immunoprecipitants Search Results


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  • 99
    Thermo Fisher dynabeads protein g
    Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with <t>Protein-G</t> <t>Dynabeads</t> (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei
    Dynabeads Protein G, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore co immunoprecipitation
    The hPer2 protein forms a ternary complex with hp53 and Mdm2, controlling the extent of hp53 ubiquitination. (A) HEK293 and HCT116 protein extracts (1 and 2 mg, respectively) were incubated with either α-p53 or α-Per2, respectively, and protein A beads. Rabbit IgG was used as a negative control. Immunoprecipitated complexes were analyzed for the presence of hPer2, hp53, and Mdm2 using specific antibodies (top for HEK293 and bottom for HCT116). Asterisk indicates a nonspecific signal. (B) HEK293 cells were transfected with pCS2+ myc -Mdm2, pCS2+ myc -hp53, pCS2+FLAG-hp53, pCS2+FLAG-hPer2, pCS2+ myc -hPer2, or a combination of plasmids and complexes immunoprecipitated using α-FLAG–coupled beads. Complex components were identified by immunoblotting using α-FLAG and α- myc antibodies (right top and bottom). Input amounts were monitored in cell lysates (20 μg) and shown in the left top and bottom. Results similar to those presented were observed in two independent experiments. (C) In vitro–synthesized myc -hPer2 and FLAG-hp53 proteins were preincubated before the addition of myc -Mdm2 (ratio 1:2:5: FLAG-hp53: myc -Mdm2: myc -hPer2). Samples were then subjected to in vitro ubiquitination, followed by <t>immunoprecipitation</t> of hp53-bound proteins using α-FLAG antibody and protein A beads. Bound proteins were detected by immunoblotting and are indicated with arrows (top and middle). FLAG-hp53(Ub) n forms of hp53 were detected using α-ubiquitin antibody (bottom). Asterisk indicates IgG heavy chain. (D) HEK293 cells were transfected with pCS2+ myc -Mdm2, pCS2+ myc -hPer2, pCS2+FLAG-hp53, or a combination of plasmids and collected 12 h after treatment with 10 μM MG132. Cell lysates (100 μg) were incubated with α-FLAG and protein A beads and hp53-ubiquitinated complexes (FLAG-hp53(Ub) n ) detected using α-ubiquitin antibody (top right). Bound proteins were visualized by immunoblotting using α- myc and α-FLAG antibodies (middle and lower right). An in vitro ubiquitination reaction was performed as described in C and is shown as control and for comparison purposes with the “in cells” result (left). Brackets denote ubiquitinated hp53. A, C, and D show immunoblot data from a single experiment that was repeated three times with similar results.
    Co Immunoprecipitation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology co immunoprecipitation
    Fig. 2. The HisRS-like region contains two independent dimerization domains. ( A ) <t>Co-immunoprecipitation</t> of GST–HisRS with LexA-HA–HisRS from yeast cell extracts. Transformants of gcn2Δ ) bearing HIS3 plasmids encoding LexA-HA–HisRS(970–1497) (pHQ588) or LexA-HA (p2247), and TRP1 plasmids encoding GST (pHQ242) or GST–HisRS(970–1497) (pHQ601), were grown in SC-His-Trp medium and whole-cell extracts were prepared. Aliquots of extracts containing 50 µg of protein were immunoprecipitated with anti-HA antibodies, and the immune complexes were resolved by SDS–PAGE and subjected to western blot analysis using anti-GST antibodies (left panels) or anti-LexA antibodies (right panels). The top panels show results from the transformant expressing GST and the LexA-HA–HisRS fusion; the middle panels derive from the transformant expressing the GST–HisRS and LexA-HA–HisRS fusions; the bottom two panels derive from the transformant expressing the GST–HisRS fusion and LexA-HA. ( B and C ) The HisRS-like region dimerizes in vitro through the N- and C-terminal subregions. The full-length HisRS-like region (aa 970–1497) or truncated segments indicated across the top of each panel were translated in vitro in the presence of [ 35 S]methionine and incubated with either GST, GST–HisRS(970–1156) (B) or GST–HisRS(1315–1497) (C) proteins, which were expressed in E.coli and immobilized on glutathione–Sepharose beads. After extensive washing of the beads, the bound proteins were resolved by SDS–PAGE and visualized by fluorography. Numbers to the left of each gel give the positions of size standards of the indicated molecular weight in kilodaltons. ( D ) Summary of results from the binding assays shown in (B and C). Rectangular boxes with the locations of conserved motifs M1–3 (indicated with diagonal hatching) represent the in vitro translated proteins with GCN2 residue numbers indicated at their N- and C-termini. The binding (+) or failure to bind (–) of these segments to GST–HisRS(970–1156) or GST–HisRS(1315–1497) is summarized to the right and left of the schematics, respectively. ND, not determined. The results of these assays indicate that dimerization by the C-terminal segment of the HisRS-like region is dependent on residues 1315–1383, as indicated by the bar above the boxes. Bars below the boxes represent HisRS-N and HisRS-C subregions, respectively.
    Co Immunoprecipitation, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam co immunoprecipitation
    The PLA assay can be used for high-throughput drug screening for targeted compounds in synovial sarcoma From a 16 000 compound small molecule drug library, a compound designated SXT1596 was recognized to significantly decrease relative co-localization of SS18-SSX/TLE1 in human synovial sarcoma cells A. SXT1596 selectively decreases cell viability in synovial sarcoma cell lines, SYO-1, FUJI, Yamato-SS, HS-SY-II, YaFuss and MoJo (white bars), when compared to negative control cell lines HEK293T, MCF7, HeLa and A673 (shaded bars) at a concentration of 5 μM B. Negative control cell lines HeLa and A673 are sensitive to SXT1596 at higher doses. SXT1596 (5-(2-Nitro-1-propenyl)-1,3-benzodioxole) (structure shown in C. ) mediated disruption of the SS18-SSX/TLE1 association was confirmed by <t>immunoprecipitation</t> D. Nuclear SS18-SSX/TLE1 PLA signal in SYO-1 cells decreased following SXT1596 treatment to a degree similar to that obtained with the HDAC inhibitor FK228 E, F. Statistical significance compared to vehicle treatment controls was determined by Student t test: * denotes p
    Co Immunoprecipitation, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 296 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc co immunoprecipitation
    DACH1 promotes adenomas organoid formation via modulating BMP signalling. a DACH1 overexpression induced upregulation of cancer stem cell marker genes. b and c. Gene Ontology (GO) analysis showed that DACH1 overexpression induced the upregulation of stem cell signature genes and the downregulation of cell cycle arrest signature genes. d and e. DACH1 induced the downregulation of ATOH8, TGFβ3, MSX1, NBL1, BMP7, and FKBP4 and the upregulation of FKBP8, ID1, and MAPK3 (experiments were performed in triplicate). f. IF images showing the colocalization of SMAD4 and DACH1 in the nuclei; scale bars=20 μm g-h. DACH1 coprecipitated endogenous SMAD4. Reverse <t>immunoprecipitation</t> was confirmed with an anti-SMAD4 antibody. i. DACH1 overexpression increased the protein level of SMAD4 and decreased the level of phosphorylated SMAD4 (Thr276) [Thr277]. j and k. DACH1 knockdown led to an increase of the mRNA levels of NBL1 and BMP7 compared with the shNC group, and siRNA mediated SMAD4 knockdown in HCT116-shDACH1 cells eliminated the increase. l. DACH1 overexpression was sufficient to compensate for the withdrawal of Noggin and supported the formation of adenoma organoids. m. Addition of Noggin into the culture medium for 24 and 36 h decreased the mRNA levels of NBL1 and BMP7, which were increased by DACH1 knockdown in HCT116 cells. n and o. Overexpression of DACH1 upregulates LGR5, Notch1 and the protein level of NICD, while did not induce significant upregulation of HES1. Scale bars=20 μm. For 5d, 5e, 5k, 5 m and 5n, ** P
    Co Immunoprecipitation, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dynabeads protein a
    EIN3 antibody reproducibly enriches DNA in chromatin immunoprecipitation. ( A ) Enrichment of the known target of EIN3, the promoter of ERF1, using <t>Dynabeads</t> <t>Protein</t> A and Dynabeads Sheep anti-Rabbit IgG to collect protein-DNA complexes. The average fold change for two technical ChIP replicates with three QPCR technical replicates each is shown. ( B ) Reproducibility in the two biological replicates for EIN3 ChIP performed upon treatment of ethylene gas for 0, 0.5, 1, and 4 hr. ( C ) Average RPKM of EIN3 binding sites 0, 0.5, 1, and 4 hr of ethylene gas treatment. ( D ) EIN3 binding preferentially occurs in the promoter regions of genes (1000 bp upstream of the TSS). DOI: http://dx.doi.org/10.7554/eLife.00675.004
    Dynabeads Protein A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6501 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore magna rip rna binding protein immunoprecipitation kit
    miR503HG physically interacts with HNRNPA2B1 in HCC cells. (A) <t>RNA</t> pull-down assays were performed in SMMC-7721 cells twice. Biotinylated miR503HG (left lane) or antisense RNA (right lane) was incubated with cell extracts and targeted with streptavidin beads; the associated proteins were resolved on a gel. The highlighted region was submitted for mass spectrometry. (B) Western blotting analysis of the specific association of HNRNPA2B1 with miR503HG in SMMC-7721 and Huh7 cells. (C) <t>RIP</t> enrichment was determined as RNA associated with the HNRNPA2B1 IP relative to an input control. The data represent the average and standard deviation of three independent experiments. (D) Deletion mapping of the HNRNPA2B1-binding domain in miR503HG. Top: graphic illustration of predicted miR503HG secondary structure (LNCipedia, http://www.lncipedia.org ), and the truncation of miR503HG according to the stem-loop structure. Middle: the in vitro transcribed full-length and deletion fragments of miR503HG. Bottom: Western blotting analysis of HNRNPA2B1 in protein samples pulled down by the different miR503HG constructs. (E) Deletion mapping of the miR503HG-binding domain in HNRNPA2B1. Top: diagrams of full-length HNRNPA2B1 and the domain-truncated fragments RNA recognition motif (RRM), hnRNP K homology domain (KH), M9 signal (MN). Bottom: qPCR analysis of HNRNPA2B1 retrieved by full-length or domain-truncated HNRNPA2B1-HA using a HA antibody. RIP assays were performed using Huh7 cells transfected with the indicated vectors.
    Magna Rip Rna Binding Protein Immunoprecipitation Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3542 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology immunoprecipitations
    Immunoprecipitation reveals interaction between Flag-MAVS and PB2-GFP. (A) Lysates from 293T cells transfected with Flag-MAVS and pcDNA-PB2-GFP or pcDNA-PB1-GFP and immunoprecipitates (IP) were analyzed by Western blotting using anti-Flag and anti-GFP antibodies. <t>Immunoprecipitations</t> of MAVS were performed with anti-Flag antibody. (B) Lysates from 293T cells transfected with Flag-MAVS and pcDNA-PB2-GFP or pcDNA-PB2 N9D-GFP and immunoprecipitates were analyzed by Western blotting using anti-Flag and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-Flag antibody. (C) Lysates from 293T cells transfected with pcDNA-PB2-GFP or pcDNA-PB2 N9D-GFP and immunoprecipitates were analyzed by Western blotting using anti-MAVS and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-MAVS antibody. α, anti.
    Immunoprecipitations, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 3492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare immunoprecipitation
    Immunoprecipitation reveals interaction between Flag-MAVS and PB2-GFP. (A) Lysates from 293T cells transfected with Flag-MAVS and pcDNA-PB2-GFP or pcDNA-PB1-GFP and immunoprecipitates (IP) were analyzed by Western blotting using anti-Flag and anti-GFP antibodies. <t>Immunoprecipitations</t> of MAVS were performed with anti-Flag antibody. (B) Lysates from 293T cells transfected with Flag-MAVS and pcDNA-PB2-GFP or pcDNA-PB2 N9D-GFP and immunoprecipitates were analyzed by Western blotting using anti-Flag and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-Flag antibody. (C) Lysates from 293T cells transfected with pcDNA-PB2-GFP or pcDNA-PB2 N9D-GFP and immunoprecipitates were analyzed by Western blotting using anti-MAVS and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-MAVS antibody. α, anti.
    Immunoprecipitation, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1618 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad immunoprecipitates
    Pulse–chase analysis of intracellular PC-wt and PC-A267T in stably transfected CHO-K1 cells. Stably transfected CHO-K1 cells were pulse-labeled for 30 min with [ 35 S]methionine and chased for 0, 1.5, 3 and 6 h. Equivalent amounts of cell extracts were immunoprecipitated using a polyclonal antibody against PC. <t>Immunoprecipitates</t> were analyzed by SDS-PAGE under reducing conditions (A). The amount of radioactivity remaining at each time point was plotted as the percentage of that measured in the immunoprecipate from unchased cells. The mean values from two independent experiments are presented. The turnover rates of the intracellular PC-wt and PC-A267T are depicted as • and ▴, respectively (B).
    Immunoprecipitates, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 2378 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology immunoprecipitates
    MUC13-Glut-1 interaction is disrupted by Sulfasalazine. a Confocal immunofluorescence of MUC13 and Glut-1 in MUC13-expressing, HPAF-II, and AsPC1 cells depicting co-localization (yellow color). Cells were cultured in 4-well chambered slides, fixed, permeabilized, stained with indicated antibodies and analyzed for confocal microscopy. DAPI was used as a counter stain for the nucleus. b Cells were treated with sulphasalazine (0.9 mM) for 24 h and processed for immunostaining and confocal microscopy. Images were captured at ×400. c , d Reciprocal co-immunoprecipitation assay between MUC13 and Glut-1 performed using protein lysates from HPAF-II cells. Cells were treated with sulfasalazine (0.9 mM) for 24 h and protein lysates prepared. The <t>immunoprecipitates</t> were resolved on a 10% gel, and followed by immunoblotting with anti-MUC13 and anti-Glut-1 antibodies. e Lactate assay was performed in MUC13-expressing and MUC13-null cells after treatment with or without sulfasalazine. Cell culture media was collected after 48 h to measure the amount of l -lactate concentration using lactate assay kit
    Immunoprecipitates, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pierce co immunoprecipitation kit
    A. Cdk2 association with PP1 binding partners. Mass spectrometric analysis of Cdk2 immunoprecipitates (lysates pooled from 3 independent experiments) from resting or thrombin exposed platelets identified cdk2 binding to a subset of PP1 regulatory proteins. Peptide counts identified for proteins listed under each incubation condition shown; PPP1R12A was most prevalent. B. <t>Co-immunoprecipitation</t> of Cdk2, Erk, PPP1CA and PP1R12A. Lysates of platelets under resting or thrombin-exposed (T, 1 minute, 0.5 U/ml) platelets immunoprecipitated with polyclonal cdk2 or monoclonal Erk1/2 or PPP1CA antibodies as indicated were subjected to Western Blotting. Molecular weight ladder lane excised (gap); PPP1R12A input run on parallel gel. C . Erk and PPP1R12a-associated phosphatase activity. Immunoprecipitates of platelets exposed to vehicle (−), thrombin (T) or roscovitine plus thrombin (RT) were reacted with a PP1-specific phosphatase substrate and liberation of phosphate measured.
    Pierce Co Immunoprecipitation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 928 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad immunoprecipitation
    A. Cdk2 association with PP1 binding partners. Mass spectrometric analysis of Cdk2 immunoprecipitates (lysates pooled from 3 independent experiments) from resting or thrombin exposed platelets identified cdk2 binding to a subset of PP1 regulatory proteins. Peptide counts identified for proteins listed under each incubation condition shown; PPP1R12A was most prevalent. B. <t>Co-immunoprecipitation</t> of Cdk2, Erk, PPP1CA and PP1R12A. Lysates of platelets under resting or thrombin-exposed (T, 1 minute, 0.5 U/ml) platelets immunoprecipitated with polyclonal cdk2 or monoclonal Erk1/2 or PPP1CA antibodies as indicated were subjected to Western Blotting. Molecular weight ladder lane excised (gap); PPP1R12A input run on parallel gel. C . Erk and PPP1R12a-associated phosphatase activity. Immunoprecipitates of platelets exposed to vehicle (−), thrombin (T) or roscovitine plus thrombin (RT) were reacted with a PP1-specific phosphatase substrate and liberation of phosphate measured.
    Immunoprecipitation, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dynabeads protein g immunoprecipitation kit
    Pif1 is acetylated both in vivo and in vitro . [A] Top Panel : S. cerevisiae lysates from WT (lane 1), esa1-414 (lane 2), and rpd3Δ (lane 3) backgrounds were immunoprecipitated with anti-acetyl lysine (ac-K) antibody-coated Protein <t>G-dynabeads</t> and immunoblotted with anti-FLAG antibody (1:1000); middle Panel : 10% of input immunoblotted with the anti-FLAG antibody and; bottom panel : PGK-1 antibody (1:10,000). [B] Immunoblot of unmodified Pif1 (lane 1), NuA4 (Esa1) in vitro -acetylated full-length Pif1 (lane 2), unmodified Pif1ΔN, and NuA4 (Esa1) in vitro -acetylated Pif1ΔN probed with anti-Ac-K antibody. [C] Schematic of the full-length Pif1 sequence. The positions of all of the lysine residues in the sequence are denoted with red lines, and all acetylated lysine residues identified by mass spectrometry are denoted with black lines and red filled circles.
    Dynabeads Protein G Immunoprecipitation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson immunoprecipitation
    Pif1 is acetylated both in vivo and in vitro . [A] Top Panel : S. cerevisiae lysates from WT (lane 1), esa1-414 (lane 2), and rpd3Δ (lane 3) backgrounds were immunoprecipitated with anti-acetyl lysine (ac-K) antibody-coated Protein <t>G-dynabeads</t> and immunoblotted with anti-FLAG antibody (1:1000); middle Panel : 10% of input immunoblotted with the anti-FLAG antibody and; bottom panel : PGK-1 antibody (1:10,000). [B] Immunoblot of unmodified Pif1 (lane 1), NuA4 (Esa1) in vitro -acetylated full-length Pif1 (lane 2), unmodified Pif1ΔN, and NuA4 (Esa1) in vitro -acetylated Pif1ΔN probed with anti-Ac-K antibody. [C] Schematic of the full-length Pif1 sequence. The positions of all of the lysine residues in the sequence are denoted with red lines, and all acetylated lysine residues identified by mass spectrometry are denoted with black lines and red filled circles.
    Immunoprecipitation, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc immunoprecipitation
    Pif1 is acetylated both in vivo and in vitro . [A] Top Panel : S. cerevisiae lysates from WT (lane 1), esa1-414 (lane 2), and rpd3Δ (lane 3) backgrounds were immunoprecipitated with anti-acetyl lysine (ac-K) antibody-coated Protein <t>G-dynabeads</t> and immunoblotted with anti-FLAG antibody (1:1000); middle Panel : 10% of input immunoblotted with the anti-FLAG antibody and; bottom panel : PGK-1 antibody (1:10,000). [B] Immunoblot of unmodified Pif1 (lane 1), NuA4 (Esa1) in vitro -acetylated full-length Pif1 (lane 2), unmodified Pif1ΔN, and NuA4 (Esa1) in vitro -acetylated Pif1ΔN probed with anti-Ac-K antibody. [C] Schematic of the full-length Pif1 sequence. The positions of all of the lysine residues in the sequence are denoted with red lines, and all acetylated lysine residues identified by mass spectrometry are denoted with black lines and red filled circles.
    Immunoprecipitation, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ChromoTek co immunoprecipitation
    YAP <t>immunoprecipitation</t> and mass-spectrometry analysis of binding partners. (A) YAP IP-MS analysis of co-precipitated proteins identifies the Engima family proteins PDLIM5 and PDLIM7 as novel YAP-associated proteins. Axes are log 10 -transformed values. (B) List of all YAP-associated proteins identified in the experiment shown in A. (C) Comparison of confidence ratios for PDLIM5/7 and known YAP interactors.
    Co Immunoprecipitation, supplied by ChromoTek, used in various techniques. Bioz Stars score: 93/100, based on 213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher magnify chromatin immunoprecipitation system
    MeCP2 binding to nucleosome is enhanced by the presence of H3K27me3. a Interactions between MeCP2 and H3K27me3 are determined by in vitro pull-down assay. Pull-down MeCP2 is shown by both MeCP2 and GST bands. Comparable levels of mononucleosomes between land 2 and 3 are shown by Ponceau S staining and H3. The presence of H3K27me3 is validated in Lane 3. b Schematic representation of MeCP2 domain constructs. c <t>Co-immunoprecipitation</t> of histone H3 with Flag-tagged MeCP2 full length or truncated fragments. From 293T cell transfected with Flag-tagged constructs in b , total protein extract was immunoprecipitated with Flag antibody and the presence of histone H3 is shown by immunoblotting. d In vitro pull-down assays of bait (purified GST, GST-tagged full length MeCP2 or MBD of MeCP2) to prey protein (recombinant mononucleosomes, either containing unmodified H3 or H3K27me3). Pull-downed H3 or H3K27me3 were visualized by SDS-PAGE and immunoblotting with anti-Histone H3 or H3K27me3. The presence of GST-fusion protein in each construct was indicated by the asterisks. e Competition assays with unmodified H3K27 or H3K27me3 peptides to MBD/nucleosome interactions. GST-tagged MBD binding to nucleosomes (top blot) containing H3K27me3 (lane 4) were challenged with H3K27 (lane 5–7) and H3K27me3 (lane 8-10) peptide. Bindings of GST-tagged MBD to histone H3 (middle blot) and H3K27me3 (bottom blot, sharp) were evaluated by GST pull-down and immunoblotting. H3K27me3 peptides in the MBD/nucleosome complexes were also revealed (bottom blot, double sharp). f , g Quantification of normalized H3K27me3 (purple, sharp) and H3K27me3 peptide (green, double sharp) levels by densitometry analysis in e . The signal strength in each band is normalized to H3K27me3 signal strength lane 4 in e . Source data are provided as a Source data file ( a , c , d and e ).
    Magnify Chromatin Immunoprecipitation System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 657 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore radio immunoprecipitation assay ripa buffer
    MeCP2 binding to nucleosome is enhanced by the presence of H3K27me3. a Interactions between MeCP2 and H3K27me3 are determined by in vitro pull-down assay. Pull-down MeCP2 is shown by both MeCP2 and GST bands. Comparable levels of mononucleosomes between land 2 and 3 are shown by Ponceau S staining and H3. The presence of H3K27me3 is validated in Lane 3. b Schematic representation of MeCP2 domain constructs. c <t>Co-immunoprecipitation</t> of histone H3 with Flag-tagged MeCP2 full length or truncated fragments. From 293T cell transfected with Flag-tagged constructs in b , total protein extract was immunoprecipitated with Flag antibody and the presence of histone H3 is shown by immunoblotting. d In vitro pull-down assays of bait (purified GST, GST-tagged full length MeCP2 or MBD of MeCP2) to prey protein (recombinant mononucleosomes, either containing unmodified H3 or H3K27me3). Pull-downed H3 or H3K27me3 were visualized by SDS-PAGE and immunoblotting with anti-Histone H3 or H3K27me3. The presence of GST-fusion protein in each construct was indicated by the asterisks. e Competition assays with unmodified H3K27 or H3K27me3 peptides to MBD/nucleosome interactions. GST-tagged MBD binding to nucleosomes (top blot) containing H3K27me3 (lane 4) were challenged with H3K27 (lane 5–7) and H3K27me3 (lane 8-10) peptide. Bindings of GST-tagged MBD to histone H3 (middle blot) and H3K27me3 (bottom blot, sharp) were evaluated by GST pull-down and immunoblotting. H3K27me3 peptides in the MBD/nucleosome complexes were also revealed (bottom blot, double sharp). f , g Quantification of normalized H3K27me3 (purple, sharp) and H3K27me3 peptide (green, double sharp) levels by densitometry analysis in e . The signal strength in each band is normalized to H3K27me3 signal strength lane 4 in e . Source data are provided as a Source data file ( a , c , d and e ).
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    Beyotime radio immunoprecipitation assay ripa lysis buffer
    MeCP2 binding to nucleosome is enhanced by the presence of H3K27me3. a Interactions between MeCP2 and H3K27me3 are determined by in vitro pull-down assay. Pull-down MeCP2 is shown by both MeCP2 and GST bands. Comparable levels of mononucleosomes between land 2 and 3 are shown by Ponceau S staining and H3. The presence of H3K27me3 is validated in Lane 3. b Schematic representation of MeCP2 domain constructs. c <t>Co-immunoprecipitation</t> of histone H3 with Flag-tagged MeCP2 full length or truncated fragments. From 293T cell transfected with Flag-tagged constructs in b , total protein extract was immunoprecipitated with Flag antibody and the presence of histone H3 is shown by immunoblotting. d In vitro pull-down assays of bait (purified GST, GST-tagged full length MeCP2 or MBD of MeCP2) to prey protein (recombinant mononucleosomes, either containing unmodified H3 or H3K27me3). Pull-downed H3 or H3K27me3 were visualized by SDS-PAGE and immunoblotting with anti-Histone H3 or H3K27me3. The presence of GST-fusion protein in each construct was indicated by the asterisks. e Competition assays with unmodified H3K27 or H3K27me3 peptides to MBD/nucleosome interactions. GST-tagged MBD binding to nucleosomes (top blot) containing H3K27me3 (lane 4) were challenged with H3K27 (lane 5–7) and H3K27me3 (lane 8-10) peptide. Bindings of GST-tagged MBD to histone H3 (middle blot) and H3K27me3 (bottom blot, sharp) were evaluated by GST pull-down and immunoblotting. H3K27me3 peptides in the MBD/nucleosome complexes were also revealed (bottom blot, double sharp). f , g Quantification of normalized H3K27me3 (purple, sharp) and H3K27me3 peptide (green, double sharp) levels by densitometry analysis in e . The signal strength in each band is normalized to H3K27me3 signal strength lane 4 in e . Source data are provided as a Source data file ( a , c , d and e ).
    Radio Immunoprecipitation Assay Ripa Lysis Buffer, supplied by Beyotime, used in various techniques. Bioz Stars score: 93/100, based on 1209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with Protein-G Dynabeads (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei

    Journal: BMC obesity

    Article Title: Adipocyte nuclei captured from VAT and SAT

    doi: 10.1186/s40608-016-0112-6

    Figure Lengend Snippet: Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with Protein-G Dynabeads (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei

    Article Snippet: Immuno-capture of nuclei Anti-RFP, anti-FLAG, anti-CD4, anti-CD8, anti-mRFP1 antibodies were coupled to 2.8 micron diameter protein G supraparamagnetic Dynabeads (ThermoFischer Scientific, #10004D).

    Techniques: Expressing, Fluorescence, Plasmid Preparation, Staining, Negative Control

    Mature MA-INTACT mouse adipocyte nuclei expressed SUN1mRFP1Flag RNA in BAT, VAT, and SAT allowing adipocyte nuclei to be captured on Immuno-paramagnetic beads. a Illustration of mouse fat deposits examined. b , c , d , e . Nuclei were efficiently captured from crude nuclear preparations prepared from inguinal SAT ( b ) retroperitioneal VAT ( c ) epididymal VAT ( d ) and scapular BAT ( e ). Protein G Dynabeads pre-adsorbed to rabbit anti-mRFP1 polyclonal antibody were used in these capture experiments. The clumping of nuclei occurs during successive rounds of washing and capture. Negative control antibodies did not capture significant numbers of nuclei (not shown). f A Western blot ( top panel ) showed that preparations of captured VAT, SAT, and BAT nuclei expressed the 131 kDa SUN1mRFP1Flag reporter protein, while uncaptured nuclei did not. In house prepared anti-mRFP1 rabbit pAb detected the mRFP1 domain. Purified 25 kDa mRFP1 was run as a positive control. A loading control ( bottom panel ) showed the approximately equal loading of nuclear proteins and an mRFP1 standard. The loading control samples were run for a short time (20 min) on an SDS PAGE system and protein front stained with Coomassie

    Journal: BMC obesity

    Article Title: Adipocyte nuclei captured from VAT and SAT

    doi: 10.1186/s40608-016-0112-6

    Figure Lengend Snippet: Mature MA-INTACT mouse adipocyte nuclei expressed SUN1mRFP1Flag RNA in BAT, VAT, and SAT allowing adipocyte nuclei to be captured on Immuno-paramagnetic beads. a Illustration of mouse fat deposits examined. b , c , d , e . Nuclei were efficiently captured from crude nuclear preparations prepared from inguinal SAT ( b ) retroperitioneal VAT ( c ) epididymal VAT ( d ) and scapular BAT ( e ). Protein G Dynabeads pre-adsorbed to rabbit anti-mRFP1 polyclonal antibody were used in these capture experiments. The clumping of nuclei occurs during successive rounds of washing and capture. Negative control antibodies did not capture significant numbers of nuclei (not shown). f A Western blot ( top panel ) showed that preparations of captured VAT, SAT, and BAT nuclei expressed the 131 kDa SUN1mRFP1Flag reporter protein, while uncaptured nuclei did not. In house prepared anti-mRFP1 rabbit pAb detected the mRFP1 domain. Purified 25 kDa mRFP1 was run as a positive control. A loading control ( bottom panel ) showed the approximately equal loading of nuclear proteins and an mRFP1 standard. The loading control samples were run for a short time (20 min) on an SDS PAGE system and protein front stained with Coomassie

    Article Snippet: Immuno-capture of nuclei Anti-RFP, anti-FLAG, anti-CD4, anti-CD8, anti-mRFP1 antibodies were coupled to 2.8 micron diameter protein G supraparamagnetic Dynabeads (ThermoFischer Scientific, #10004D).

    Techniques: Negative Control, Western Blot, Purification, Positive Control, SDS Page, Staining

    ITGA5 trafficking in ECs is NRP2 dependent. ( A ): Label-free quantitative mass spectrometry peptide hits identified using MaxQuant software from the Andromeda peptide database. Panel depicts peptide hits detected at a higher fold-change in two control siRNA treated EC lines than those detected from two NRP2 siRNA treated EC lines. NRP2 silencing was confirmed by subjecting EC extracts to Western blot analysis. ( B ): siRNA-transfected ECs were seeded onto FN and incubated for 48 hours. EC extracts were immunoprecipitated by incubation with protein-G Dynabeads® coupled to a NRP2 antibody. Immunoprecipitated complexes were subjected to Western blot analysis using antibodies against clathrin heavy chain-1 (top) and caveolin-1 (bottom). ( C ): siRNA-transfected ECs were seeded onto FN and incubated for 48 hours. ECs were subsequently starved in serum-free media, before being placed on ice. EC cell surface proteins were labelled with 0.3 mg/ml biotin. ECs were then incubated for the indicated timepoints at 37°C, 5% CO 2 . A sample of ECs were maintained at 4°C for use as positive/negative (+/-Mesna) controls. Following incubation at 37°C, ECs were placed on ice and incubated with 100 mM Mesna. EC lysates were then immunoprecipitated with protein-G Dynabeads® coupled to an anti-biotin antibody. Immunoprecipitated biotin-labelled proteins were separated by SDS-PAGE and subjected to Western bolt analysis. The level of internalised ITGA5 at each time of incubation was normalised to the (-) Mesna control. Left panel shows Western blot image, right panel shows the mean densitometric analysis obtained using ImageJ TM . Also shown is confirmation of NRP-2 silencing. N=3 independent experiments. ( D ): Co-immunoprecipitation study as described in B . Immunoprecipitated complexes were subjected to Western blot analysis using an anti-Rab11 antibody. ( E ): siRNA-transfected ECs were prepared as described in C . After biotin surface labelling, ECs were incubated in serum free media for 20 minutes at 37°C to allow for internalisation. A sample of ECs were maintained at 4°C for use as positive/negative controls. The remaining ECs were then placed on ice, and any un-internalised biotin-labelled proteins stripped off using 100 mM Mesna. The internalised protein fraction was allowed to recycle by incubating the ECs for the indicated timepoints at 37°C. ECs were then returned to ice and incubated in 100 mM Mesna. No Mesna treatment dishes at each timepoint were used as comparative controls. All subsequent stages were performed in the same manner as described in C . The level of the recycled ITGA5 was determined by normalising the amount of ITGA5 quantified from dishes treated with Mesna to the total ITGA5 on the membranes of the Mesna-untreated cells in the same period of incubation. N=3 independent experiments.

    Journal: bioRxiv

    Article Title: NRP2 as an emerging angiogenic player; promoting endothelial cell adhesion and migration by regulating recycling of α5 integrin

    doi: 10.1101/744763

    Figure Lengend Snippet: ITGA5 trafficking in ECs is NRP2 dependent. ( A ): Label-free quantitative mass spectrometry peptide hits identified using MaxQuant software from the Andromeda peptide database. Panel depicts peptide hits detected at a higher fold-change in two control siRNA treated EC lines than those detected from two NRP2 siRNA treated EC lines. NRP2 silencing was confirmed by subjecting EC extracts to Western blot analysis. ( B ): siRNA-transfected ECs were seeded onto FN and incubated for 48 hours. EC extracts were immunoprecipitated by incubation with protein-G Dynabeads® coupled to a NRP2 antibody. Immunoprecipitated complexes were subjected to Western blot analysis using antibodies against clathrin heavy chain-1 (top) and caveolin-1 (bottom). ( C ): siRNA-transfected ECs were seeded onto FN and incubated for 48 hours. ECs were subsequently starved in serum-free media, before being placed on ice. EC cell surface proteins were labelled with 0.3 mg/ml biotin. ECs were then incubated for the indicated timepoints at 37°C, 5% CO 2 . A sample of ECs were maintained at 4°C for use as positive/negative (+/-Mesna) controls. Following incubation at 37°C, ECs were placed on ice and incubated with 100 mM Mesna. EC lysates were then immunoprecipitated with protein-G Dynabeads® coupled to an anti-biotin antibody. Immunoprecipitated biotin-labelled proteins were separated by SDS-PAGE and subjected to Western bolt analysis. The level of internalised ITGA5 at each time of incubation was normalised to the (-) Mesna control. Left panel shows Western blot image, right panel shows the mean densitometric analysis obtained using ImageJ TM . Also shown is confirmation of NRP-2 silencing. N=3 independent experiments. ( D ): Co-immunoprecipitation study as described in B . Immunoprecipitated complexes were subjected to Western blot analysis using an anti-Rab11 antibody. ( E ): siRNA-transfected ECs were prepared as described in C . After biotin surface labelling, ECs were incubated in serum free media for 20 minutes at 37°C to allow for internalisation. A sample of ECs were maintained at 4°C for use as positive/negative controls. The remaining ECs were then placed on ice, and any un-internalised biotin-labelled proteins stripped off using 100 mM Mesna. The internalised protein fraction was allowed to recycle by incubating the ECs for the indicated timepoints at 37°C. ECs were then returned to ice and incubated in 100 mM Mesna. No Mesna treatment dishes at each timepoint were used as comparative controls. All subsequent stages were performed in the same manner as described in C . The level of the recycled ITGA5 was determined by normalising the amount of ITGA5 quantified from dishes treated with Mesna to the total ITGA5 on the membranes of the Mesna-untreated cells in the same period of incubation. N=3 independent experiments.

    Article Snippet: 100 µg protein from each sample was immunoprecipitated by incubation with protein-G Dynabeads® (Invitrogen) coupled to a rabbit anti-NRP2 antibody (clone 3366, Cell Signalling Technology) on a rotator overnight at 4°C.

    Techniques: Mass Spectrometry, Software, Western Blot, Transfection, Incubation, Immunoprecipitation, SDS Page

    FMRP promotes AKAP Rugose expression in the MB learning/memory circuit. (A) RNA immunoprecipitation (RIP) comparing w 1118 genetic background control and dfmr1 null brains ( dfmr1 50M / dfmr1 50M ). Amplified cDNA transcribed from input lysates and RNA pulled down with anti-FMRP-conjugated Dynabeads; futsch is the positive control and α- tubulin is the negative control. Molecular weights are indicated to the right. (B) Western blots of control ( w 1118 ) vs. dfmr1 null ( dfmr1 50M )(top), and driver control ( elav- Gal4/+) vs. UAS- dfmr1 rescue ( elav- Gal4/+; UAS- dfmr1 9557−3 /+; dfmr1 50M /dfmr1 50M ) bottom) brains at 1 day post-eclosion (1dpe). Proteins indicated on left, molecular weights on the right. (C-D) Dot plots of the normalized Rugose band intensities, showing the mean ± SEM. (E) Representative confocal images of anti-Rugose in w 1118 control and dfmr1 null brains. Dashed lines separate MB Kenyon cell somata from surrounding brain tissue. (F) Dot plots of normalized Rugose intensity, showing mean ± SEM. (G) Representative confocal images of anti-Rugose in MB-specific driver control (OK107-Gal4/+) and FMRP overexpression (OE) in MB (UAS- dfmr1 9557−3 /+; OK107-G4/+). Dashed lines separate the MB Kenyon cell somata from surrounding brain tissue. (H) Dot plots of normalized Rugose intensity, showing mean ± SEM. Statistics were done with unpaired Welch’s t-tests. Statistical significance is indicated as n.s. (not significant) and *** (p

    Journal: Neurobiology of disease

    Article Title: Fragile X Mental Retardation Protein Positively Regulates PKA Anchor Rugose and PKA Activity to Control Actin Assembly in Learning/Memory Circuitry

    doi: 10.1016/j.nbd.2019.02.004

    Figure Lengend Snippet: FMRP promotes AKAP Rugose expression in the MB learning/memory circuit. (A) RNA immunoprecipitation (RIP) comparing w 1118 genetic background control and dfmr1 null brains ( dfmr1 50M / dfmr1 50M ). Amplified cDNA transcribed from input lysates and RNA pulled down with anti-FMRP-conjugated Dynabeads; futsch is the positive control and α- tubulin is the negative control. Molecular weights are indicated to the right. (B) Western blots of control ( w 1118 ) vs. dfmr1 null ( dfmr1 50M )(top), and driver control ( elav- Gal4/+) vs. UAS- dfmr1 rescue ( elav- Gal4/+; UAS- dfmr1 9557−3 /+; dfmr1 50M /dfmr1 50M ) bottom) brains at 1 day post-eclosion (1dpe). Proteins indicated on left, molecular weights on the right. (C-D) Dot plots of the normalized Rugose band intensities, showing the mean ± SEM. (E) Representative confocal images of anti-Rugose in w 1118 control and dfmr1 null brains. Dashed lines separate MB Kenyon cell somata from surrounding brain tissue. (F) Dot plots of normalized Rugose intensity, showing mean ± SEM. (G) Representative confocal images of anti-Rugose in MB-specific driver control (OK107-Gal4/+) and FMRP overexpression (OE) in MB (UAS- dfmr1 9557−3 /+; OK107-G4/+). Dashed lines separate the MB Kenyon cell somata from surrounding brain tissue. (H) Dot plots of normalized Rugose intensity, showing mean ± SEM. Statistics were done with unpaired Welch’s t-tests. Statistical significance is indicated as n.s. (not significant) and *** (p

    Article Snippet: Briefly: 100 staged 1dpe heads from w 1118 control and dfmr1 50M null animals were homogenized in lysis buffer (20 mM HEPES, 100 mM NaCl, 2.5 mM EDTA, 0.05% Triton X, 5% glycerol, 1X SigmaFast Protease inhibitor (Sigma S8820), 120 Units/mL Protector RNase inhibitor (Roche 03335399001), then incubated overnight at 4°C with Dynabeads (Invitrogen 10003D) pre-conjugated to mouse anti-FMRP (Abcam 6A15) following the manufacturer’s protocols.

    Techniques: Expressing, Immunoprecipitation, Amplification, Positive Control, Negative Control, Western Blot, Over Expression

    The hPer2 protein forms a ternary complex with hp53 and Mdm2, controlling the extent of hp53 ubiquitination. (A) HEK293 and HCT116 protein extracts (1 and 2 mg, respectively) were incubated with either α-p53 or α-Per2, respectively, and protein A beads. Rabbit IgG was used as a negative control. Immunoprecipitated complexes were analyzed for the presence of hPer2, hp53, and Mdm2 using specific antibodies (top for HEK293 and bottom for HCT116). Asterisk indicates a nonspecific signal. (B) HEK293 cells were transfected with pCS2+ myc -Mdm2, pCS2+ myc -hp53, pCS2+FLAG-hp53, pCS2+FLAG-hPer2, pCS2+ myc -hPer2, or a combination of plasmids and complexes immunoprecipitated using α-FLAG–coupled beads. Complex components were identified by immunoblotting using α-FLAG and α- myc antibodies (right top and bottom). Input amounts were monitored in cell lysates (20 μg) and shown in the left top and bottom. Results similar to those presented were observed in two independent experiments. (C) In vitro–synthesized myc -hPer2 and FLAG-hp53 proteins were preincubated before the addition of myc -Mdm2 (ratio 1:2:5: FLAG-hp53: myc -Mdm2: myc -hPer2). Samples were then subjected to in vitro ubiquitination, followed by immunoprecipitation of hp53-bound proteins using α-FLAG antibody and protein A beads. Bound proteins were detected by immunoblotting and are indicated with arrows (top and middle). FLAG-hp53(Ub) n forms of hp53 were detected using α-ubiquitin antibody (bottom). Asterisk indicates IgG heavy chain. (D) HEK293 cells were transfected with pCS2+ myc -Mdm2, pCS2+ myc -hPer2, pCS2+FLAG-hp53, or a combination of plasmids and collected 12 h after treatment with 10 μM MG132. Cell lysates (100 μg) were incubated with α-FLAG and protein A beads and hp53-ubiquitinated complexes (FLAG-hp53(Ub) n ) detected using α-ubiquitin antibody (top right). Bound proteins were visualized by immunoblotting using α- myc and α-FLAG antibodies (middle and lower right). An in vitro ubiquitination reaction was performed as described in C and is shown as control and for comparison purposes with the “in cells” result (left). Brackets denote ubiquitinated hp53. A, C, and D show immunoblot data from a single experiment that was repeated three times with similar results.

    Journal: Molecular Biology of the Cell

    Article Title: The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells

    doi: 10.1091/mbc.E14-05-0993

    Figure Lengend Snippet: The hPer2 protein forms a ternary complex with hp53 and Mdm2, controlling the extent of hp53 ubiquitination. (A) HEK293 and HCT116 protein extracts (1 and 2 mg, respectively) were incubated with either α-p53 or α-Per2, respectively, and protein A beads. Rabbit IgG was used as a negative control. Immunoprecipitated complexes were analyzed for the presence of hPer2, hp53, and Mdm2 using specific antibodies (top for HEK293 and bottom for HCT116). Asterisk indicates a nonspecific signal. (B) HEK293 cells were transfected with pCS2+ myc -Mdm2, pCS2+ myc -hp53, pCS2+FLAG-hp53, pCS2+FLAG-hPer2, pCS2+ myc -hPer2, or a combination of plasmids and complexes immunoprecipitated using α-FLAG–coupled beads. Complex components were identified by immunoblotting using α-FLAG and α- myc antibodies (right top and bottom). Input amounts were monitored in cell lysates (20 μg) and shown in the left top and bottom. Results similar to those presented were observed in two independent experiments. (C) In vitro–synthesized myc -hPer2 and FLAG-hp53 proteins were preincubated before the addition of myc -Mdm2 (ratio 1:2:5: FLAG-hp53: myc -Mdm2: myc -hPer2). Samples were then subjected to in vitro ubiquitination, followed by immunoprecipitation of hp53-bound proteins using α-FLAG antibody and protein A beads. Bound proteins were detected by immunoblotting and are indicated with arrows (top and middle). FLAG-hp53(Ub) n forms of hp53 were detected using α-ubiquitin antibody (bottom). Asterisk indicates IgG heavy chain. (D) HEK293 cells were transfected with pCS2+ myc -Mdm2, pCS2+ myc -hPer2, pCS2+FLAG-hp53, or a combination of plasmids and collected 12 h after treatment with 10 μM MG132. Cell lysates (100 μg) were incubated with α-FLAG and protein A beads and hp53-ubiquitinated complexes (FLAG-hp53(Ub) n ) detected using α-ubiquitin antibody (top right). Bound proteins were visualized by immunoblotting using α- myc and α-FLAG antibodies (middle and lower right). An in vitro ubiquitination reaction was performed as described in C and is shown as control and for comparison purposes with the “in cells” result (left). Brackets denote ubiquitinated hp53. A, C, and D show immunoblot data from a single experiment that was repeated three times with similar results.

    Article Snippet: Immunoprecipitation and immunoblot assays For (co)immunoprecipitation experiments, transfected cells were harvested in lysis buffer, and extracts (∼100 μg) were incubated with either α-FLAG M2 agarose beads (Sigma-Aldrich) or α-myc (9E10) beads (Santa Cruz Biotechnology, Dallas, TX) either for 2 h or overnight at 4°C with rotation before washing.

    Techniques: Incubation, Negative Control, Immunoprecipitation, Transfection, In Vitro, Synthesized

    The circadian factor hPer2 interacts with hp53. (A) Two-hybrid protein–protein interaction between hPer2, hp53, TCTP, and Cry. Each pair of plasmids—i) pBT-LGF2 + pTRG-Gal11 P , ii) pBT-hPer2 + pTRG, iii) pBT-hPer2 + pTRG-Gal11 P , iv) pBT-hPer2 + pTRG-TCTP, v) pBT-hPer2 + pTRG-Cry, and vi) pTRG-hp53—was grown on nonselective medium plus antibiotics (LB tetracycline [Tet]/chloramphenicol [Cam]) and later patched on both selective screening minimum medium (MM Tet/Cam/5 mM 3-AT) and dual-selective minimum medium containing MM Tet/Cam/5 mM 3-AT/streptomycin (Strep). Positive controls were i and v, whereas ii and iii were negative. (B) Pellets from CHO cells transfected with pCS2+ myc -hp53 were lysed in 25 mM Tris-phosphate pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane- N , N , N ′, N ′-tetraacetic acid, 10% glycerol, and 1% Triton X-100, and extracts (∼300 μg) were incubated with α- myc beads. Endogenous mPer2 and recombinantly expressed hp53 were detected by using either α-Per2 (top) or - myc antibodies (bottom). Control indicates 20 μg of total extract. (C) Samples from CHO cells cotransfected with pCS2+ myc -hPer2 and pCS2+FLAG-hp53 were immunoprecipitated using α- myc beads and immunoblotted using α-FLAG (top) and - myc antibodies (bottom). (D) One milligram of HEK293 and HCT116 extracts was incubated with either α-Per2 or IgG. Complexes were immunoprecipitated using protein A beads and immunoblotted for endogenous proteins using α-Per2 (top) and -p53 antibodies (bottom). For the positive control, HEK293 cells were transfected with pCS2+hPer2 (hPer2-tf), and total cell extracts (20 μg [1/1] and 2 μg [1/10]) were loaded. (E) Recombinant GST-tagged fragments of hPer2 were purified using affinity chromatography, and bound beads were incubated with 35 S-labeled myc -hp53 and assayed for binding as described in the Supplemental Material. Bound complexes were visualized by Coomassie staining (bottom) and the radiolabeled protein detected by autoradiography (top). (F) Mapping of hPer2-binding regions in hp53. Schematic representation of hp53 architecture (393 residues), including the transactivation (TAD; residues 1–42), proline-rich (PRD; residues 61–92), DNA-binding (residues 101–300), and tetramerization domains (TD; residues 326–356). The binding site in hPer2 is indicated with a solid line. Bead-bound GST-hp53 and recombinant proteins were incubated with [ 35 S] myc -hPer2 and analyzed for complex formation as described. In all cases, GST beads were used as a negative control; beads, matrix sample with no antibody bound; EV, empty vector; Supernat, supernatant fraction after immunoprecipitation (IP); transf, transfected cells; B–F show immunoblot data from a single experiment that was repeated three times with similar results.

    Journal: Molecular Biology of the Cell

    Article Title: The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells

    doi: 10.1091/mbc.E14-05-0993

    Figure Lengend Snippet: The circadian factor hPer2 interacts with hp53. (A) Two-hybrid protein–protein interaction between hPer2, hp53, TCTP, and Cry. Each pair of plasmids—i) pBT-LGF2 + pTRG-Gal11 P , ii) pBT-hPer2 + pTRG, iii) pBT-hPer2 + pTRG-Gal11 P , iv) pBT-hPer2 + pTRG-TCTP, v) pBT-hPer2 + pTRG-Cry, and vi) pTRG-hp53—was grown on nonselective medium plus antibiotics (LB tetracycline [Tet]/chloramphenicol [Cam]) and later patched on both selective screening minimum medium (MM Tet/Cam/5 mM 3-AT) and dual-selective minimum medium containing MM Tet/Cam/5 mM 3-AT/streptomycin (Strep). Positive controls were i and v, whereas ii and iii were negative. (B) Pellets from CHO cells transfected with pCS2+ myc -hp53 were lysed in 25 mM Tris-phosphate pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane- N , N , N ′, N ′-tetraacetic acid, 10% glycerol, and 1% Triton X-100, and extracts (∼300 μg) were incubated with α- myc beads. Endogenous mPer2 and recombinantly expressed hp53 were detected by using either α-Per2 (top) or - myc antibodies (bottom). Control indicates 20 μg of total extract. (C) Samples from CHO cells cotransfected with pCS2+ myc -hPer2 and pCS2+FLAG-hp53 were immunoprecipitated using α- myc beads and immunoblotted using α-FLAG (top) and - myc antibodies (bottom). (D) One milligram of HEK293 and HCT116 extracts was incubated with either α-Per2 or IgG. Complexes were immunoprecipitated using protein A beads and immunoblotted for endogenous proteins using α-Per2 (top) and -p53 antibodies (bottom). For the positive control, HEK293 cells were transfected with pCS2+hPer2 (hPer2-tf), and total cell extracts (20 μg [1/1] and 2 μg [1/10]) were loaded. (E) Recombinant GST-tagged fragments of hPer2 were purified using affinity chromatography, and bound beads were incubated with 35 S-labeled myc -hp53 and assayed for binding as described in the Supplemental Material. Bound complexes were visualized by Coomassie staining (bottom) and the radiolabeled protein detected by autoradiography (top). (F) Mapping of hPer2-binding regions in hp53. Schematic representation of hp53 architecture (393 residues), including the transactivation (TAD; residues 1–42), proline-rich (PRD; residues 61–92), DNA-binding (residues 101–300), and tetramerization domains (TD; residues 326–356). The binding site in hPer2 is indicated with a solid line. Bead-bound GST-hp53 and recombinant proteins were incubated with [ 35 S] myc -hPer2 and analyzed for complex formation as described. In all cases, GST beads were used as a negative control; beads, matrix sample with no antibody bound; EV, empty vector; Supernat, supernatant fraction after immunoprecipitation (IP); transf, transfected cells; B–F show immunoblot data from a single experiment that was repeated three times with similar results.

    Article Snippet: Immunoprecipitation and immunoblot assays For (co)immunoprecipitation experiments, transfected cells were harvested in lysis buffer, and extracts (∼100 μg) were incubated with either α-FLAG M2 agarose beads (Sigma-Aldrich) or α-myc (9E10) beads (Santa Cruz Biotechnology, Dallas, TX) either for 2 h or overnight at 4°C with rotation before washing.

    Techniques: Chick Chorioallantoic Membrane Assay, Transfection, Incubation, Immunoprecipitation, Positive Control, Recombinant, Purification, Affinity Chromatography, Labeling, Binding Assay, Staining, Autoradiography, Negative Control, Plasmid Preparation

    Fig. 2. The HisRS-like region contains two independent dimerization domains. ( A ) Co-immunoprecipitation of GST–HisRS with LexA-HA–HisRS from yeast cell extracts. Transformants of gcn2Δ ) bearing HIS3 plasmids encoding LexA-HA–HisRS(970–1497) (pHQ588) or LexA-HA (p2247), and TRP1 plasmids encoding GST (pHQ242) or GST–HisRS(970–1497) (pHQ601), were grown in SC-His-Trp medium and whole-cell extracts were prepared. Aliquots of extracts containing 50 µg of protein were immunoprecipitated with anti-HA antibodies, and the immune complexes were resolved by SDS–PAGE and subjected to western blot analysis using anti-GST antibodies (left panels) or anti-LexA antibodies (right panels). The top panels show results from the transformant expressing GST and the LexA-HA–HisRS fusion; the middle panels derive from the transformant expressing the GST–HisRS and LexA-HA–HisRS fusions; the bottom two panels derive from the transformant expressing the GST–HisRS fusion and LexA-HA. ( B and C ) The HisRS-like region dimerizes in vitro through the N- and C-terminal subregions. The full-length HisRS-like region (aa 970–1497) or truncated segments indicated across the top of each panel were translated in vitro in the presence of [ 35 S]methionine and incubated with either GST, GST–HisRS(970–1156) (B) or GST–HisRS(1315–1497) (C) proteins, which were expressed in E.coli and immobilized on glutathione–Sepharose beads. After extensive washing of the beads, the bound proteins were resolved by SDS–PAGE and visualized by fluorography. Numbers to the left of each gel give the positions of size standards of the indicated molecular weight in kilodaltons. ( D ) Summary of results from the binding assays shown in (B and C). Rectangular boxes with the locations of conserved motifs M1–3 (indicated with diagonal hatching) represent the in vitro translated proteins with GCN2 residue numbers indicated at their N- and C-termini. The binding (+) or failure to bind (–) of these segments to GST–HisRS(970–1156) or GST–HisRS(1315–1497) is summarized to the right and left of the schematics, respectively. ND, not determined. The results of these assays indicate that dimerization by the C-terminal segment of the HisRS-like region is dependent on residues 1315–1383, as indicated by the bar above the boxes. Bars below the boxes represent HisRS-N and HisRS-C subregions, respectively.

    Journal: The EMBO Journal

    Article Title: The tRNA-binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation

    doi: 10.1093/emboj/20.6.1425

    Figure Lengend Snippet: Fig. 2. The HisRS-like region contains two independent dimerization domains. ( A ) Co-immunoprecipitation of GST–HisRS with LexA-HA–HisRS from yeast cell extracts. Transformants of gcn2Δ ) bearing HIS3 plasmids encoding LexA-HA–HisRS(970–1497) (pHQ588) or LexA-HA (p2247), and TRP1 plasmids encoding GST (pHQ242) or GST–HisRS(970–1497) (pHQ601), were grown in SC-His-Trp medium and whole-cell extracts were prepared. Aliquots of extracts containing 50 µg of protein were immunoprecipitated with anti-HA antibodies, and the immune complexes were resolved by SDS–PAGE and subjected to western blot analysis using anti-GST antibodies (left panels) or anti-LexA antibodies (right panels). The top panels show results from the transformant expressing GST and the LexA-HA–HisRS fusion; the middle panels derive from the transformant expressing the GST–HisRS and LexA-HA–HisRS fusions; the bottom two panels derive from the transformant expressing the GST–HisRS fusion and LexA-HA. ( B and C ) The HisRS-like region dimerizes in vitro through the N- and C-terminal subregions. The full-length HisRS-like region (aa 970–1497) or truncated segments indicated across the top of each panel were translated in vitro in the presence of [ 35 S]methionine and incubated with either GST, GST–HisRS(970–1156) (B) or GST–HisRS(1315–1497) (C) proteins, which were expressed in E.coli and immobilized on glutathione–Sepharose beads. After extensive washing of the beads, the bound proteins were resolved by SDS–PAGE and visualized by fluorography. Numbers to the left of each gel give the positions of size standards of the indicated molecular weight in kilodaltons. ( D ) Summary of results from the binding assays shown in (B and C). Rectangular boxes with the locations of conserved motifs M1–3 (indicated with diagonal hatching) represent the in vitro translated proteins with GCN2 residue numbers indicated at their N- and C-termini. The binding (+) or failure to bind (–) of these segments to GST–HisRS(970–1156) or GST–HisRS(1315–1497) is summarized to the right and left of the schematics, respectively. ND, not determined. The results of these assays indicate that dimerization by the C-terminal segment of the HisRS-like region is dependent on residues 1315–1383, as indicated by the bar above the boxes. Bars below the boxes represent HisRS-N and HisRS-C subregions, respectively.

    Article Snippet: Co-immunoprecipitation and immunoblotting were conducted as described previously ( ) using GCN2 antibodies described previously ( ) and GST antibodies purchased from Santa Cruz (1:2000 dilution).

    Techniques: Immunoprecipitation, SDS Page, Western Blot, Expressing, In Vitro, Incubation, Molecular Weight, Binding Assay

    Fig. 5. Effects of mutations in the HisRS-N region on dimerization, tRNA binding and ribosome association by full-length GCN2 in vivo . ( A ) Co-immunoprecipitation assay of dimerization by wild-type or mutant GCN2 proteins with LexA-HA–GCN2. Whole-cell extracts were prepared from transformants of gcn2Δ strain HQY132 bearing high copy-number plasmid pHQ400 encoding LexA-HA–GCN2 and plasmids bearing wild-type GCN2 (p630), gcn2-Δ1018–1047 (pHQ1044), gcn2-Δ1048–1071 (pHQ1045), gcn2-as4 (pHQ1081), gcn2-as3/4 (pHQ1082), gcn2-m2 (p332) or gcn2-Δ1536–1659 ). Lanes 1, 4, 7, 10, 13, 16 and 19: 1/10 of the input (I) amounts of extract used for immunoprecipitations; lanes 2, 5, 8, 11, 14, 17 and 20: 1/2 of the pellet (P) fractions from the immunoprecipitations; lanes 3, 6, 9, 12, 15, 18 and 21: 1/10 of the supernatant (S) fractions from the immunoprecipitations. ( B ) GMSA of GCN2–tRNA complexes. The following affinity-purified FL-tagged GCN2 proteins were incubated with 32 P-labeled total yeast tRNA (0.5 pmol) in 20 µl of RNA-binding buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 7.5 mM MgCl 2 , 10% glycerol) at 30°C for 25 min: FL-GCN2 (2 pmol), FL-gcn2-m2 (2 pmol), FL-gcn2-as3/4 (2 pmol), FL-gcn2-Δ1018–1047 (∼0.4 pmol) and FL-gcn2-Δ1048–1071 (∼0.4 pmol). The [ 32 P]tRNA–GCN2 complexes were resolved by electrophoresis in a 5% polyacrylamide gel in 0.5× Tris-borate-EDTA buffer (3 h, 130 V), dried under vacuum and visualized by autoradiography, with the results shown in the upper panel. The same amounts of each protein used in the binding reactions were resolved by SDS–PAGE and stained with Coomassie Brilliant Blue, with the results shown in the right panel. ( C ) Ribosome binding analysis. Cell extracts were prepared in a buffer lacking Mg 2+ ) harboring high-copy plasmids containing wild-type GCN2 (p630), gcn2-Δ1018–1047 (pHQ1044), gcn2-Δ1048–1071 (pHQ1045), gcn2-as4 (pHQ1081), gcn2-as3/4 (pHQ1082) or gcn2-Δ1536–1659 (p2461). Extracts were resolved by velocity sedimentation in sucrose density gradients and fractions were collected while scanning for A 254 ). Fraction 1 is from the top of the gradient. The first and last lanes contain 1% of the input (I) amounts of extracts separated on the gradients. Positions of free 40S and 60S ribosomal subunits are indicated by bars above the gels.

    Journal: The EMBO Journal

    Article Title: The tRNA-binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation

    doi: 10.1093/emboj/20.6.1425

    Figure Lengend Snippet: Fig. 5. Effects of mutations in the HisRS-N region on dimerization, tRNA binding and ribosome association by full-length GCN2 in vivo . ( A ) Co-immunoprecipitation assay of dimerization by wild-type or mutant GCN2 proteins with LexA-HA–GCN2. Whole-cell extracts were prepared from transformants of gcn2Δ strain HQY132 bearing high copy-number plasmid pHQ400 encoding LexA-HA–GCN2 and plasmids bearing wild-type GCN2 (p630), gcn2-Δ1018–1047 (pHQ1044), gcn2-Δ1048–1071 (pHQ1045), gcn2-as4 (pHQ1081), gcn2-as3/4 (pHQ1082), gcn2-m2 (p332) or gcn2-Δ1536–1659 ). Lanes 1, 4, 7, 10, 13, 16 and 19: 1/10 of the input (I) amounts of extract used for immunoprecipitations; lanes 2, 5, 8, 11, 14, 17 and 20: 1/2 of the pellet (P) fractions from the immunoprecipitations; lanes 3, 6, 9, 12, 15, 18 and 21: 1/10 of the supernatant (S) fractions from the immunoprecipitations. ( B ) GMSA of GCN2–tRNA complexes. The following affinity-purified FL-tagged GCN2 proteins were incubated with 32 P-labeled total yeast tRNA (0.5 pmol) in 20 µl of RNA-binding buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 7.5 mM MgCl 2 , 10% glycerol) at 30°C for 25 min: FL-GCN2 (2 pmol), FL-gcn2-m2 (2 pmol), FL-gcn2-as3/4 (2 pmol), FL-gcn2-Δ1018–1047 (∼0.4 pmol) and FL-gcn2-Δ1048–1071 (∼0.4 pmol). The [ 32 P]tRNA–GCN2 complexes were resolved by electrophoresis in a 5% polyacrylamide gel in 0.5× Tris-borate-EDTA buffer (3 h, 130 V), dried under vacuum and visualized by autoradiography, with the results shown in the upper panel. The same amounts of each protein used in the binding reactions were resolved by SDS–PAGE and stained with Coomassie Brilliant Blue, with the results shown in the right panel. ( C ) Ribosome binding analysis. Cell extracts were prepared in a buffer lacking Mg 2+ ) harboring high-copy plasmids containing wild-type GCN2 (p630), gcn2-Δ1018–1047 (pHQ1044), gcn2-Δ1048–1071 (pHQ1045), gcn2-as4 (pHQ1081), gcn2-as3/4 (pHQ1082) or gcn2-Δ1536–1659 (p2461). Extracts were resolved by velocity sedimentation in sucrose density gradients and fractions were collected while scanning for A 254 ). Fraction 1 is from the top of the gradient. The first and last lanes contain 1% of the input (I) amounts of extracts separated on the gradients. Positions of free 40S and 60S ribosomal subunits are indicated by bars above the gels.

    Article Snippet: Co-immunoprecipitation and immunoblotting were conducted as described previously ( ) using GCN2 antibodies described previously ( ) and GST antibodies purchased from Santa Cruz (1:2000 dilution).

    Techniques: Binding Assay, In Vivo, Co-Immunoprecipitation Assay, Mutagenesis, Plasmid Preparation, Affinity Purification, Incubation, Labeling, RNA Binding Assay, Electrophoresis, Autoradiography, SDS Page, Staining, Sedimentation

    The PLA assay can be used for high-throughput drug screening for targeted compounds in synovial sarcoma From a 16 000 compound small molecule drug library, a compound designated SXT1596 was recognized to significantly decrease relative co-localization of SS18-SSX/TLE1 in human synovial sarcoma cells A. SXT1596 selectively decreases cell viability in synovial sarcoma cell lines, SYO-1, FUJI, Yamato-SS, HS-SY-II, YaFuss and MoJo (white bars), when compared to negative control cell lines HEK293T, MCF7, HeLa and A673 (shaded bars) at a concentration of 5 μM B. Negative control cell lines HeLa and A673 are sensitive to SXT1596 at higher doses. SXT1596 (5-(2-Nitro-1-propenyl)-1,3-benzodioxole) (structure shown in C. ) mediated disruption of the SS18-SSX/TLE1 association was confirmed by immunoprecipitation D. Nuclear SS18-SSX/TLE1 PLA signal in SYO-1 cells decreased following SXT1596 treatment to a degree similar to that obtained with the HDAC inhibitor FK228 E, F. Statistical significance compared to vehicle treatment controls was determined by Student t test: * denotes p

    Journal: Oncotarget

    Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay

    doi: 10.18632/oncotarget.8882

    Figure Lengend Snippet: The PLA assay can be used for high-throughput drug screening for targeted compounds in synovial sarcoma From a 16 000 compound small molecule drug library, a compound designated SXT1596 was recognized to significantly decrease relative co-localization of SS18-SSX/TLE1 in human synovial sarcoma cells A. SXT1596 selectively decreases cell viability in synovial sarcoma cell lines, SYO-1, FUJI, Yamato-SS, HS-SY-II, YaFuss and MoJo (white bars), when compared to negative control cell lines HEK293T, MCF7, HeLa and A673 (shaded bars) at a concentration of 5 μM B. Negative control cell lines HeLa and A673 are sensitive to SXT1596 at higher doses. SXT1596 (5-(2-Nitro-1-propenyl)-1,3-benzodioxole) (structure shown in C. ) mediated disruption of the SS18-SSX/TLE1 association was confirmed by immunoprecipitation D. Nuclear SS18-SSX/TLE1 PLA signal in SYO-1 cells decreased following SXT1596 treatment to a degree similar to that obtained with the HDAC inhibitor FK228 E, F. Statistical significance compared to vehicle treatment controls was determined by Student t test: * denotes p

    Article Snippet: Co-immunoprecipitation and western blots According to the manufacturers protocol, 6 μg of antibody (TLE1: Abcam, ab125183; normal rabbit IgG: Santa-Cruz Biotechnologies, sc-2027) in PBST was added to 50 μL of Dynabeads® Protein G magnetic beads (Life Technologies) for 30 minutes with rotation at room temperature.

    Techniques: Proximity Ligation Assay, High Throughput Screening Assay, Negative Control, Concentration Assay, Immunoprecipitation

    HDAC inhibitors disrupt SS18-SSX/TLE1 co-localization A significant decrease in detectable PLA signal following HDAC inhibition in SYO-1 cells A, B. is also confirmed by immunoprecipitation C. The decrease in PLA co-localization signal correlates with apoptosis induction by HDAC inhibitor FK228 in SYO-1 cells D. A panel of cytotoxic compounds and HDAC1/3 inhibitors were screened on SYO-1 cell at IC 50 doses for 12 hours. While all compounds were effective in decreasing cell viability, only compounds inhibiting class I HDACs were found to decrease the PLA nuclear signal, as a result of disrupting SS18-SSX/TLE1 co-localization E. Scale bars represent 20 μm. Statistical significance compared to vehicle treatment controls was determined by Student t test: * denotes p

    Journal: Oncotarget

    Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay

    doi: 10.18632/oncotarget.8882

    Figure Lengend Snippet: HDAC inhibitors disrupt SS18-SSX/TLE1 co-localization A significant decrease in detectable PLA signal following HDAC inhibition in SYO-1 cells A, B. is also confirmed by immunoprecipitation C. The decrease in PLA co-localization signal correlates with apoptosis induction by HDAC inhibitor FK228 in SYO-1 cells D. A panel of cytotoxic compounds and HDAC1/3 inhibitors were screened on SYO-1 cell at IC 50 doses for 12 hours. While all compounds were effective in decreasing cell viability, only compounds inhibiting class I HDACs were found to decrease the PLA nuclear signal, as a result of disrupting SS18-SSX/TLE1 co-localization E. Scale bars represent 20 μm. Statistical significance compared to vehicle treatment controls was determined by Student t test: * denotes p

    Article Snippet: Co-immunoprecipitation and western blots According to the manufacturers protocol, 6 μg of antibody (TLE1: Abcam, ab125183; normal rabbit IgG: Santa-Cruz Biotechnologies, sc-2027) in PBST was added to 50 μL of Dynabeads® Protein G magnetic beads (Life Technologies) for 30 minutes with rotation at room temperature.

    Techniques: Proximity Ligation Assay, Inhibition, Immunoprecipitation

    SXT1596 decreases cell viability and reactivates EGR1 expression in synovial sarcoma The small molecule SXT1596 induces apoptosis at a similar rate to that of HDAC inhibitor FK228 in SYO-1 cells A, B. and brings about a consistent decrease in SS18-SSX/TLE1 PLA co-localization signal in SYO-1 cells C. The induction of apoptosis correlates with the decrease in PLA co-localization signal D. Chromatin immunoprecipitation at the EGR1 promoter reveals a decrease in H3K27me3 enrichment following SXT1596 treatment E. SXT1596 reactivates EGR1 expression, at the RNA F. and protein G. level. Error bars represent standard error of mean from conditions performed in triplicate. Linear regression was measured by goodness of fit calculations in Prism Graphpad.

    Journal: Oncotarget

    Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay

    doi: 10.18632/oncotarget.8882

    Figure Lengend Snippet: SXT1596 decreases cell viability and reactivates EGR1 expression in synovial sarcoma The small molecule SXT1596 induces apoptosis at a similar rate to that of HDAC inhibitor FK228 in SYO-1 cells A, B. and brings about a consistent decrease in SS18-SSX/TLE1 PLA co-localization signal in SYO-1 cells C. The induction of apoptosis correlates with the decrease in PLA co-localization signal D. Chromatin immunoprecipitation at the EGR1 promoter reveals a decrease in H3K27me3 enrichment following SXT1596 treatment E. SXT1596 reactivates EGR1 expression, at the RNA F. and protein G. level. Error bars represent standard error of mean from conditions performed in triplicate. Linear regression was measured by goodness of fit calculations in Prism Graphpad.

    Article Snippet: Co-immunoprecipitation and western blots According to the manufacturers protocol, 6 μg of antibody (TLE1: Abcam, ab125183; normal rabbit IgG: Santa-Cruz Biotechnologies, sc-2027) in PBST was added to 50 μL of Dynabeads® Protein G magnetic beads (Life Technologies) for 30 minutes with rotation at room temperature.

    Techniques: Expressing, Proximity Ligation Assay, Chromatin Immunoprecipitation

    The proximity ligation assay demonstrates SS18-SSX/TLE1 co-localization selectively in synovial sarcoma cell lines SS18-SSX positive cell lines (SYO-1, FUJI, MoJo) demonstrate SS18-SSX/TLE1 proximity by visualization of fluorescent nuclear foci while control cell lines (MCF7, HeLa, A673) showed little nuclear staining A. The intensity of the nuclear signal was quantified and shown to be significantly greater in SS18-SSX confirmed-positive cell lines B. The SS18-SSX/TLE1 protein-protein interaction was confirmed to be specific for synovial sarcoma cell lines by co-immunoprecipitation C. Scale bars represent 20 μm. Error bars represent standard error of mean from three independent studies.

    Journal: Oncotarget

    Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay

    doi: 10.18632/oncotarget.8882

    Figure Lengend Snippet: The proximity ligation assay demonstrates SS18-SSX/TLE1 co-localization selectively in synovial sarcoma cell lines SS18-SSX positive cell lines (SYO-1, FUJI, MoJo) demonstrate SS18-SSX/TLE1 proximity by visualization of fluorescent nuclear foci while control cell lines (MCF7, HeLa, A673) showed little nuclear staining A. The intensity of the nuclear signal was quantified and shown to be significantly greater in SS18-SSX confirmed-positive cell lines B. The SS18-SSX/TLE1 protein-protein interaction was confirmed to be specific for synovial sarcoma cell lines by co-immunoprecipitation C. Scale bars represent 20 μm. Error bars represent standard error of mean from three independent studies.

    Article Snippet: Co-immunoprecipitation and western blots According to the manufacturers protocol, 6 μg of antibody (TLE1: Abcam, ab125183; normal rabbit IgG: Santa-Cruz Biotechnologies, sc-2027) in PBST was added to 50 μL of Dynabeads® Protein G magnetic beads (Life Technologies) for 30 minutes with rotation at room temperature.

    Techniques: Proximity Ligation Assay, Staining, Immunoprecipitation

    DACH1 promotes adenomas organoid formation via modulating BMP signalling. a DACH1 overexpression induced upregulation of cancer stem cell marker genes. b and c. Gene Ontology (GO) analysis showed that DACH1 overexpression induced the upregulation of stem cell signature genes and the downregulation of cell cycle arrest signature genes. d and e. DACH1 induced the downregulation of ATOH8, TGFβ3, MSX1, NBL1, BMP7, and FKBP4 and the upregulation of FKBP8, ID1, and MAPK3 (experiments were performed in triplicate). f. IF images showing the colocalization of SMAD4 and DACH1 in the nuclei; scale bars=20 μm g-h. DACH1 coprecipitated endogenous SMAD4. Reverse immunoprecipitation was confirmed with an anti-SMAD4 antibody. i. DACH1 overexpression increased the protein level of SMAD4 and decreased the level of phosphorylated SMAD4 (Thr276) [Thr277]. j and k. DACH1 knockdown led to an increase of the mRNA levels of NBL1 and BMP7 compared with the shNC group, and siRNA mediated SMAD4 knockdown in HCT116-shDACH1 cells eliminated the increase. l. DACH1 overexpression was sufficient to compensate for the withdrawal of Noggin and supported the formation of adenoma organoids. m. Addition of Noggin into the culture medium for 24 and 36 h decreased the mRNA levels of NBL1 and BMP7, which were increased by DACH1 knockdown in HCT116 cells. n and o. Overexpression of DACH1 upregulates LGR5, Notch1 and the protein level of NICD, while did not induce significant upregulation of HES1. Scale bars=20 μm. For 5d, 5e, 5k, 5 m and 5n, ** P

    Journal: EBioMedicine

    Article Title: Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling

    doi: 10.1016/j.ebiom.2020.102800

    Figure Lengend Snippet: DACH1 promotes adenomas organoid formation via modulating BMP signalling. a DACH1 overexpression induced upregulation of cancer stem cell marker genes. b and c. Gene Ontology (GO) analysis showed that DACH1 overexpression induced the upregulation of stem cell signature genes and the downregulation of cell cycle arrest signature genes. d and e. DACH1 induced the downregulation of ATOH8, TGFβ3, MSX1, NBL1, BMP7, and FKBP4 and the upregulation of FKBP8, ID1, and MAPK3 (experiments were performed in triplicate). f. IF images showing the colocalization of SMAD4 and DACH1 in the nuclei; scale bars=20 μm g-h. DACH1 coprecipitated endogenous SMAD4. Reverse immunoprecipitation was confirmed with an anti-SMAD4 antibody. i. DACH1 overexpression increased the protein level of SMAD4 and decreased the level of phosphorylated SMAD4 (Thr276) [Thr277]. j and k. DACH1 knockdown led to an increase of the mRNA levels of NBL1 and BMP7 compared with the shNC group, and siRNA mediated SMAD4 knockdown in HCT116-shDACH1 cells eliminated the increase. l. DACH1 overexpression was sufficient to compensate for the withdrawal of Noggin and supported the formation of adenoma organoids. m. Addition of Noggin into the culture medium for 24 and 36 h decreased the mRNA levels of NBL1 and BMP7, which were increased by DACH1 knockdown in HCT116 cells. n and o. Overexpression of DACH1 upregulates LGR5, Notch1 and the protein level of NICD, while did not induce significant upregulation of HES1. Scale bars=20 μm. For 5d, 5e, 5k, 5 m and 5n, ** P

    Article Snippet: 2.8 Western blotting (WB) and co-immunoprecipitation (CoIP)For WB, briefly, primary antibodies were purchased from Cell signalling Technology, Affinity Biosciences (OH, USA), Abclonal, Proteintech, or MBL (Janpan).

    Techniques: Over Expression, Marker, Immunoprecipitation

    EIN3 antibody reproducibly enriches DNA in chromatin immunoprecipitation. ( A ) Enrichment of the known target of EIN3, the promoter of ERF1, using Dynabeads Protein A and Dynabeads Sheep anti-Rabbit IgG to collect protein-DNA complexes. The average fold change for two technical ChIP replicates with three QPCR technical replicates each is shown. ( B ) Reproducibility in the two biological replicates for EIN3 ChIP performed upon treatment of ethylene gas for 0, 0.5, 1, and 4 hr. ( C ) Average RPKM of EIN3 binding sites 0, 0.5, 1, and 4 hr of ethylene gas treatment. ( D ) EIN3 binding preferentially occurs in the promoter regions of genes (1000 bp upstream of the TSS). DOI: http://dx.doi.org/10.7554/eLife.00675.004

    Journal: eLife

    Article Title: Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis

    doi: 10.7554/eLife.00675

    Figure Lengend Snippet: EIN3 antibody reproducibly enriches DNA in chromatin immunoprecipitation. ( A ) Enrichment of the known target of EIN3, the promoter of ERF1, using Dynabeads Protein A and Dynabeads Sheep anti-Rabbit IgG to collect protein-DNA complexes. The average fold change for two technical ChIP replicates with three QPCR technical replicates each is shown. ( B ) Reproducibility in the two biological replicates for EIN3 ChIP performed upon treatment of ethylene gas for 0, 0.5, 1, and 4 hr. ( C ) Average RPKM of EIN3 binding sites 0, 0.5, 1, and 4 hr of ethylene gas treatment. ( D ) EIN3 binding preferentially occurs in the promoter regions of genes (1000 bp upstream of the TSS). DOI: http://dx.doi.org/10.7554/eLife.00675.004

    Article Snippet: We then substituted Dynabeads Protein A (Invitrogen, cat#100-1D) and Dynabeads M-280 Sheep anti-Rabbit IgG (Invitrogen, cat#112-04D) for the salmon sperm DNA blocked Protein A agarose beads recommended in the protocol (4), as to avoid sequencing of salmon sperm DNA.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    miR503HG physically interacts with HNRNPA2B1 in HCC cells. (A) RNA pull-down assays were performed in SMMC-7721 cells twice. Biotinylated miR503HG (left lane) or antisense RNA (right lane) was incubated with cell extracts and targeted with streptavidin beads; the associated proteins were resolved on a gel. The highlighted region was submitted for mass spectrometry. (B) Western blotting analysis of the specific association of HNRNPA2B1 with miR503HG in SMMC-7721 and Huh7 cells. (C) RIP enrichment was determined as RNA associated with the HNRNPA2B1 IP relative to an input control. The data represent the average and standard deviation of three independent experiments. (D) Deletion mapping of the HNRNPA2B1-binding domain in miR503HG. Top: graphic illustration of predicted miR503HG secondary structure (LNCipedia, http://www.lncipedia.org ), and the truncation of miR503HG according to the stem-loop structure. Middle: the in vitro transcribed full-length and deletion fragments of miR503HG. Bottom: Western blotting analysis of HNRNPA2B1 in protein samples pulled down by the different miR503HG constructs. (E) Deletion mapping of the miR503HG-binding domain in HNRNPA2B1. Top: diagrams of full-length HNRNPA2B1 and the domain-truncated fragments RNA recognition motif (RRM), hnRNP K homology domain (KH), M9 signal (MN). Bottom: qPCR analysis of HNRNPA2B1 retrieved by full-length or domain-truncated HNRNPA2B1-HA using a HA antibody. RIP assays were performed using Huh7 cells transfected with the indicated vectors.

    Journal: Theranostics

    Article Title: Long noncoding RNA miR503HG, a prognostic indicator, inhibits tumor metastasis by regulating the HNRNPA2B1/NF-κB pathway in hepatocellular carcinoma

    doi: 10.7150/thno.23012

    Figure Lengend Snippet: miR503HG physically interacts with HNRNPA2B1 in HCC cells. (A) RNA pull-down assays were performed in SMMC-7721 cells twice. Biotinylated miR503HG (left lane) or antisense RNA (right lane) was incubated with cell extracts and targeted with streptavidin beads; the associated proteins were resolved on a gel. The highlighted region was submitted for mass spectrometry. (B) Western blotting analysis of the specific association of HNRNPA2B1 with miR503HG in SMMC-7721 and Huh7 cells. (C) RIP enrichment was determined as RNA associated with the HNRNPA2B1 IP relative to an input control. The data represent the average and standard deviation of three independent experiments. (D) Deletion mapping of the HNRNPA2B1-binding domain in miR503HG. Top: graphic illustration of predicted miR503HG secondary structure (LNCipedia, http://www.lncipedia.org ), and the truncation of miR503HG according to the stem-loop structure. Middle: the in vitro transcribed full-length and deletion fragments of miR503HG. Bottom: Western blotting analysis of HNRNPA2B1 in protein samples pulled down by the different miR503HG constructs. (E) Deletion mapping of the miR503HG-binding domain in HNRNPA2B1. Top: diagrams of full-length HNRNPA2B1 and the domain-truncated fragments RNA recognition motif (RRM), hnRNP K homology domain (KH), M9 signal (MN). Bottom: qPCR analysis of HNRNPA2B1 retrieved by full-length or domain-truncated HNRNPA2B1-HA using a HA antibody. RIP assays were performed using Huh7 cells transfected with the indicated vectors.

    Article Snippet: RNA immunoprecipitation RNA immunoprecipitation (RIP) experiments were performed with a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions.

    Techniques: Incubation, Mass Spectrometry, Western Blot, Standard Deviation, Binding Assay, In Vitro, Construct, Real-time Polymerase Chain Reaction, Transfection

    N-Terminal DNA Binding Domain of GTL1 Binds to PHYA and SDD1 Promoters. (A) Schematic diagram of the GTL1 protein, showing the four helical, N- and C-terminal DNA binding domains (represented as gray cylinders). Two protein fragments (Nt1 and GTL1N) were fused with MBP. (B) Nt1 and GTL1N polypeptides and a biotin-labeled rice PHYA promoter fragment (400 ng) were used for EMSA. In (B) to (E) , free probe and GTL1N-probe complexes are indicated by an asterisk and arrows, respectively. (C) Unlabeled PHYA promoter (2 μg) was used as a competitor to determine the specificity of DNA binding activity for GTL1N. (D) GTL1N polypeptide was used for EMSA with a biotin-labeled SDD1 promoter fragment (250 ng). Unlabeled probes (250 ng [ + ] and 1 μg [ ++ ]) were used as competitors. An unlabeled mutant version of the SDD1 promoter (GG → CC) was used as a noncompetitor. (E) The mutant version of the SDD1 promoter (GG → CC) was labeled with biotin (250 ng) and used for EMSA with GTL1N polypeptides. (F) ChIP analysis was conducted to determine the in vivo interaction between GTL1 with the SDD1 promoter. Input is chromatin before immunoprecipitation. Anti-GFP antibody was used to precipitate chromatin associated with GTL1-GFP. Mouse IgG was used as a negative control for the specificity of immunoprecipitation. The SDD1 promoter region associated with GTL1 was amplified by PCR using SDD1 promoter-specific primers for three different regions (1, 2, and 3). Region 1 (−1136 to −1400) is distal to the GT3 box. Region 2 (−538 to −824) is adjacent to, but does not contain, the GT3 box. Region 3 (−279 to −556) includes the GT3 box in the SDD1 promoter.

    Journal: The Plant Cell

    Article Title: The Arabidopsis GTL1 Transcription Factor Regulates Water Use Efficiency and Drought Tolerance by Modulating Stomatal Density via Transrepression of SDD1 [W] [W] [OA]

    doi: 10.1105/tpc.110.078691

    Figure Lengend Snippet: N-Terminal DNA Binding Domain of GTL1 Binds to PHYA and SDD1 Promoters. (A) Schematic diagram of the GTL1 protein, showing the four helical, N- and C-terminal DNA binding domains (represented as gray cylinders). Two protein fragments (Nt1 and GTL1N) were fused with MBP. (B) Nt1 and GTL1N polypeptides and a biotin-labeled rice PHYA promoter fragment (400 ng) were used for EMSA. In (B) to (E) , free probe and GTL1N-probe complexes are indicated by an asterisk and arrows, respectively. (C) Unlabeled PHYA promoter (2 μg) was used as a competitor to determine the specificity of DNA binding activity for GTL1N. (D) GTL1N polypeptide was used for EMSA with a biotin-labeled SDD1 promoter fragment (250 ng). Unlabeled probes (250 ng [ + ] and 1 μg [ ++ ]) were used as competitors. An unlabeled mutant version of the SDD1 promoter (GG → CC) was used as a noncompetitor. (E) The mutant version of the SDD1 promoter (GG → CC) was labeled with biotin (250 ng) and used for EMSA with GTL1N polypeptides. (F) ChIP analysis was conducted to determine the in vivo interaction between GTL1 with the SDD1 promoter. Input is chromatin before immunoprecipitation. Anti-GFP antibody was used to precipitate chromatin associated with GTL1-GFP. Mouse IgG was used as a negative control for the specificity of immunoprecipitation. The SDD1 promoter region associated with GTL1 was amplified by PCR using SDD1 promoter-specific primers for three different regions (1, 2, and 3). Region 1 (−1136 to −1400) is distal to the GT3 box. Region 2 (−538 to −824) is adjacent to, but does not contain, the GT3 box. Region 3 (−279 to −556) includes the GT3 box in the SDD1 promoter.

    Article Snippet: Anti-GFP antibody bound to GTL-GFP-chromatin complexes was incubated with protein G agarose beads for 1 h at 4°C, washed several times, and eluted with elution buffer according to the manufacturer’s protocol (EZ ChIP chromatin immunoprecipitation kit; Millipore).

    Techniques: Binding Assay, Labeling, Activity Assay, Mutagenesis, Chromatin Immunoprecipitation, In Vivo, Immunoprecipitation, Negative Control, Amplification, Polymerase Chain Reaction

    Immunoprecipitation reveals interaction between Flag-MAVS and PB2-GFP. (A) Lysates from 293T cells transfected with Flag-MAVS and pcDNA-PB2-GFP or pcDNA-PB1-GFP and immunoprecipitates (IP) were analyzed by Western blotting using anti-Flag and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-Flag antibody. (B) Lysates from 293T cells transfected with Flag-MAVS and pcDNA-PB2-GFP or pcDNA-PB2 N9D-GFP and immunoprecipitates were analyzed by Western blotting using anti-Flag and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-Flag antibody. (C) Lysates from 293T cells transfected with pcDNA-PB2-GFP or pcDNA-PB2 N9D-GFP and immunoprecipitates were analyzed by Western blotting using anti-MAVS and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-MAVS antibody. α, anti.

    Journal: Journal of Virology

    Article Title: The PB2 Subunit of the Influenza Virus RNA Polymerase Affects Virulence by Interacting with the Mitochondrial Antiviral Signaling Protein and Inhibiting Expression of Beta Interferon ▿

    doi: 10.1128/JVI.00879-10

    Figure Lengend Snippet: Immunoprecipitation reveals interaction between Flag-MAVS and PB2-GFP. (A) Lysates from 293T cells transfected with Flag-MAVS and pcDNA-PB2-GFP or pcDNA-PB1-GFP and immunoprecipitates (IP) were analyzed by Western blotting using anti-Flag and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-Flag antibody. (B) Lysates from 293T cells transfected with Flag-MAVS and pcDNA-PB2-GFP or pcDNA-PB2 N9D-GFP and immunoprecipitates were analyzed by Western blotting using anti-Flag and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-Flag antibody. (C) Lysates from 293T cells transfected with pcDNA-PB2-GFP or pcDNA-PB2 N9D-GFP and immunoprecipitates were analyzed by Western blotting using anti-MAVS and anti-GFP antibodies. Immunoprecipitations of MAVS were performed with anti-MAVS antibody. α, anti.

    Article Snippet: Clarified cell lysate (150 μl) was used for immunoprecipitations with 5 μl of rabbit anti-OctA-Probe (Flag) (Santa Cruz) or rabbit anti-MAVS (Abcam) and 6 mg of protein A-Sepharose (Sigma Aldrich).

    Techniques: Immunoprecipitation, Transfection, Western Blot

    Pulse–chase analysis of intracellular PC-wt and PC-A267T in stably transfected CHO-K1 cells. Stably transfected CHO-K1 cells were pulse-labeled for 30 min with [ 35 S]methionine and chased for 0, 1.5, 3 and 6 h. Equivalent amounts of cell extracts were immunoprecipitated using a polyclonal antibody against PC. Immunoprecipitates were analyzed by SDS-PAGE under reducing conditions (A). The amount of radioactivity remaining at each time point was plotted as the percentage of that measured in the immunoprecipate from unchased cells. The mean values from two independent experiments are presented. The turnover rates of the intracellular PC-wt and PC-A267T are depicted as • and ▴, respectively (B).

    Journal: PLoS ONE

    Article Title: Protein C Mutation (A267T) Results in ER Retention and Unfolded Protein Response Activation

    doi: 10.1371/journal.pone.0024009

    Figure Lengend Snippet: Pulse–chase analysis of intracellular PC-wt and PC-A267T in stably transfected CHO-K1 cells. Stably transfected CHO-K1 cells were pulse-labeled for 30 min with [ 35 S]methionine and chased for 0, 1.5, 3 and 6 h. Equivalent amounts of cell extracts were immunoprecipitated using a polyclonal antibody against PC. Immunoprecipitates were analyzed by SDS-PAGE under reducing conditions (A). The amount of radioactivity remaining at each time point was plotted as the percentage of that measured in the immunoprecipate from unchased cells. The mean values from two independent experiments are presented. The turnover rates of the intracellular PC-wt and PC-A267T are depicted as • and ▴, respectively (B).

    Article Snippet: The immunoprecipitates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) and the bands were visualized using autoradiography.

    Techniques: Pulse Chase, Stable Transfection, Transfection, Labeling, Immunoprecipitation, SDS Page, Radioactivity

    MUC13-Glut-1 interaction is disrupted by Sulfasalazine. a Confocal immunofluorescence of MUC13 and Glut-1 in MUC13-expressing, HPAF-II, and AsPC1 cells depicting co-localization (yellow color). Cells were cultured in 4-well chambered slides, fixed, permeabilized, stained with indicated antibodies and analyzed for confocal microscopy. DAPI was used as a counter stain for the nucleus. b Cells were treated with sulphasalazine (0.9 mM) for 24 h and processed for immunostaining and confocal microscopy. Images were captured at ×400. c , d Reciprocal co-immunoprecipitation assay between MUC13 and Glut-1 performed using protein lysates from HPAF-II cells. Cells were treated with sulfasalazine (0.9 mM) for 24 h and protein lysates prepared. The immunoprecipitates were resolved on a 10% gel, and followed by immunoblotting with anti-MUC13 and anti-Glut-1 antibodies. e Lactate assay was performed in MUC13-expressing and MUC13-null cells after treatment with or without sulfasalazine. Cell culture media was collected after 48 h to measure the amount of l -lactate concentration using lactate assay kit

    Journal: Oncogenesis

    Article Title: MUC13 contributes to rewiring of glucose metabolism in pancreatic cancer

    doi: 10.1038/s41389-018-0031-0

    Figure Lengend Snippet: MUC13-Glut-1 interaction is disrupted by Sulfasalazine. a Confocal immunofluorescence of MUC13 and Glut-1 in MUC13-expressing, HPAF-II, and AsPC1 cells depicting co-localization (yellow color). Cells were cultured in 4-well chambered slides, fixed, permeabilized, stained with indicated antibodies and analyzed for confocal microscopy. DAPI was used as a counter stain for the nucleus. b Cells were treated with sulphasalazine (0.9 mM) for 24 h and processed for immunostaining and confocal microscopy. Images were captured at ×400. c , d Reciprocal co-immunoprecipitation assay between MUC13 and Glut-1 performed using protein lysates from HPAF-II cells. Cells were treated with sulfasalazine (0.9 mM) for 24 h and protein lysates prepared. The immunoprecipitates were resolved on a 10% gel, and followed by immunoblotting with anti-MUC13 and anti-Glut-1 antibodies. e Lactate assay was performed in MUC13-expressing and MUC13-null cells after treatment with or without sulfasalazine. Cell culture media was collected after 48 h to measure the amount of l -lactate concentration using lactate assay kit

    Article Snippet: Immunoprecipitates were eluted with SDS sample buffer obtained from Santa Cruz Biotechnology, TX, USA following the procedures as described earlier [ , ,].

    Techniques: Immunofluorescence, Expressing, Cell Culture, Staining, Confocal Microscopy, Immunostaining, Co-Immunoprecipitation Assay, Lactate Assay, Concentration Assay

    A. Cdk2 association with PP1 binding partners. Mass spectrometric analysis of Cdk2 immunoprecipitates (lysates pooled from 3 independent experiments) from resting or thrombin exposed platelets identified cdk2 binding to a subset of PP1 regulatory proteins. Peptide counts identified for proteins listed under each incubation condition shown; PPP1R12A was most prevalent. B. Co-immunoprecipitation of Cdk2, Erk, PPP1CA and PP1R12A. Lysates of platelets under resting or thrombin-exposed (T, 1 minute, 0.5 U/ml) platelets immunoprecipitated with polyclonal cdk2 or monoclonal Erk1/2 or PPP1CA antibodies as indicated were subjected to Western Blotting. Molecular weight ladder lane excised (gap); PPP1R12A input run on parallel gel. C . Erk and PPP1R12a-associated phosphatase activity. Immunoprecipitates of platelets exposed to vehicle (−), thrombin (T) or roscovitine plus thrombin (RT) were reacted with a PP1-specific phosphatase substrate and liberation of phosphate measured.

    Journal: bioRxiv

    Article Title: Cyclin-dependent kinase 2 (Cdk2) controls phosphatase-regulated signaling and function in platelets

    doi: 10.1101/2020.05.31.126953

    Figure Lengend Snippet: A. Cdk2 association with PP1 binding partners. Mass spectrometric analysis of Cdk2 immunoprecipitates (lysates pooled from 3 independent experiments) from resting or thrombin exposed platelets identified cdk2 binding to a subset of PP1 regulatory proteins. Peptide counts identified for proteins listed under each incubation condition shown; PPP1R12A was most prevalent. B. Co-immunoprecipitation of Cdk2, Erk, PPP1CA and PP1R12A. Lysates of platelets under resting or thrombin-exposed (T, 1 minute, 0.5 U/ml) platelets immunoprecipitated with polyclonal cdk2 or monoclonal Erk1/2 or PPP1CA antibodies as indicated were subjected to Western Blotting. Molecular weight ladder lane excised (gap); PPP1R12A input run on parallel gel. C . Erk and PPP1R12a-associated phosphatase activity. Immunoprecipitates of platelets exposed to vehicle (−), thrombin (T) or roscovitine plus thrombin (RT) were reacted with a PP1-specific phosphatase substrate and liberation of phosphate measured.

    Article Snippet: Immunoprecipitation of Proteins in human platelets To limit the interference of IgG from immunoprecipitation the Thermo-Fisher Pierce CO-Immunoprecipitation kit (Catalog #26149, ThermoFisher, Rockford, IL) was used to perform co-immunoprecipitations.

    Techniques: Binding Assay, Incubation, Immunoprecipitation, Western Blot, Molecular Weight, Activity Assay

    Cdk2 associates with and phosphorylates PPP1CA in platelets, inhibiting its activity. A. Phosphorylation of PPP1CA in thrombin activated platelets. 2 exposures of total and phospho-PPP1CA shown; the lower PPP1CA band is non-phosphorylated. B . Co-immunoprecipitation of PPP1CA with cdk2; - (DMSO vehicle), T (0.5 U/ml thrombin), R (roscovitine), 2i (cdk2 peptide inhibitor); 10 minute exposure to thrombin before pulldown. C . Immunofluorescence staining of platelets for PPP1CA (PP1) and Cdk2. Absence of DAPI signal confirmed absence of nucleated cells. 250x, bar 10 um. D . Kinase assay. Cdk2 (or Ig control) immunoprecipitates were incubated with recombinant PPP1CA (input, 0.5 ug/Rx) followed by WB of reaction mixture for phospho-PPP1CA. E . Phosphatase assay. Immunoprecipitates of platelets exposed to vehicle (−), thrombin (T) or roscovitine plus thrombin (RT) were reacted with a PP1/2a-specific phosphatase substrate and liberation of phosphate measured.

    Journal: bioRxiv

    Article Title: Cyclin-dependent kinase 2 (Cdk2) controls phosphatase-regulated signaling and function in platelets

    doi: 10.1101/2020.05.31.126953

    Figure Lengend Snippet: Cdk2 associates with and phosphorylates PPP1CA in platelets, inhibiting its activity. A. Phosphorylation of PPP1CA in thrombin activated platelets. 2 exposures of total and phospho-PPP1CA shown; the lower PPP1CA band is non-phosphorylated. B . Co-immunoprecipitation of PPP1CA with cdk2; - (DMSO vehicle), T (0.5 U/ml thrombin), R (roscovitine), 2i (cdk2 peptide inhibitor); 10 minute exposure to thrombin before pulldown. C . Immunofluorescence staining of platelets for PPP1CA (PP1) and Cdk2. Absence of DAPI signal confirmed absence of nucleated cells. 250x, bar 10 um. D . Kinase assay. Cdk2 (or Ig control) immunoprecipitates were incubated with recombinant PPP1CA (input, 0.5 ug/Rx) followed by WB of reaction mixture for phospho-PPP1CA. E . Phosphatase assay. Immunoprecipitates of platelets exposed to vehicle (−), thrombin (T) or roscovitine plus thrombin (RT) were reacted with a PP1/2a-specific phosphatase substrate and liberation of phosphate measured.

    Article Snippet: Immunoprecipitation of Proteins in human platelets To limit the interference of IgG from immunoprecipitation the Thermo-Fisher Pierce CO-Immunoprecipitation kit (Catalog #26149, ThermoFisher, Rockford, IL) was used to perform co-immunoprecipitations.

    Techniques: Activity Assay, Immunoprecipitation, Immunofluorescence, Staining, Kinase Assay, Incubation, Recombinant, Western Blot, Phosphatase Assay

    Platelets were unexposed or exposed to thrombin (0.25 U/ml) as indicated for 10 minutes following a 5 minute preincubation with DMSO vehicle (resting), palbociclib (150 ng/ml), roscovitine (5 ug/ml) or cdk2 inhibitory peptide (10 uM, cdk2i). Following immunoprecipitation with polyclonal cdk2 antibody ( ), a kinase assay was performed using recombinant p-RB amino acids -. A recombinant cdk2/cyclinE complex was used as a positive control, and kinase reactions were immunoblotted with anti-Phospho-Rb 807/811 antibody (Cell Signaling Technologies, #9308). Results show Rb phosphorylation by immunoprecipitated Cdk2 from thrombin exposed platelets, and blockade of phosphorylation by cdk2 (roscovitine, cdk2i) but not cdk4/6 (palbociclib) inhibitor.

    Journal: bioRxiv

    Article Title: Cyclin-dependent kinase 2 (Cdk2) controls phosphatase-regulated signaling and function in platelets

    doi: 10.1101/2020.05.31.126953

    Figure Lengend Snippet: Platelets were unexposed or exposed to thrombin (0.25 U/ml) as indicated for 10 minutes following a 5 minute preincubation with DMSO vehicle (resting), palbociclib (150 ng/ml), roscovitine (5 ug/ml) or cdk2 inhibitory peptide (10 uM, cdk2i). Following immunoprecipitation with polyclonal cdk2 antibody ( ), a kinase assay was performed using recombinant p-RB amino acids -. A recombinant cdk2/cyclinE complex was used as a positive control, and kinase reactions were immunoblotted with anti-Phospho-Rb 807/811 antibody (Cell Signaling Technologies, #9308). Results show Rb phosphorylation by immunoprecipitated Cdk2 from thrombin exposed platelets, and blockade of phosphorylation by cdk2 (roscovitine, cdk2i) but not cdk4/6 (palbociclib) inhibitor.

    Article Snippet: Immunoprecipitation of Proteins in human platelets To limit the interference of IgG from immunoprecipitation the Thermo-Fisher Pierce CO-Immunoprecipitation kit (Catalog #26149, ThermoFisher, Rockford, IL) was used to perform co-immunoprecipitations.

    Techniques: Immunoprecipitation, Kinase Assay, Recombinant, Positive Control

    CtBP effects on p300 and NF-κB acetylation. a Chromatin immunoprecipitation (ChIP) with antibody to HDAC1, acetyl H3, and p65 was performed in RAW264.7 cells to evaluate binding to the IL-6 promoter regions. LPS increased all three signals, but only the effect of p65 binding was reversed by 2DG (conditions as in Fig. 1c ); n = 3, * p

    Journal: Nature Communications

    Article Title: Bioenergetic state regulates innate inflammatory responses through the transcriptional co-repressor CtBP

    doi: 10.1038/s41467-017-00707-0

    Figure Lengend Snippet: CtBP effects on p300 and NF-κB acetylation. a Chromatin immunoprecipitation (ChIP) with antibody to HDAC1, acetyl H3, and p65 was performed in RAW264.7 cells to evaluate binding to the IL-6 promoter regions. LPS increased all three signals, but only the effect of p65 binding was reversed by 2DG (conditions as in Fig. 1c ); n = 3, * p

    Article Snippet: Co-immunoprecipitation and western blots Cells were lysed with M-PER reagent (Pierce) supplemented with benzonase nuclease (Novagen) and protease inhibitors (Roche).

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    Direct inhibition of CtBP dimerization blocks LPS-induced pro-inflammatory gene expression. a Schematic of CtBP1 protein showing functional domains and alignment with the CtBP peptide used to block CtBP dimerization. PLDLS indicates substrate binding domain. The CtBP blocking peptide includes an N-terminal TAT sequence for cellular internalization. b Immunoprecipitation assay showing ability of CtBP peptide (50 µm) to block dimerization of tagged CtBP proteins incubated with 10 mM lactate + 10 µm NADH. Full length immunoblots are shown in Supplementary Fig. 3 . Immunoprecipitation was performed with anti-Flag antibody, and CtBP1-Flag/CtBP1-HA heterodimers were detected by western blots using anti-HA antibody. Lysate control lane = 5% of input used in immunoprecipitated samples. n = 4. ** p

    Journal: Nature Communications

    Article Title: Bioenergetic state regulates innate inflammatory responses through the transcriptional co-repressor CtBP

    doi: 10.1038/s41467-017-00707-0

    Figure Lengend Snippet: Direct inhibition of CtBP dimerization blocks LPS-induced pro-inflammatory gene expression. a Schematic of CtBP1 protein showing functional domains and alignment with the CtBP peptide used to block CtBP dimerization. PLDLS indicates substrate binding domain. The CtBP blocking peptide includes an N-terminal TAT sequence for cellular internalization. b Immunoprecipitation assay showing ability of CtBP peptide (50 µm) to block dimerization of tagged CtBP proteins incubated with 10 mM lactate + 10 µm NADH. Full length immunoblots are shown in Supplementary Fig. 3 . Immunoprecipitation was performed with anti-Flag antibody, and CtBP1-Flag/CtBP1-HA heterodimers were detected by western blots using anti-HA antibody. Lysate control lane = 5% of input used in immunoprecipitated samples. n = 4. ** p

    Article Snippet: Co-immunoprecipitation and western blots Cells were lysed with M-PER reagent (Pierce) supplemented with benzonase nuclease (Novagen) and protease inhibitors (Roche).

    Techniques: Inhibition, Expressing, Functional Assay, Blocking Assay, Binding Assay, Sequencing, Immunoprecipitation, Incubation, Western Blot

    Establishment of our drug screening model. (A) Schematic representation of the drug screening model. Beclin1 plays several important roles in autophagy, the dissociation of Beclin1 from Bcl2 is essential for autophagy. Autophagy either supports IAV replication or induces autophagic cell death which may result in acute lung injury (left). Bcl2 binds with Beclin1 to form Beclin1-Bcl2 heterodimer and inhibits autophagy (right). Beclin1-binding proteins (such as MyD88, TRIF and HMGB1), Bcl2-binding proteins (such as BNIP3, Bad, Noxa, Puma, BimEL and Bik) and the activations of ERK1/2, JNK1, IKK, TLRs and DAPK signal pathways under oxidative stress, endoplasmic reticulum (ER) stress and energy stress can promote the dissociation of Beclin1 from Bcl2, and finally promote autophagy. IAV infection can elevate the expressions of many aforementioned proteins and activate aforementioned signal pathways. Drugs inhibiting oxidative stress, ER stress, energy stress, and the activations of ERK1/2, JNK1, IKK, TLRs and DAPK signal pathways may inhibit the dissociation of Beclin1 from Bcl2, sequentially inhibit autophagy, and finally impair IAV replication or inhibit autophagic cell death and acute lung injury. In our study, Beclin1 and Bcl2 were fused with C’- and N’- fragments of a red fluorescence protein (RFP), respectively. The amino acid sequence of the linker was RPACKIPNDLKQKVMNH. After cotransfection, the intact RFP would reconstitute through the interaction of Beclin1 and Bcl2. The fluorescence intensity (FI) of the reconstituted RFP, which was determined at 610 nm after excitation at 587 nm using a microplate reader (Tecan infinite M1000), represents the level of the Beclin1-Bcl2 heterodimer, which is positively relevant with the degree of autophagy inhibition and antiviral activity. (B) Reconstitution of RFP. A549 cells were transfected or cotransfected with pMN-Bcl2 and pMC-Beclin1, after 8 h, only cotransfected cells appeared a lot of red fluorescence. (C) The influence of HMGB1 and MyD88 on the Beclin1-Bcl2 heterodimer. Beclin1-binding proteins HMGB1 and MyD88 were expected to disrupt the Beclin1-Bcl2 heterodimer, after cotransfection as the graph indicated, the FI was really significantly decreased, and the cells of same batch were subjected to the co-immunoprecipitation (co-IP) assay (right), the results satisfied our expectation. (D) The influence of ERK1/2 inhibitor (U0126, 10 µM), ERK1/2 activator (EGF, 100 ng/ml), JNK/p38 inhibitor (SB203580, 40 µM), p38 MAPK activator (anisomycin, 10 µM), antioxidant (NAC, 2 mM) and oxidant (H2O2, 100 µM) on the dissociation of Beclin1-Bcl2 heterodimer. After cotransfection, A549 cells were treated with these inhibitors and activators, after 8 h, the FI was measured, the inhibitors (U0126, SB203580) and antioxidant (NAC) could significantly elevate the FI, which meant that they could inhibit the dissociation of Beclin1 from Bcl2, as comparing with their corresponding activators (EGF, anisomycin) and oxidant (H 2 O 2 ), the cells of the same batch were also subjected to the co-IP assay (right), the results also satisfied our expectation. Normal rabbit IgG was used as a control in co-IP assay. Data shown were the mean ± SD of three independent experiments and shown as the fold change to the corresponding control. * P

    Journal: PLoS ONE

    Article Title: Drug Screening for Autophagy Inhibitors Based on the Dissociation of Beclin1-Bcl2 Complex Using BiFC Technique and Mechanism of Eugenol on Anti-Influenza A Virus Activity

    doi: 10.1371/journal.pone.0061026

    Figure Lengend Snippet: Establishment of our drug screening model. (A) Schematic representation of the drug screening model. Beclin1 plays several important roles in autophagy, the dissociation of Beclin1 from Bcl2 is essential for autophagy. Autophagy either supports IAV replication or induces autophagic cell death which may result in acute lung injury (left). Bcl2 binds with Beclin1 to form Beclin1-Bcl2 heterodimer and inhibits autophagy (right). Beclin1-binding proteins (such as MyD88, TRIF and HMGB1), Bcl2-binding proteins (such as BNIP3, Bad, Noxa, Puma, BimEL and Bik) and the activations of ERK1/2, JNK1, IKK, TLRs and DAPK signal pathways under oxidative stress, endoplasmic reticulum (ER) stress and energy stress can promote the dissociation of Beclin1 from Bcl2, and finally promote autophagy. IAV infection can elevate the expressions of many aforementioned proteins and activate aforementioned signal pathways. Drugs inhibiting oxidative stress, ER stress, energy stress, and the activations of ERK1/2, JNK1, IKK, TLRs and DAPK signal pathways may inhibit the dissociation of Beclin1 from Bcl2, sequentially inhibit autophagy, and finally impair IAV replication or inhibit autophagic cell death and acute lung injury. In our study, Beclin1 and Bcl2 were fused with C’- and N’- fragments of a red fluorescence protein (RFP), respectively. The amino acid sequence of the linker was RPACKIPNDLKQKVMNH. After cotransfection, the intact RFP would reconstitute through the interaction of Beclin1 and Bcl2. The fluorescence intensity (FI) of the reconstituted RFP, which was determined at 610 nm after excitation at 587 nm using a microplate reader (Tecan infinite M1000), represents the level of the Beclin1-Bcl2 heterodimer, which is positively relevant with the degree of autophagy inhibition and antiviral activity. (B) Reconstitution of RFP. A549 cells were transfected or cotransfected with pMN-Bcl2 and pMC-Beclin1, after 8 h, only cotransfected cells appeared a lot of red fluorescence. (C) The influence of HMGB1 and MyD88 on the Beclin1-Bcl2 heterodimer. Beclin1-binding proteins HMGB1 and MyD88 were expected to disrupt the Beclin1-Bcl2 heterodimer, after cotransfection as the graph indicated, the FI was really significantly decreased, and the cells of same batch were subjected to the co-immunoprecipitation (co-IP) assay (right), the results satisfied our expectation. (D) The influence of ERK1/2 inhibitor (U0126, 10 µM), ERK1/2 activator (EGF, 100 ng/ml), JNK/p38 inhibitor (SB203580, 40 µM), p38 MAPK activator (anisomycin, 10 µM), antioxidant (NAC, 2 mM) and oxidant (H2O2, 100 µM) on the dissociation of Beclin1-Bcl2 heterodimer. After cotransfection, A549 cells were treated with these inhibitors and activators, after 8 h, the FI was measured, the inhibitors (U0126, SB203580) and antioxidant (NAC) could significantly elevate the FI, which meant that they could inhibit the dissociation of Beclin1 from Bcl2, as comparing with their corresponding activators (EGF, anisomycin) and oxidant (H 2 O 2 ), the cells of the same batch were also subjected to the co-IP assay (right), the results also satisfied our expectation. Normal rabbit IgG was used as a control in co-IP assay. Data shown were the mean ± SD of three independent experiments and shown as the fold change to the corresponding control. * P

    Article Snippet: The influence of drugs on the dissociation of Beclin1-Bcl2 heterodimer and of Beclin1-IAV M2 heterodimer were detect by co-IP assay, A549 cells were seeded into a 6-well plate for 24 h, after cotransfection with corresponding plasmids for 6 h, the drugs were added, and the cells were collected after 24 h. The interactions were determined following the instrument of the Co-Immunoprecipitation Kit (Thermo scientific, #23600).

    Techniques: Binding Assay, Infection, Fluorescence, Sequencing, Cotransfection, Inhibition, Activity Assay, Transfection, Co-Immunoprecipitation Assay

    Pif1 is acetylated both in vivo and in vitro . [A] Top Panel : S. cerevisiae lysates from WT (lane 1), esa1-414 (lane 2), and rpd3Δ (lane 3) backgrounds were immunoprecipitated with anti-acetyl lysine (ac-K) antibody-coated Protein G-dynabeads and immunoblotted with anti-FLAG antibody (1:1000); middle Panel : 10% of input immunoblotted with the anti-FLAG antibody and; bottom panel : PGK-1 antibody (1:10,000). [B] Immunoblot of unmodified Pif1 (lane 1), NuA4 (Esa1) in vitro -acetylated full-length Pif1 (lane 2), unmodified Pif1ΔN, and NuA4 (Esa1) in vitro -acetylated Pif1ΔN probed with anti-Ac-K antibody. [C] Schematic of the full-length Pif1 sequence. The positions of all of the lysine residues in the sequence are denoted with red lines, and all acetylated lysine residues identified by mass spectrometry are denoted with black lines and red filled circles.

    Journal: bioRxiv

    Article Title: Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

    doi: 10.1101/2020.07.06.189761

    Figure Lengend Snippet: Pif1 is acetylated both in vivo and in vitro . [A] Top Panel : S. cerevisiae lysates from WT (lane 1), esa1-414 (lane 2), and rpd3Δ (lane 3) backgrounds were immunoprecipitated with anti-acetyl lysine (ac-K) antibody-coated Protein G-dynabeads and immunoblotted with anti-FLAG antibody (1:1000); middle Panel : 10% of input immunoblotted with the anti-FLAG antibody and; bottom panel : PGK-1 antibody (1:10,000). [B] Immunoblot of unmodified Pif1 (lane 1), NuA4 (Esa1) in vitro -acetylated full-length Pif1 (lane 2), unmodified Pif1ΔN, and NuA4 (Esa1) in vitro -acetylated Pif1ΔN probed with anti-Ac-K antibody. [C] Schematic of the full-length Pif1 sequence. The positions of all of the lysine residues in the sequence are denoted with red lines, and all acetylated lysine residues identified by mass spectrometry are denoted with black lines and red filled circles.

    Article Snippet: Specifically, Protein G Dynabeads (Invitrogen 10007D) were incubated with anti-acetyl lysine antibody (CST 9441) with end-over-end rotation for 4 h at 4°C, followed by the addition of 1 mg of cell lysate, which was then rotated overnight at 4°C.

    Techniques: In Vivo, In Vitro, Immunoprecipitation, Sequencing, Mass Spectrometry

    YAP immunoprecipitation and mass-spectrometry analysis of binding partners. (A) YAP IP-MS analysis of co-precipitated proteins identifies the Engima family proteins PDLIM5 and PDLIM7 as novel YAP-associated proteins. Axes are log 10 -transformed values. (B) List of all YAP-associated proteins identified in the experiment shown in A. (C) Comparison of confidence ratios for PDLIM5/7 and known YAP interactors.

    Journal: Journal of Cell Science

    Article Title: Enigma proteins regulate YAP mechanotransduction

    doi: 10.1242/jcs.221788

    Figure Lengend Snippet: YAP immunoprecipitation and mass-spectrometry analysis of binding partners. (A) YAP IP-MS analysis of co-precipitated proteins identifies the Engima family proteins PDLIM5 and PDLIM7 as novel YAP-associated proteins. Axes are log 10 -transformed values. (B) List of all YAP-associated proteins identified in the experiment shown in A. (C) Comparison of confidence ratios for PDLIM5/7 and known YAP interactors.

    Article Snippet: The sample was then lysed and subjected to co-immunoprecipitation using a GFP Trap Kit containing lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40 and 0.5 mM EDTA) (Chromotek).

    Techniques: Immunoprecipitation, Mass Spectrometry, Binding Assay, Transformation Assay

    PDLIM5/7 proteins localize in the cytoplasm in dense cells but to basal stress fibers in sparse cells and bind directly to the stress fiber component α-actinin 1 . (A) PDLIM5 localizes to the cytoplasm in dense cells but translocates, in part, to F-actin stress fibers and adherens junctions in sparsely plated cells. (B) PDLIM7 localizes to the cytoplasm in dense cells but translocates in part to F-actin stress fibers and adherens junctions in sparsely plated cells. (C) Co-immunoprecipitation of PDLIM5/7 proteins with GFP-tagged α-actinin 1 from human Caco2 cells. Results are representative of n =3 biological replicates. (D) Schematic diagram of YAP recruitment via the Enigma PDLIM5/7 proteins to integrin–Src signaling complexes to sense mechanical forces basally. Scale bar: 20 µm (A,B).

    Journal: Journal of Cell Science

    Article Title: Enigma proteins regulate YAP mechanotransduction

    doi: 10.1242/jcs.221788

    Figure Lengend Snippet: PDLIM5/7 proteins localize in the cytoplasm in dense cells but to basal stress fibers in sparse cells and bind directly to the stress fiber component α-actinin 1 . (A) PDLIM5 localizes to the cytoplasm in dense cells but translocates, in part, to F-actin stress fibers and adherens junctions in sparsely plated cells. (B) PDLIM7 localizes to the cytoplasm in dense cells but translocates in part to F-actin stress fibers and adherens junctions in sparsely plated cells. (C) Co-immunoprecipitation of PDLIM5/7 proteins with GFP-tagged α-actinin 1 from human Caco2 cells. Results are representative of n =3 biological replicates. (D) Schematic diagram of YAP recruitment via the Enigma PDLIM5/7 proteins to integrin–Src signaling complexes to sense mechanical forces basally. Scale bar: 20 µm (A,B).

    Article Snippet: The sample was then lysed and subjected to co-immunoprecipitation using a GFP Trap Kit containing lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40 and 0.5 mM EDTA) (Chromotek).

    Techniques: Immunoprecipitation

    The Enigma family proteins PDLIM5 /7 bind to the YAP C-terminal PBM and promote YAP nuclear localization and transcriptional activity. (A) Confirmation of YAP–PDLIM5 and YAP–PDLIM7 interaction by co-immunoprecipitation (IP) of GFP-tagged YAP, and immunoblotting with anti-PDLIM5 and anti-PDLIM7 antibodies. Both Enigma family proteins PDLIM5 and PDLIM7 bind to the YAP C-terminal PBM, as deletion of this motif (YAPΔC) abolishes the interaction in co-immunoprecipitation experiments. (B) Endogenous YAP co-immunoprecipitates with PDLIM5 and PDLIM7. (C) Deletion of the C-terminal PBM (YAPΔC) reduces nuclear localization of GFP-tagged YAP in human Caco2 cells plated at low density. Results in A–C are representative of n =3 biological replicates. (D) Quantification (mean±s.d., n =3) of YAP localization for experiments as shown in C. **** P

    Journal: Journal of Cell Science

    Article Title: Enigma proteins regulate YAP mechanotransduction

    doi: 10.1242/jcs.221788

    Figure Lengend Snippet: The Enigma family proteins PDLIM5 /7 bind to the YAP C-terminal PBM and promote YAP nuclear localization and transcriptional activity. (A) Confirmation of YAP–PDLIM5 and YAP–PDLIM7 interaction by co-immunoprecipitation (IP) of GFP-tagged YAP, and immunoblotting with anti-PDLIM5 and anti-PDLIM7 antibodies. Both Enigma family proteins PDLIM5 and PDLIM7 bind to the YAP C-terminal PBM, as deletion of this motif (YAPΔC) abolishes the interaction in co-immunoprecipitation experiments. (B) Endogenous YAP co-immunoprecipitates with PDLIM5 and PDLIM7. (C) Deletion of the C-terminal PBM (YAPΔC) reduces nuclear localization of GFP-tagged YAP in human Caco2 cells plated at low density. Results in A–C are representative of n =3 biological replicates. (D) Quantification (mean±s.d., n =3) of YAP localization for experiments as shown in C. **** P

    Article Snippet: The sample was then lysed and subjected to co-immunoprecipitation using a GFP Trap Kit containing lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40 and 0.5 mM EDTA) (Chromotek).

    Techniques: Activity Assay, Immunoprecipitation

    MeCP2 binding to nucleosome is enhanced by the presence of H3K27me3. a Interactions between MeCP2 and H3K27me3 are determined by in vitro pull-down assay. Pull-down MeCP2 is shown by both MeCP2 and GST bands. Comparable levels of mononucleosomes between land 2 and 3 are shown by Ponceau S staining and H3. The presence of H3K27me3 is validated in Lane 3. b Schematic representation of MeCP2 domain constructs. c Co-immunoprecipitation of histone H3 with Flag-tagged MeCP2 full length or truncated fragments. From 293T cell transfected with Flag-tagged constructs in b , total protein extract was immunoprecipitated with Flag antibody and the presence of histone H3 is shown by immunoblotting. d In vitro pull-down assays of bait (purified GST, GST-tagged full length MeCP2 or MBD of MeCP2) to prey protein (recombinant mononucleosomes, either containing unmodified H3 or H3K27me3). Pull-downed H3 or H3K27me3 were visualized by SDS-PAGE and immunoblotting with anti-Histone H3 or H3K27me3. The presence of GST-fusion protein in each construct was indicated by the asterisks. e Competition assays with unmodified H3K27 or H3K27me3 peptides to MBD/nucleosome interactions. GST-tagged MBD binding to nucleosomes (top blot) containing H3K27me3 (lane 4) were challenged with H3K27 (lane 5–7) and H3K27me3 (lane 8-10) peptide. Bindings of GST-tagged MBD to histone H3 (middle blot) and H3K27me3 (bottom blot, sharp) were evaluated by GST pull-down and immunoblotting. H3K27me3 peptides in the MBD/nucleosome complexes were also revealed (bottom blot, double sharp). f , g Quantification of normalized H3K27me3 (purple, sharp) and H3K27me3 peptide (green, double sharp) levels by densitometry analysis in e . The signal strength in each band is normalized to H3K27me3 signal strength lane 4 in e . Source data are provided as a Source data file ( a , c , d and e ).

    Journal: Nature Communications

    Article Title: MeCP2 regulates gene expression through recognition of H3K27me3

    doi: 10.1038/s41467-020-16907-0

    Figure Lengend Snippet: MeCP2 binding to nucleosome is enhanced by the presence of H3K27me3. a Interactions between MeCP2 and H3K27me3 are determined by in vitro pull-down assay. Pull-down MeCP2 is shown by both MeCP2 and GST bands. Comparable levels of mononucleosomes between land 2 and 3 are shown by Ponceau S staining and H3. The presence of H3K27me3 is validated in Lane 3. b Schematic representation of MeCP2 domain constructs. c Co-immunoprecipitation of histone H3 with Flag-tagged MeCP2 full length or truncated fragments. From 293T cell transfected with Flag-tagged constructs in b , total protein extract was immunoprecipitated with Flag antibody and the presence of histone H3 is shown by immunoblotting. d In vitro pull-down assays of bait (purified GST, GST-tagged full length MeCP2 or MBD of MeCP2) to prey protein (recombinant mononucleosomes, either containing unmodified H3 or H3K27me3). Pull-downed H3 or H3K27me3 were visualized by SDS-PAGE and immunoblotting with anti-Histone H3 or H3K27me3. The presence of GST-fusion protein in each construct was indicated by the asterisks. e Competition assays with unmodified H3K27 or H3K27me3 peptides to MBD/nucleosome interactions. GST-tagged MBD binding to nucleosomes (top blot) containing H3K27me3 (lane 4) were challenged with H3K27 (lane 5–7) and H3K27me3 (lane 8-10) peptide. Bindings of GST-tagged MBD to histone H3 (middle blot) and H3K27me3 (bottom blot, sharp) were evaluated by GST pull-down and immunoblotting. H3K27me3 peptides in the MBD/nucleosome complexes were also revealed (bottom blot, double sharp). f , g Quantification of normalized H3K27me3 (purple, sharp) and H3K27me3 peptide (green, double sharp) levels by densitometry analysis in e . The signal strength in each band is normalized to H3K27me3 signal strength lane 4 in e . Source data are provided as a Source data file ( a , c , d and e ).

    Article Snippet: ChIP-seq in olfactory neuroepitheliaChromatin immunoprecipitation assays were performed using MAGnify chromatin immunoprecipitation system (Invitrogen, 49-2024).

    Techniques: Binding Assay, In Vitro, Pull Down Assay, Staining, Construct, Immunoprecipitation, Transfection, Purification, Recombinant, SDS Page