in peptide-binding buffer (50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 1 m m EDTA, 2 m m dithiothreitol, 0.5% Nonidet P-40) overnight at 4 °C and washed three times with binding buffer to remove unbound peptides.

    Techniques: In Vitro, Incubation, Staining, Construct, Purification, Recombinant, Transfection, Expressing, Mutagenesis

    Surface biotinylation results show that KChIP4a KISD is transmembrane where N2, but not E4, is exposed on the HEK293T cell surface. A , streptavidin pull-down of surface biotinylated KChIP4a wild-type and N-terminal cysteine mutants (N2C, E4C, and N2C/E4C).

    Journal:

    Article Title: Multiple Kv Channel-interacting Proteins Contain an N-terminal Transmembrane Domain That Regulates Kv4 Channel Trafficking and Gating *

    doi: 10.1074/jbc.M806852200

    Figure Lengend Snippet: Surface biotinylation results show that KChIP4a KISD is transmembrane where N2, but not E4, is exposed on the HEK293T cell surface. A , streptavidin pull-down of surface biotinylated KChIP4a wild-type and N-terminal cysteine mutants (N2C, E4C, and N2C/E4C).

    Article Snippet: Equilibrated streptavidin-agarose beads (50 μl; Novagen) were added to the lysate, and the mixture was incubated overnight at 4 °C with constant mixing.

    Techniques:

    Binding of unmodified and acetylated WRN to 2-stranded fork and 4-stranded replication fork substrates a) As described in Methods, individual reactions containing equimolar amounts of unmodified (lanes 1–3) or acetylated (lanes 4–6) FLAG-tagged WRN were incubated without or with biotin-tagged 2-stranded fork or 4-stranded replication fork substrate (2 pmol) as indicated and added to streptavidin-agarose beads to bind biotin-tagged DNA. After washing away unbound WRN, bound proteins were released, separated by SDS-PAGE, detected by Western blotting using anti-WRN antibody, and visualized using chemiluminescence. b) For experiments performed as in A , signal intensity for amounts of bound WRN in DNA-containing samples was quantified and normalized to signal intensity for unmodified WRN bound to the 2-stranded fork. Data are mean and standard deviation from 5 independent experiments

    Journal: Biogerontology

    Article Title: Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    doi: 10.1007/s10522-014-9506-3

    Figure Lengend Snippet: Binding of unmodified and acetylated WRN to 2-stranded fork and 4-stranded replication fork substrates a) As described in Methods, individual reactions containing equimolar amounts of unmodified (lanes 1–3) or acetylated (lanes 4–6) FLAG-tagged WRN were incubated without or with biotin-tagged 2-stranded fork or 4-stranded replication fork substrate (2 pmol) as indicated and added to streptavidin-agarose beads to bind biotin-tagged DNA. After washing away unbound WRN, bound proteins were released, separated by SDS-PAGE, detected by Western blotting using anti-WRN antibody, and visualized using chemiluminescence. b) For experiments performed as in A , signal intensity for amounts of bound WRN in DNA-containing samples was quantified and normalized to signal intensity for unmodified WRN bound to the 2-stranded fork. Data are mean and standard deviation from 5 independent experiments

    Article Snippet: Reactions were subsequently incubated with constant mixing with 20 ul of pre-equilibrated Streptavidin-Agarose beads (Sigma) for 30 min at 25°C.

    Techniques: Binding Assay, Incubation, SDS Page, Western Blot, Standard Deviation

    MuSK antibodies specifically activate MuSK and the downstream cascade. Specific phosphorylation of MuSK and AChR β subunit (AChRβ) by MuSK antibodies. C2C12 myotubes were treated with agrin or MuSK antibodies for 30 minutes and then immunoprecipitated with ( A ) MuSK antibodies or ( B ) biotinylated BTX using streptavidin-Sepharose. Immunoblots of immunoprecipitates were probed with antibodies to MuSK ( A ), AChRβ ( B ), or phosphotyrosine (PY, A and B ). Both MuSK and AChRβ were phosphorylated after the treatment of agrin or MuSK antibodies. Phosphorylation of MuSK and AChRβ was inhibited by the absorption of MuSK antibodies with MuSK-AP protein.

    Journal: Journal of Clinical Investigation

    Article Title: Induction of myasthenia by immunization against muscle-specific kinase

    doi: 10.1172/JCI21545

    Figure Lengend Snippet: MuSK antibodies specifically activate MuSK and the downstream cascade. Specific phosphorylation of MuSK and AChR β subunit (AChRβ) by MuSK antibodies. C2C12 myotubes were treated with agrin or MuSK antibodies for 30 minutes and then immunoprecipitated with ( A ) MuSK antibodies or ( B ) biotinylated BTX using streptavidin-Sepharose. Immunoblots of immunoprecipitates were probed with antibodies to MuSK ( A ), AChRβ ( B ), or phosphotyrosine (PY, A and B ). Both MuSK and AChRβ were phosphorylated after the treatment of agrin or MuSK antibodies. Phosphorylation of MuSK and AChRβ was inhibited by the absorption of MuSK antibodies with MuSK-AP protein.

    Article Snippet: The cells were then washed with cold phosphate-buffered saline, followed by lysis and precipitation with streptavidin-coupled agarose beads (Sigma-Aldrich).

    Techniques: Immunoprecipitation, Western Blot

    N63Y mutant CVB3 has a growth defect in cell culture and reduced glycan-mediated cell attachment. Single-cycle assays of viral replication in HeLa (A) and Huh7 (B) cells were performed. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. n = 3. (C) Cell attachment of 35 S-labeled WT or N63Y mutant CVB3. Virus was incubated with cells at 4°C for 40 min. Cells were washed and trypsinized, and cell-associated 35 S was quantified. (D) 35 S-labeled WT or N63Y mutant CVB3 was incubated with CHO cells (CHO-K1, pgsA745, pgsD677, and pgsB761) that vary in GAG expression. Plus and minus signs indicate the relative levels of GAGs on the cell surface. (E) Effect of heparinase treatment on CVB3 cell attachment. Huh7 cells were treated with or without heparinase I for 90 min prior to quantification of 35 S CVB3 attachment. n = 7. (F) Heparin-agarose pulldown assay. 35 S-labeled WT or N63Y mutant CVB3 was incubated with heparin-agarose resin or streptavidin-agarose resin (control). Resin was washed, and bound 35 S-labeled CVB3 was quantified. n = 3. *, P

    Journal: mBio

    Article Title: Emergence of a Large-Plaque Variant in Mice Infected with Coxsackievirus B3

    doi: 10.1128/mBio.00119-16

    Figure Lengend Snippet: N63Y mutant CVB3 has a growth defect in cell culture and reduced glycan-mediated cell attachment. Single-cycle assays of viral replication in HeLa (A) and Huh7 (B) cells were performed. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. n = 3. (C) Cell attachment of 35 S-labeled WT or N63Y mutant CVB3. Virus was incubated with cells at 4°C for 40 min. Cells were washed and trypsinized, and cell-associated 35 S was quantified. (D) 35 S-labeled WT or N63Y mutant CVB3 was incubated with CHO cells (CHO-K1, pgsA745, pgsD677, and pgsB761) that vary in GAG expression. Plus and minus signs indicate the relative levels of GAGs on the cell surface. (E) Effect of heparinase treatment on CVB3 cell attachment. Huh7 cells were treated with or without heparinase I for 90 min prior to quantification of 35 S CVB3 attachment. n = 7. (F) Heparin-agarose pulldown assay. 35 S-labeled WT or N63Y mutant CVB3 was incubated with heparin-agarose resin or streptavidin-agarose resin (control). Resin was washed, and bound 35 S-labeled CVB3 was quantified. n = 3. *, P

    Article Snippet: 35 S-labeled CVB3 (5 × 106 PFU/5,000 cpm) was mixed with heparin agarose resin (Sigma Aldrich) or streptavidin agarose resin (Sigma Aldrich).

    Techniques: Mutagenesis, Cell Culture, Cell Attachment Assay, Plaque Assay, Labeling, Incubation, Expressing

    DDX3 enhances HNF4 binding to the MTP promoter. ( a ) Western blot analysis of subcellular localization of DDX3. HuH7 cells were transfected with plasmid expressing HA-HNF4 alone or together with HA-DDX3 expression construct as indicated. After 48 hr, cytosolic fractions and nuclear extracts (20 μg of each) were prepared and subjected to SDS-PAGE then immunoblotting with anti-HA antibody. ( b ) HNF4 DNA binding ability is enhanced by DDX3. The nuclear extracts of transfected HuH7 cells were incubated with annealed biotinylated probe containing HNF4-responsive element and subjected to binding with streptavidin agarose. After extensive washing, the bound fractions were analyzed by SDS-PAGE, followed by immunoblotting with anti-HA antibody. ( c ) Western blot analysis of the ectopically expressed Flag-HNF4 and HA-DDX3. HuH7 cells were transfected with plasmids expressing HA-DDX3 and Flag-HNF4 as indicated. Western blot analysis was performed 48 hr posttransfection. The expression level of HA-DDX3 and Flag-HNF4 was detected using anti-HA and anti-Flag antibodies, respectively. ( d ) Overexpression of DDX3 enhances the DNA binding activity of Flag-HNF4 on MTP promoter. Chromatin immunoprecipitation assay was performed by isolation of soluble chromatin fragments prepared from transfected-HuH7 cells and then immunoprecipitated with anti-FLAG M2 agarose resins. Immunoprecipitates were analyzed by quantitative real-time PCR with specific primers for MTP promoter (as described in Materials and Methods). Results are expressed as means ± S.D. for at least three independent experiments and ** p

    Journal: Scientific Reports

    Article Title: RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

    doi: 10.1038/srep41452

    Figure Lengend Snippet: DDX3 enhances HNF4 binding to the MTP promoter. ( a ) Western blot analysis of subcellular localization of DDX3. HuH7 cells were transfected with plasmid expressing HA-HNF4 alone or together with HA-DDX3 expression construct as indicated. After 48 hr, cytosolic fractions and nuclear extracts (20 μg of each) were prepared and subjected to SDS-PAGE then immunoblotting with anti-HA antibody. ( b ) HNF4 DNA binding ability is enhanced by DDX3. The nuclear extracts of transfected HuH7 cells were incubated with annealed biotinylated probe containing HNF4-responsive element and subjected to binding with streptavidin agarose. After extensive washing, the bound fractions were analyzed by SDS-PAGE, followed by immunoblotting with anti-HA antibody. ( c ) Western blot analysis of the ectopically expressed Flag-HNF4 and HA-DDX3. HuH7 cells were transfected with plasmids expressing HA-DDX3 and Flag-HNF4 as indicated. Western blot analysis was performed 48 hr posttransfection. The expression level of HA-DDX3 and Flag-HNF4 was detected using anti-HA and anti-Flag antibodies, respectively. ( d ) Overexpression of DDX3 enhances the DNA binding activity of Flag-HNF4 on MTP promoter. Chromatin immunoprecipitation assay was performed by isolation of soluble chromatin fragments prepared from transfected-HuH7 cells and then immunoprecipitated with anti-FLAG M2 agarose resins. Immunoprecipitates were analyzed by quantitative real-time PCR with specific primers for MTP promoter (as described in Materials and Methods). Results are expressed as means ± S.D. for at least three independent experiments and ** p

    Article Snippet: In vitro biotinylated probe binding assay The WT (5′-CAGTGAACTTAGGTCCTGATT-3′) and mutant form (5′-CAGTG TTG TTAGGTCCTGATT-3′) of the 5′ biotinylated probes containing HNF4 responsive element were annealed with their complementary oligonucleotides in 20 μl of annealing buffer (40 mM Tris-HCl pH7.5, 20 mM MgCl2 and 50 mM NaCl) at 80 °C for 5 min. Nuclear extracts (300 μg) of transfected HuH7 cells were precleared with streptavidin-agarose resins (Sigma) and then incubated with 125 mg/ml poly(dI.dC) (Sigma) at 4 °C for 30 min in 300 μl binding buffer (18 mM HEPES pH 7.9, 40 mM KCl, 2 mM MgCl2 , 10 mM DTT and protease inhibitor).

    Techniques: Binding Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Construct, SDS Page, Incubation, Over Expression, Activity Assay, Chromatin Immunoprecipitation, Isolation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Specific binding of RAG-2 to histone H3 containing di- or trimethylated lysine 4. (A) Whole-cell lysates of 293T cells expressing full-length, wild-type RAG-2 were incubated with streptavidin bead-bound, biotinylated peptides corresponding to residues

    Journal: Immunity

    Article Title: A PHD FINGER DOMAIN IN RAG-2 THAT BINDS HYPERMETHYLATED LYSINE 4 OF HISTONE H3 IS NECESSARY FOR EFFICIENT V(D)J REARRANGEMENT

    doi: 10.1016/j.immuni.2007.09.005

    Figure Lengend Snippet: Specific binding of RAG-2 to histone H3 containing di- or trimethylated lysine 4. (A) Whole-cell lysates of 293T cells expressing full-length, wild-type RAG-2 were incubated with streptavidin bead-bound, biotinylated peptides corresponding to residues

    Article Snippet: The diluted supernatant, containing 300 µg protein, was precleared with 50 µl of a 50% streptavidin agarose slurry (Novagen) and incubated with 5 µg peptide prebound to streptavidin agarose for 4 hr at 4°C.

    Techniques: Binding Assay, Expressing, Incubation

    Sam68 regulates alternative splicing of Sgce exon 8 in male germ cells. RT–PCR analysis of Sgce exon 8 inclusion in total RNA extracted from Sam68 wild-type or knockout testes at 8, 16 and 30 days post partum (dpp) ( A ) or from isolated wild-type or knockout spermatocytes and spermatids. ( B ) Bands corresponding to mRNAs containing or not, exon 8 are indicated on the right of the panels. ( C and D ) Chromatin immunoprecipitation (ChIP) analysis of Sam68 wild-type and knockout germ cells. Sonicated chromatin (100 µg of DNA/sample) was immunoprecipitated with 2µg of H5, H14 antibodies or control rabbit IgGs and co-precipitated DNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( E ) CLIP analysis of the binding of Sam68 to Sgce pre-mRNA. After UV crosslink, Sam68 wild-type and knockout germ cell extracts were sonicated and treated with DNase and RNase to yield RNA fragments of ∼200 to 250 nt. Wild-type and knockout germ cell extracts were immunoprecipitated with 2 µg of rabbit IgGs or anti-Sam68 antibodies and co-precipitated RNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( F ) Electrophoretic mobility shift assays (EMSAs) of the binding of purified GST-Sam68 1–277 to a labelled Sgce probe containing the sequences encoded at the intron 7/exon 8 boundary. The position of the free probe and the probe complexed with GST-Sam68 1–277 are shown on the left side. Competition with the wild-type (middle panel) or mutated (right panel) cold probes is shown. The scheme above the gels shows the wild-type and mutated sequence used in the EMSAs. The mutated bases that interfere with the Sam68 consensus are shown in violet. ( G ) The western blot analysis of RNA pulldown assay of U2AF65 binding to Sgce exon 8. Biotinylated RNAs encoding the Sgce wild-type and mutated exon 8 sequences (from −32 to +63) were bound to Streptavidine agarose beads and incubated with testicular nuclear extract (100 µg) in the presence of 1 µg of purified GST or GST-Sam68 1–277, as indicated.

    Journal: Nucleic Acids Research

    Article Title: Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells

    doi: 10.1093/nar/gkr085

    Figure Lengend Snippet: Sam68 regulates alternative splicing of Sgce exon 8 in male germ cells. RT–PCR analysis of Sgce exon 8 inclusion in total RNA extracted from Sam68 wild-type or knockout testes at 8, 16 and 30 days post partum (dpp) ( A ) or from isolated wild-type or knockout spermatocytes and spermatids. ( B ) Bands corresponding to mRNAs containing or not, exon 8 are indicated on the right of the panels. ( C and D ) Chromatin immunoprecipitation (ChIP) analysis of Sam68 wild-type and knockout germ cells. Sonicated chromatin (100 µg of DNA/sample) was immunoprecipitated with 2µg of H5, H14 antibodies or control rabbit IgGs and co-precipitated DNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( E ) CLIP analysis of the binding of Sam68 to Sgce pre-mRNA. After UV crosslink, Sam68 wild-type and knockout germ cell extracts were sonicated and treated with DNase and RNase to yield RNA fragments of ∼200 to 250 nt. Wild-type and knockout germ cell extracts were immunoprecipitated with 2 µg of rabbit IgGs or anti-Sam68 antibodies and co-precipitated RNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( F ) Electrophoretic mobility shift assays (EMSAs) of the binding of purified GST-Sam68 1–277 to a labelled Sgce probe containing the sequences encoded at the intron 7/exon 8 boundary. The position of the free probe and the probe complexed with GST-Sam68 1–277 are shown on the left side. Competition with the wild-type (middle panel) or mutated (right panel) cold probes is shown. The scheme above the gels shows the wild-type and mutated sequence used in the EMSAs. The mutated bases that interfere with the Sam68 consensus are shown in violet. ( G ) The western blot analysis of RNA pulldown assay of U2AF65 binding to Sgce exon 8. Biotinylated RNAs encoding the Sgce wild-type and mutated exon 8 sequences (from −32 to +63) were bound to Streptavidine agarose beads and incubated with testicular nuclear extract (100 µg) in the presence of 1 µg of purified GST or GST-Sam68 1–277, as indicated.

    Article Snippet: The 23 dpp testis nuclear extract (100 µg) were pre-cleared on Streptavidine agarose beads (Sigma-Aldrich) in the presence of 1 µg of purified GST or GST-Sam681–277, 0.01% BSA and yeast tRNA for 90 min at 4°C under rotation and then incubated for 30 min at 30°C with Streptavidine beads pre-adsorbed with 500 ng of biotinylated RNA, 0.01% BSA and yeast tRNA.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Knock-Out, Isolation, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Real-time Polymerase Chain Reaction, Cross-linking Immunoprecipitation, Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Sequencing, Western Blot, Incubation

    P cr -400 interacts with the coiled-coil of MBP-Fishhook. (A) MBP (lanes 2 and 3), MBP-Hairpin (lanes 4 and 5), or MBP-Fishhook (lanes 6 and 7) was incubated without (−) or with (+) P cr -400, and the complexes were analyzed by native PAGE and stained with Coomasie blue. P cr -400 induces a shift in the electrophoretic mobility of the trimeric MBP-Fishhook (T F ), causing it to migrate similarly to trimeric MBP-Hairpin (T H ). A, aggregate. Lane M, molecular-mass markers. (B) Increasing amounts of P cr -400 were incubated with MBP-Fishhook, and the complexes were analyzed by native PAGE. Lanes 2 and 3, MBP-Hairpin and MBP-Fishhook controls. The amount of MBP-Fishhook (T F ) shifting to a complex that mimics MBP-Hairpin (T H ) increases with the amount of P cr -400 added (lanes 4 to 12). (C) Increasing amounts of biotinylated P cr -400 were incubated with MBP-Fishhook (lanes 4 to 8). As a control, MBP was also incubated with biotinylated P cr -400 (lane 9). Streptavidin-agarose was then added to the reaction mixture, and the bound complexes were precipitated, heat denatured, and analyzed by SDS-PAGE.

    Journal: Journal of Virology

    Article Title: An Antiviral Peptide Targets a Coiled-Coil Domain of the Human T-Cell Leukemia Virus Envelope Glycoprotein

    doi: 10.1128/JVI.77.5.3281-3290.2003

    Figure Lengend Snippet: P cr -400 interacts with the coiled-coil of MBP-Fishhook. (A) MBP (lanes 2 and 3), MBP-Hairpin (lanes 4 and 5), or MBP-Fishhook (lanes 6 and 7) was incubated without (−) or with (+) P cr -400, and the complexes were analyzed by native PAGE and stained with Coomasie blue. P cr -400 induces a shift in the electrophoretic mobility of the trimeric MBP-Fishhook (T F ), causing it to migrate similarly to trimeric MBP-Hairpin (T H ). A, aggregate. Lane M, molecular-mass markers. (B) Increasing amounts of P cr -400 were incubated with MBP-Fishhook, and the complexes were analyzed by native PAGE. Lanes 2 and 3, MBP-Hairpin and MBP-Fishhook controls. The amount of MBP-Fishhook (T F ) shifting to a complex that mimics MBP-Hairpin (T H ) increases with the amount of P cr -400 added (lanes 4 to 12). (C) Increasing amounts of biotinylated P cr -400 were incubated with MBP-Fishhook (lanes 4 to 8). As a control, MBP was also incubated with biotinylated P cr -400 (lane 9). Streptavidin-agarose was then added to the reaction mixture, and the bound complexes were precipitated, heat denatured, and analyzed by SDS-PAGE.

    Article Snippet: Nine hundred microliters of RIPA buffer (PBS-0.25% Triton X-100) and 50 μl of streptavidin-agarose solution (Sigma) were added, and the reaction mixture was incubated at 4°C for 1 h with gentle rotation.

    Techniques: Incubation, Clear Native PAGE, Staining, SDS Page

    Cys37 in VPAC1 is S-palmitoylated determined by acyl-biotin exchange assay and click chemistry palmitolaytion assay ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by streptavidin-agarose and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.

    Journal: Oncotarget

    Article Title: The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity

    doi: 10.18632/oncotarget.17449

    Figure Lengend Snippet: Cys37 in VPAC1 is S-palmitoylated determined by acyl-biotin exchange assay and click chemistry palmitolaytion assay ( A ) After the biotinylated proteins from VPAC1-CHO and VPAC1-C37/A-CHO subjected to the acyl-biotin exchange assay with (HA+) or without (HA-) hydroxylamine treatment were pulled down by streptavidin-agarose and electrophoresed by SDS-PAGE (left) and then detected using anti-EYFP antibody (right), VPAC1-EYFP was significantly detectable, while VPAC1-C37/A-EYFP is almost undetectable, indicating that Cys37 was palmitoylated. And the negative interference of HA showed that the acyl-biotin exchange assay was HA depended and the palmitoylation of Cys37 is via hydroxylamine-sensitive thioester bond. ( B ) VPAC1-CHO cells were incubated for 12 hours with (Odya+) or without (Odya-) palmitate ortholog. Samples were divided and either not biotinylated through the click reaction (No RXN), reduced prior to the reaction with HA, or biotinylated and not reduced (RXN). Only samples treated with RXN without HA showed significant signal, supporting the specificity of the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( C ) After VPAC1-CHO cells were incubated with Odya for 30, 60, 120 and 240 min hours, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. The time-dependent signals confirmed the technique's validity. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. ( D ) After VPAC1-CHO cells and VPAC1-C37/A-CHO cells were incubated with (Odya+) or without (Odya-) palmitate ortholog, click reaction was performed followed by biotin pull-down and western blot analysis for EYFP. VPAC1-EYFP was significant detectable, while VPAC1-C37/A-EYFP was almost undetectable, indicating the palmitoylation of Cys37 was determined by the click chemistry palmitolaytion assay. Relative equal inputs without click chemistry palmitolaytion assay were detected using anti-EYFP antibody. Representative blots from at least three independent experiments are shown.

    Article Snippet: The biotinylated protein were pulled down from the cells lysates using streptavidin-agarose mini-column (Sigma, USA), and then were subjected to western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).

    Techniques: SDS Page, Incubation, Western Blot

    H3K27m3 and DNA methylation co‐exist at ATCOPIA93 IGB (integrative genome browser) views showing H3K9m2 levels and H3K27m3 levels in WT and met1 rosette leaves, at ATCOPIA93 EVD and ATR (ChIP‐chip public data, Deleris et al , 2012 ). Orange horizontal bars: protein‐coding genes; horizontal green bars: transposable elements. The LTRs are delineated by pink bars. Vertical blue bars: H3K9m2 signal relative to H3 (two top lanes); vertical purple bars: H3K27m3 signal relative to H3 for each probe. Analysis of H3K27m3 marks at ATCOPIA93 EVD and ATR by ChIP on rosette leaves, followed by qPCR, in wild‐type plants and in clf plants mutated for the H3K27 methyltransferase CURLY LEAF. Data were normalized to the input DNA. ATCOPIA93 CDS is a region in ATCOPIA93 GAG common to EVD and ATR . AT5g17120 is a region in the protein‐coding gene located upstream of EVD . FLC is a region located in the first intron of FLOWERING LOCUS C which shows high levels of H3K27m3 in vegetative tissues and serves as a positive control. TA3 is a transposon and serves as a negative control. Because of technical variability in the ChIP efficiency, one ChIP experiment is presented here and two other independent experiments are presented in Fig EV3 B. ChIPs were performed on a pool of rosette leaves from eight to 10 plants/genotype. Genomic distribution of H3K27m3 marks between EVD and ATR loci by ChIP‐PCR pyrosequencing. Upper panel: Depiction of the pyrosequenced region (in yellow) within the GAG biotinylated qPCR amplicon obtained after H3K27m3 ChIP‐qPCR and purification with streptavidin beads. The position interrogated corresponds to the discriminating SNP between EVD (C/G) and ATR (A/T). Lower panel: The % indicated represents the % of G ( EVD , dark blue bar) or T ( ATR , light blue bar) at that position. The PCR and sequencing primers were designed so that other ATCOPIA93 ‐derived sequences (divergent and presumably nonfunctional) such as AT4G04410 and AT1G43775 cannot be amplified and so that the allelic ratio between the two active ATCOPIA93 copies EVD and ATR only can be evaluated. To verify this, the qPCR GAG product is also amplified from the Input gDNA as a control where a 50–50% ratio is expected. For clarity, an average of two experiments performed on two independent Input and ChIPs samples is shown (error bars represent standard error (SE) of the mean) and individual datasets presented in Fig EV3 C. Methylation status of the DNA captured with H3K27m3 by Sau96I Chop‐qPCR. H3K27m3 ChIP‐DNA from two independent ChIPs was digested with the methylation‐sensitive restriction endonuclease Sau96I which is sensitive to the methylation of the second C at the GGGCCG site in the LTR (as in Fig EV1 ). The values plotted correspond to the ratio between the amount of amplified DNA in the Sau96I digestion and the amount of amplified DNA in the undigested control, as calculated by the formula 2 −(Ct.digestedDNA–Ct.undigestedDNA) and using primers specific for a region of EVD‐ LTR spanning this Sau96I restriction site. Dark and light symbols are used for the first and second experiments, respectively. Results show that the WT ChIP‐DNA had significantly less digestion compared with the ddm1 control; thus, there was more methylation. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Transcriptional control and exploitation of an immune‐responsive family of plant retrotransposons

    doi: 10.15252/embj.201798482

    Figure Lengend Snippet: H3K27m3 and DNA methylation co‐exist at ATCOPIA93 IGB (integrative genome browser) views showing H3K9m2 levels and H3K27m3 levels in WT and met1 rosette leaves, at ATCOPIA93 EVD and ATR (ChIP‐chip public data, Deleris et al , 2012 ). Orange horizontal bars: protein‐coding genes; horizontal green bars: transposable elements. The LTRs are delineated by pink bars. Vertical blue bars: H3K9m2 signal relative to H3 (two top lanes); vertical purple bars: H3K27m3 signal relative to H3 for each probe. Analysis of H3K27m3 marks at ATCOPIA93 EVD and ATR by ChIP on rosette leaves, followed by qPCR, in wild‐type plants and in clf plants mutated for the H3K27 methyltransferase CURLY LEAF. Data were normalized to the input DNA. ATCOPIA93 CDS is a region in ATCOPIA93 GAG common to EVD and ATR . AT5g17120 is a region in the protein‐coding gene located upstream of EVD . FLC is a region located in the first intron of FLOWERING LOCUS C which shows high levels of H3K27m3 in vegetative tissues and serves as a positive control. TA3 is a transposon and serves as a negative control. Because of technical variability in the ChIP efficiency, one ChIP experiment is presented here and two other independent experiments are presented in Fig EV3 B. ChIPs were performed on a pool of rosette leaves from eight to 10 plants/genotype. Genomic distribution of H3K27m3 marks between EVD and ATR loci by ChIP‐PCR pyrosequencing. Upper panel: Depiction of the pyrosequenced region (in yellow) within the GAG biotinylated qPCR amplicon obtained after H3K27m3 ChIP‐qPCR and purification with streptavidin beads. The position interrogated corresponds to the discriminating SNP between EVD (C/G) and ATR (A/T). Lower panel: The % indicated represents the % of G ( EVD , dark blue bar) or T ( ATR , light blue bar) at that position. The PCR and sequencing primers were designed so that other ATCOPIA93 ‐derived sequences (divergent and presumably nonfunctional) such as AT4G04410 and AT1G43775 cannot be amplified and so that the allelic ratio between the two active ATCOPIA93 copies EVD and ATR only can be evaluated. To verify this, the qPCR GAG product is also amplified from the Input gDNA as a control where a 50–50% ratio is expected. For clarity, an average of two experiments performed on two independent Input and ChIPs samples is shown (error bars represent standard error (SE) of the mean) and individual datasets presented in Fig EV3 C. Methylation status of the DNA captured with H3K27m3 by Sau96I Chop‐qPCR. H3K27m3 ChIP‐DNA from two independent ChIPs was digested with the methylation‐sensitive restriction endonuclease Sau96I which is sensitive to the methylation of the second C at the GGGCCG site in the LTR (as in Fig EV1 ). The values plotted correspond to the ratio between the amount of amplified DNA in the Sau96I digestion and the amount of amplified DNA in the undigested control, as calculated by the formula 2 −(Ct.digestedDNA–Ct.undigestedDNA) and using primers specific for a region of EVD‐ LTR spanning this Sau96I restriction site. Dark and light symbols are used for the first and second experiments, respectively. Results show that the WT ChIP‐DNA had significantly less digestion compared with the ddm1 control; thus, there was more methylation. Source data are available online for this figure.

    Article Snippet: Pyrosequencing ATCOPIA93 DNA (ChIP‐DNA, cDNA, or gDNA as a control) was amplified with a biotinylated (forward) primer in the same region where RNA levels were analyzed and containing a SNP between EVD and ATR ; the biotinylated PCR product (40 μl reaction) was pulled down with streptavidin beads (sigma GE17‐5113‐01) and the sense biotinylated strand sequenced with a Pyromark Q24 (Qiagen) on the sequencing mode.

    Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Polymerase Chain Reaction, Amplification, Purification, Sequencing, Derivative Assay, Methylation

    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with streptavidin–agarose beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.

    Journal: EMBO Molecular Medicine

    Article Title: Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors

    doi: 10.1002/emmm.201100124

    Figure Lengend Snippet: Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with streptavidin–agarose beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.

    Article Snippet: Reagents, proteasome inhibition and EndoH treatment The anti-human CMG2 monoclonal antibody 2F6 was obtained by genetic immunization of rats with an hCMG2 construct (Genovac, Germany); polyclonal goat anti human CMG2 antibodies were from R & D (Ref. AF2940), PA of anthrax toxin, anti-PA and anti-CMG2 antibodies were provided from the Leppla laboratory; goat anti-human CMG2 antibody was from R & D Systems; anti-HA-HRP antibody, anti-HA beads were from Roche (Basel, Switzerland); anti human-transferin receptor from Invitrogen (Carlsbad, CA); protein G-agarose-conjugated beads were from GE Healthcare; anti-V5 antibody was from Invitrogen; streptavidin–agarose conjugated beads from Sigma–Aldrich (St. Louis, MO); anti-ubiquitin antibody from Santa Cruz; HRP secondary antibodies from Pierce Chemical Co. (Rockford, IL); Alexa-conjugated secondary antibodies from Jackson Immunoresearch.

    Techniques: Expressing, Derivative Assay, Incubation, Immunoprecipitation, SDS Page, Western Blot, Concentration Assay

    CARM1 is in association with Sox2. (A) Co-immunoprecipitation of Sox2 with CARM1. Whole cell extracts (WCEs) of MCF7 cells transfected with (+) or without (-) FLAG-CARM1 or GFP-Sox2, were subjected to immunoprecipitation with anti-FLAG antibody and blotted with antibodies against GFP or FLAG. (B) Co-precipitation of endogenous CARM1 with Sox2 by DNA pull-down. Biotin-labeled probes covering Sox2-binding sites in fgf4 and utf1 respectively were incubated with WCEs of P19 cells and streptavidin-conjugated agarose beads. The proteins precipitated with beads were denatured and resolved in SDS-PAGE and detected by immunoblotting with antibodies against Sox2 or CARM1. NC: negative control, no probe added. (C) Schematic drawing of the wild type and truncated CARM1 fragments. (D) GST pull-down assays to detect the interaction of Sox2 with CARM1 derivates. GST or GST-Sox2 was first incubated with WCEs of HEK293T cells ectopically expressing FLAG-CARM1 derivates. The GST pull-down products were immunoblotted with anti-FLAG antibody. Input: WCE control. (E) Schematic drawing of the wild type and truncated Sox2 fragments. (F) The interaction of Sox2 derivates with CARM1 in GST pull-down assays. GST or GST-Sox2 derivates were first incubated with whole cell extracts (WCEs) of HEK293T cells ectopically expressing FLAG-CARM1. The GST pull-down products were immunoblotted with anti-FLAG antibody.

    Journal: PLoS ONE

    Article Title: CARM1 Mediates Modulation of Sox2

    doi: 10.1371/journal.pone.0027026

    Figure Lengend Snippet: CARM1 is in association with Sox2. (A) Co-immunoprecipitation of Sox2 with CARM1. Whole cell extracts (WCEs) of MCF7 cells transfected with (+) or without (-) FLAG-CARM1 or GFP-Sox2, were subjected to immunoprecipitation with anti-FLAG antibody and blotted with antibodies against GFP or FLAG. (B) Co-precipitation of endogenous CARM1 with Sox2 by DNA pull-down. Biotin-labeled probes covering Sox2-binding sites in fgf4 and utf1 respectively were incubated with WCEs of P19 cells and streptavidin-conjugated agarose beads. The proteins precipitated with beads were denatured and resolved in SDS-PAGE and detected by immunoblotting with antibodies against Sox2 or CARM1. NC: negative control, no probe added. (C) Schematic drawing of the wild type and truncated CARM1 fragments. (D) GST pull-down assays to detect the interaction of Sox2 with CARM1 derivates. GST or GST-Sox2 was first incubated with WCEs of HEK293T cells ectopically expressing FLAG-CARM1 derivates. The GST pull-down products were immunoblotted with anti-FLAG antibody. Input: WCE control. (E) Schematic drawing of the wild type and truncated Sox2 fragments. (F) The interaction of Sox2 derivates with CARM1 in GST pull-down assays. GST or GST-Sox2 derivates were first incubated with whole cell extracts (WCEs) of HEK293T cells ectopically expressing FLAG-CARM1. The GST pull-down products were immunoblotted with anti-FLAG antibody.

    Article Snippet: In brief, 50 nmol 5′-end biotin-labeled oligo-DNA probes covering the Sox2-binding sites in fgf4 or utf1 were incubated with 1 mg whole cell extracts of P19 cells and streptavidin-conjugated agarose beads (Millipore) overnight at 4°C.

    Techniques: Immunoprecipitation, Transfection, Labeling, Binding Assay, Incubation, SDS Page, Negative Control, Expressing

    Comparison of the inhibitory rate on the proliferation of U87MG and U87-EGFRvIII cells following c-Met gene silencing. The inhibitory rate was calculated according to the OD values of the control and experimental groups obtained from the MTT assay. OD, optical density; BA, 5′-biotin-labeled aptamer 32; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + streptavidin; lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Data show the means ± standard deviation of three independent experiments. ** P

    Journal: Biomedical Reports

    Article Title: A U87-EGFRvIII cell-specific aptamer mediates small interfering RNA delivery

    doi: 10.3892/br.2014.276

    Figure Lengend Snippet: Comparison of the inhibitory rate on the proliferation of U87MG and U87-EGFRvIII cells following c-Met gene silencing. The inhibitory rate was calculated according to the OD values of the control and experimental groups obtained from the MTT assay. OD, optical density; BA, 5′-biotin-labeled aptamer 32; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + streptavidin; lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Data show the means ± standard deviation of three independent experiments. ** P

    Article Snippet: Subsequently, the two solutions were mixed with 2.5 μl/well of 20 μM streptavidin (Sigma-Aldrich, St. Louis, MO, USA) and left to stand for 20 min prior to being added to the culture media.

    Techniques: MTT Assay, Labeling, Small Interfering RNA, Standard Deviation

    Flow cytometry detection of apoptotic changes of U87-EGFRvIII cells following c-Met gene silencing. (A–D) Flow cytometry results of U87-EGFRvIII apoptosis in each group. Q2 and Q4 show late and early apoptotic rates, respectively. (E) Comparison of apoptotic rate of U87MG and U87-EGFRvIII cells in each group. BA, 5′-biotin-labeled aptamer 32; FITC, fluorescein isothiocyanate; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + streptavidin. lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Error bars show the means ± standard deviation (n=3). * P

    Journal: Biomedical Reports

    Article Title: A U87-EGFRvIII cell-specific aptamer mediates small interfering RNA delivery

    doi: 10.3892/br.2014.276

    Figure Lengend Snippet: Flow cytometry detection of apoptotic changes of U87-EGFRvIII cells following c-Met gene silencing. (A–D) Flow cytometry results of U87-EGFRvIII apoptosis in each group. Q2 and Q4 show late and early apoptotic rates, respectively. (E) Comparison of apoptotic rate of U87MG and U87-EGFRvIII cells in each group. BA, 5′-biotin-labeled aptamer 32; FITC, fluorescein isothiocyanate; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + streptavidin. lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Error bars show the means ± standard deviation (n=3). * P

    Article Snippet: Subsequently, the two solutions were mixed with 2.5 μl/well of 20 μM streptavidin (Sigma-Aldrich, St. Louis, MO, USA) and left to stand for 20 min prior to being added to the culture media.

    Techniques: Flow Cytometry, Cytometry, Labeling, Small Interfering RNA, Standard Deviation

    BA-mediated delivery of c-Met siRNA into U87-EGFRvIII cells downregulated c-Met protein expression. (A) Determination of the amount of BA, c-Met siRNA (small interfering RNA) and streptavidin used for conjugation was performed by agarose gel electrophoresis. A32, biotin-aptamer 32 (BA); B-siRNA, biotin-c-Met siRNA; B-siRNA + s = biotin-c-Met siRNA + streptavidin. A32 + S + si = biotin-A32 + streptavidin + biotin-c-Met siRNA. (B) Western blotting and (C) quantitative analysis of the changes in c-Met protein expression following BA-delivered c-Met siRNA into U87-EGFRvIII cells. Transfected and lipo, liposome-mediated group; no transfection and BA group, BA-mediated group; NCsi = liposome or BA + control siRNA + streptavidin; Bsi + S = liposome or BA + c-Met siRNA + streptavidin; Bsi = liposome or BA + c-Met siRNA; BA = BA alone. Error bars show the means ± standard deviation (n=3). * P

    Journal: Biomedical Reports

    Article Title: A U87-EGFRvIII cell-specific aptamer mediates small interfering RNA delivery

    doi: 10.3892/br.2014.276

    Figure Lengend Snippet: BA-mediated delivery of c-Met siRNA into U87-EGFRvIII cells downregulated c-Met protein expression. (A) Determination of the amount of BA, c-Met siRNA (small interfering RNA) and streptavidin used for conjugation was performed by agarose gel electrophoresis. A32, biotin-aptamer 32 (BA); B-siRNA, biotin-c-Met siRNA; B-siRNA + s = biotin-c-Met siRNA + streptavidin. A32 + S + si = biotin-A32 + streptavidin + biotin-c-Met siRNA. (B) Western blotting and (C) quantitative analysis of the changes in c-Met protein expression following BA-delivered c-Met siRNA into U87-EGFRvIII cells. Transfected and lipo, liposome-mediated group; no transfection and BA group, BA-mediated group; NCsi = liposome or BA + control siRNA + streptavidin; Bsi + S = liposome or BA + c-Met siRNA + streptavidin; Bsi = liposome or BA + c-Met siRNA; BA = BA alone. Error bars show the means ± standard deviation (n=3). * P

    Article Snippet: Subsequently, the two solutions were mixed with 2.5 μl/well of 20 μM streptavidin (Sigma-Aldrich, St. Louis, MO, USA) and left to stand for 20 min prior to being added to the culture media.

    Techniques: Expressing, Small Interfering RNA, Conjugation Assay, Agarose Gel Electrophoresis, Western Blot, Transfection, Standard Deviation

    DNA binding activity of RUNX2 or RUNX2 R131G HA tagged RUNX2 or RUNX2 R131G proteins were expressed in HeLa cells. Nuclear extracts from transfected HeLa cells were incubated with biotinylated wild type (Wt) or mutant (Mu) RUNX2 binding oligonucleotides for 1h at RT. Streptavidin coated Sepharose CL4B beads were added to nuclear protein/DNA complexes and incubated at RT for 1 hr with rotation. Reacted (A) or non-reacted (B) nuclear extracts were separated in 10% SDS-polyacrylamide gels, transferred, and immunoblotted with antibodies to RUNX2. Lamin B antibody was used as internal loading control.

    Journal: Journal of cellular biochemistry

    Article Title: The cleidocranial dysplasia related R131G mutation in the Runt-related transcription factor RUNX2 disrupts binding to DNA but not CBF-?

    doi: 10.1002/jcb.22516

    Figure Lengend Snippet: DNA binding activity of RUNX2 or RUNX2 R131G HA tagged RUNX2 or RUNX2 R131G proteins were expressed in HeLa cells. Nuclear extracts from transfected HeLa cells were incubated with biotinylated wild type (Wt) or mutant (Mu) RUNX2 binding oligonucleotides for 1h at RT. Streptavidin coated Sepharose CL4B beads were added to nuclear protein/DNA complexes and incubated at RT for 1 hr with rotation. Reacted (A) or non-reacted (B) nuclear extracts were separated in 10% SDS-polyacrylamide gels, transferred, and immunoblotted with antibodies to RUNX2. Lamin B antibody was used as internal loading control.

    Article Snippet: Mouse IgG monoclonal antibodies for hemagglutinin (HA) (BAbCO, Richmond, CA, USA), Myc (Invitrogen), Lamin B1 (Santa Cruz, City, CA USA), β-actin, as well as anti-mouse IgG (H+L) conjugated with Alexa Fluor® 555 (Invitrogen), goat anti-mouse IgG (Santa Cruz), Protein G sepharose (Santa Cruz) and Streptavidin-conjugated agarose CL4B (Sigma, Springfield, Missouri, USA) were acquired from the indicated suppliers.

    Techniques: Binding Assay, Activity Assay, Transfection, Incubation, Mutagenesis

    Purification of a larger NRE-associated protein complex. ( A ) Comparison of complete HeLa cell nuclear extract (lane 1) with extracts depleted by preincubation with biotinylated NRE–RNA and adsorption on streptavidin beads (lanes 2 and 4) or depleted by preincubation with poly(U) Sepharose in the presence of 2 M KCl (lane 3). Release of NRE-bound proteins from streptavidin beads by RNase treatment (lane 5). ▸, 65-kDa protein; ○, depleted 65-kDa protein; [cirf], enriched proteins; R, RNase A.

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

    Article Title: A cellular 65-kDa protein recognizes the negative regulatory element of human papillomavirus late mRNA

    doi:

    Figure Lengend Snippet: Purification of a larger NRE-associated protein complex. ( A ) Comparison of complete HeLa cell nuclear extract (lane 1) with extracts depleted by preincubation with biotinylated NRE–RNA and adsorption on streptavidin beads (lanes 2 and 4) or depleted by preincubation with poly(U) Sepharose in the presence of 2 M KCl (lane 3). Release of NRE-bound proteins from streptavidin beads by RNase treatment (lane 5). ▸, 65-kDa protein; ○, depleted 65-kDa protein; [cirf], enriched proteins; R, RNase A.

    Article Snippet: The RNA and associated proteins were then bound for 20 min at 20°C to 200 μl 4% streptavidin-beaded agarose (Sigma) equilibrated in binding buffer.

    Techniques: Purification, Adsorption