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  • 98
    Thermo Fisher dynabeads protein g immunoprecipiation kit
    Dynabeads Protein G Immunoprecipiation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore protein g immunoprecipiation kit
    AhSLF-S 2 Interacts with ASK1- and CUL1-Like Proteins. (A) Extracts of styles of S 1 S 4 or S 2 S 5 lines (50 μg each) and pollen of an S 2 S 5 line (50 μg) were coimmunoprecipitated with anti-AhSLF-S 2 -C antibody (3 μL) (lanes 1 and 3) or control preimmune serum (3 μL) (lanes 2 and 4), respectively. As additional controls, the extracts of styles of S 1 S 4 (lane 5) and S 2 S 5 (lane 6) lines or pollen of S 1 S 4 (lane 7) and S 2 S 5 (lane 8) lines also were coimmunoprecipitated with anti-AhSLF-S 2 -C antibody (3 μL). After <t>immunoprecipitation,</t> the proteins were subjected to 12% SDS-PAGE followed by protein gel blotting with specific antibodies against AtASK1 of Arabidopsis (top panel). Total proteins of Antirrhinum pollen (lane 9) and Arabidopsis leaf (lane 10) also were used as positive controls. The bottom panel represents the Coomassie blue–stained gel before protein gel blotting for equal loading of proteins. (B) Immunoblot detection of CUL1-like protein from total pollen proteins of Antirrhinum pollen (lane 1) and Arabidopsis (lane 2). (C) A pull-down assay for an interaction between AhSLF-S 2 and CUL1-like proteins. His-NAT resin or the purified fusion proteins of His-AhSLF-S 2 -C were incubated with the extracts of Antirrhinum styles from S 2 S 5 and pollen of S 2 S 5 or S 1 S 4 (lanes 5 and 10), respectively. The extracts of the Antirrhinum styles from S 2 S 5 and pollen of S 2 S 5 or S 1 S 4 were incubated without His-AhSLF-S 2 -C as negative controls (lanes 4 and 9), and style and pollen extracts were used as positive controls (lanes 1 and 6). As additional controls, the extracts of the styles of S 2 S 5 (lane 2) and S 1 S 4 (lane 7) lines or pollen of S 2 S 5 (lane 3) and S 1 S 4 (lane 8) lines were incubated with anti-AhSLF-S 2 -C antibody (3 μL). Proteins bound were pulled down with His-NAT resin, eluted with lysis buffer, separated by 12% SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with Arabidopsis anti-CUL1 antibody.
    Protein G Immunoprecipiation Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore co immunoprecipitation
    Infusion of HSV-RGS4 into the dSTR results in over-expression of RGS4 protein as well as binding of recombinant RGS4 to mGluR5 receptors Animals received bilateral intra-dSTR infusion of HSV-RGS4- FLAG or a control vector HSV-LacZ (2μl/side) and brains were analyzed 3 days later. A , Upper panel: Outline of the rat brain coronal section depicting a representative placement of HSV infusions; adapted from Paxinos and Watson (2005). Lower panels: Immunohistochemical detection of RGS4 and β-galactosidase over-expression in the dSTR near the infusion site (black box represents a tip of the injector). Representative image of Nissl-stained striatal section showing no necrosis or abnormal cytoarchitecture in the proximity of the infusion site. B , Protein levels of β-galactosidase, RGS4, RGS4- FLAG and calnexin as measured by immunoblotting in tissue lysates prepared from the dSTR. C , FLAG -tagged RGS4 co-immunoprecipitates with mGluR5, but not with, dopamine D1 or D2 receptors in the dSTR ( D ). Immunoprecipitates (IP) were analyzed by immunoblotting (IB) using antibodies against FLAG -tag, mGluR5 or D1 and D2 receptors. T - total lysate used as an <t>immunoprecipitation</t> input. co-IP analysis included negative controls in which lysate, FLAG IP beads or RGS4- FLAG over-expression was omitted.
    Co Immunoprecipitation, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore imprint chromatin immunopreciptitation kit
    Infusion of HSV-RGS4 into the dSTR results in over-expression of RGS4 protein as well as binding of recombinant RGS4 to mGluR5 receptors Animals received bilateral intra-dSTR infusion of HSV-RGS4- FLAG or a control vector HSV-LacZ (2μl/side) and brains were analyzed 3 days later. A , Upper panel: Outline of the rat brain coronal section depicting a representative placement of HSV infusions; adapted from Paxinos and Watson (2005). Lower panels: Immunohistochemical detection of RGS4 and β-galactosidase over-expression in the dSTR near the infusion site (black box represents a tip of the injector). Representative image of Nissl-stained striatal section showing no necrosis or abnormal cytoarchitecture in the proximity of the infusion site. B , Protein levels of β-galactosidase, RGS4, RGS4- FLAG and calnexin as measured by immunoblotting in tissue lysates prepared from the dSTR. C , FLAG -tagged RGS4 co-immunoprecipitates with mGluR5, but not with, dopamine D1 or D2 receptors in the dSTR ( D ). Immunoprecipitates (IP) were analyzed by immunoblotting (IB) using antibodies against FLAG -tag, mGluR5 or D1 and D2 receptors. T - total lysate used as an <t>immunoprecipitation</t> input. co-IP analysis included negative controls in which lysate, FLAG IP beads or RGS4- FLAG over-expression was omitted.
    Imprint Chromatin Immunopreciptitation Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore immunoprecipitation assays
    EZH2 interacts with DNMT3A in imatinib-resistant EOL-1 cells. Western blot analysis. ( a ) EOL-1 cells were cultured with imatinib (1 n) for 3 or 4 months. Cells were harvested and subjected to western blot analysis to monitor the levels of tri-methyl-histone H3 (Lys27), histone H3, EZH2 and DNMT3A. Each lane was loaded with 20 μg of nuclear protein lysates. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured as a loading control. Band intensities were quantified with ImageJ software (Wayne Rasband, NIH). <t>Immunoprecipitation.</t> ( b ) EOL-1 cells were cultured with imatinib (1 n) for 3 or 4 months. Cells were harvested and proteins were extracted. The EZH2 proteins were immunoprecipitated and subjected to western blot analysis. The membrane was probed sequentially with anti-DNMT3A (top) and anti-EZH2 antibodies (bottom). Binding of EZH2 and DNMT3A in PTEN promoter region. Chromatin immunoprecipitation assay. ( c ) Binding of DNMT3A and EZH2 in the PTEN promoter was analyzed by ChIP assay. To quantify acetylated DNA precisely, we employed real-time PCR. The amplified sequences were normalized to those from input (the cross-linked DNA/protein complexes, which were not immunoprecipitated with anti-DNMT3A and -EZH2 antibodies). Results represent the mean±s.d. of two experiments performed in duplicate. The statistical significance was assessed by a paired t -test. * P
    Immunoprecipitation Assays, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 781 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam immunoprecipitation kit
    RagC complexes isolated by <t>immunoprecipitation</t> from Huh7 cells. ( A ) Huh7 cultured with Si-OH, Si-NH2, or PS-NH2 NPs (all 50 µg/ml) bearing BSA or RNase (both 50 µM) as hard protein corona or bare NPs for 4 h. After 4 h post NP treatment, cells were lysed with lysis buffer from immunoprecipitation kit (Abcam). RagC-mTOR complexes were co-immunoprecipitated from the precleared cell lysates with appropriate Ab as described in the manufacturer’s instruction. The resulting protein complex was eluted from the beads with Laemmli protein sample buffer for SDS-PAGE (Bio-Rad) and resolved on SDS-PAGE with specific antibody against mTOR (Cell Signaling) (full blots are presented in Supporting Information). ( B ) Scheme of district biochemical signaling activation in cells after stimulation with amino-functionalized non-biodegradable PS and biodegradable silica NPs. ∆mΦ – mitochondrial membrane potential; LMP – lysosomal membrane permeabilization.
    Immunoprecipitation Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam co immunoprecipitation
    Validation of interaction between Shisa9 and its putative interactors by means of <t>co-immunoprecipitation</t> from HEK293T cells. A. Schematic view of Shisa9-constructs used in co-immunoprecipitations. SP, signal sequence; TM, transmembrane domain; HA, HA-tag; EVTV, PDZ-ligand motif. B. Co-immunoprecipitation of Shisa9-interactor complexes from HEK293T cells. HA-Shisa9WT and HA-Shisa9ΔEVTV were overexpressed in HEK293T cells in combination with interacting proteins (one at a time). Anti-HA-tag antibody was added to immunoprecipitate HA-Shisa9-interactor complexes. Obtained samples were resolved on SDS-PAGE, western blotted and immunostained with anti-V5 antibody against V5-tagged interactors. Shisa9WT co-immunoprecipitates with PSD95, PSD93, GRIP1, PICK1 and Lin7b proteins, whereas Shisa9ΔEVTV lost the possibility to establish the interaction with named proteins (left panel). The right panel shows the same membranes as in the left panel stained with the anti-HA antibody in order to visualize the presence of Shisa9 in the immunoprecipitated samples. The 50 kDa band is indicated.
    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
    Becton Dickinson immunoprecipitations
    Validation of interaction between Shisa9 and its putative interactors by means of <t>co-immunoprecipitation</t> from HEK293T cells. A. Schematic view of Shisa9-constructs used in co-immunoprecipitations. SP, signal sequence; TM, transmembrane domain; HA, HA-tag; EVTV, PDZ-ligand motif. B. Co-immunoprecipitation of Shisa9-interactor complexes from HEK293T cells. HA-Shisa9WT and HA-Shisa9ΔEVTV were overexpressed in HEK293T cells in combination with interacting proteins (one at a time). Anti-HA-tag antibody was added to immunoprecipitate HA-Shisa9-interactor complexes. Obtained samples were resolved on SDS-PAGE, western blotted and immunostained with anti-V5 antibody against V5-tagged interactors. Shisa9WT co-immunoprecipitates with PSD95, PSD93, GRIP1, PICK1 and Lin7b proteins, whereas Shisa9ΔEVTV lost the possibility to establish the interaction with named proteins (left panel). The right panel shows the same membranes as in the left panel stained with the anti-HA antibody in order to visualize the presence of Shisa9 in the immunoprecipitated samples. The 50 kDa band is indicated.
    Immunoprecipitations, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex immunoprecipitations
    KIAA1199 connects Semaphorin 3A signalling to EGFR phosphorylation. ( a ) Semaphorin 3A-mediated EGFR phosphorylation requires KIAA1199. Control or KIAA1199-depleted CaSki cells were untreated or stimulated with Semaphorin 3A (100 ng ml −1 ) and WB analyses were carried out on the resulting cell extracts (lysis in SDS 1%). ( b ) KIAA1199 deficiency does not have an impact on EGFR mRNA levels in cervical cancer cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR, to assess EGFR mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from two independent experiments (means±s.d.) are shown. ( c ) Plexin A2 deficiency potentiates Semaphorin 3A-mediated EGFR phosphorylation. Control or Plexin A2-depleted CaSki cells were left untreated or stimulated with Semaphorin 3A for the indicated periods of time. The resulting cell extracts (lysis in SDS 1%) were subjected to WBs using the indicated antibodies. ( d ) Plexin A2 deficiency prolongs the binding of KIAA1199 to EGFR on EGF stimulation. Control or Plexin A2-deficient cells were untreated or stimulated with EGF for the indicated periods of time. Cell extracts were subjected to anti-FLAG (negative control) or -EGFR <t>immunoprecipitations</t> followed by anti- KIAA1199 or -EGFR WBs (top panels). Crude cell extracts were subjected to anti-pEGFR (Y845 and Y1068) (to validate the triggering of the EGF-dependent pathway), -EGFR, -KIAA1199, -Plexin A2 and -HSP90 WBs, as indicated. ( e ) KIAA1199 binds EGFR through its N-terminal domain. Cells were transfected with the indicated expression plasmids and protein extracts were subjected to anti-HA (negative control) or -FLAG IPs followed by an anti-EGFR WB (top panel). Crude cell extracts were also subjected to anti-EGFR and -FLAG WB analyses (bottom panels).
    Immunoprecipitations, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam immunoprecipitations
    Occupancy of Sp1 and κB Sites in Bright and Off States. (A) Flow cytometry histograms of expanded populations of Off- and Bright-sorted Jurkats infected with LGIT and each LGIT mutant (as in Figure 3A , panels 5–6). 10 6 cells were initially sorted from Off and Bright regions, and seven days of expansion was conducted to achieve 5×10 7 cells necessary for this ChIP protocol. We observed a moderate extent of Bright→Off and Off→Bright dynamic switching over this one-week expansion. (B) RelA ChIP results for Off- and Bright-sorted populations of LGIT , mutI Sp1 , mutII Sp1 , mutIII Sp1 , mutI NF-κB , and mutII NF-κB . <t>Immunoprecipitations</t> were performed using RelA antibody, and immunoprecipitated DNA was quantified using QPCR with primers against the HIV LTR. For analysis of input DNA and RelA immunoprecipitation, all LTR QPCR measurements were normalized by with ChIP-QPCR measurements for the endogenous TAP1/LMP2 regulatory domain [86] , which contains single κB and Sp1 sites that recruit RelA and p50 (refer to Figure S4A and S4B for non-normalized results). Primer sequences and QPCR conditions for HIV LTR and TAP1/LMP2 are supplied in Materials and Methods and Table S2 . The QPCR measurements for LTR and control TAP1/LMP2 were performed in triplicate, and error bars are standard deviations. Statistically significant differences from WT LGIT are denoted by black single asterisks (*, p
    Immunoprecipitations, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 1034 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche immunoprecipitations
    Phosphorylation of NS1 T49 leads to reduced vRNA, RIG‐I and TRIM25 binding as well as structural destabilization of the RBD. A. A549 cells were transfected with pcDNA3 plasmids containing wt NS1 or NS1 with indicated mutations or were mock transfected. Cell lysates were subjected to <t>immunoprecipitations</t> 24 h p.i. using mouse anti‐NS1 antibody. Washed beads were incubated with vRNA and RNA bound to immunocomplexes was extracted. The relative amount of viral NS1 mRNA was determined by qRT‐PCR as described previously (Habjan et al ., 2008 ). Data represents mean ± SD of two independently repeated experiments. B. Comparative binding scores of RNA to RBD T49E, T49A and T49 as calculated using random forest model from the RNA–protein interaction prediction (RPISeq) tool (Muppirala et al ., 2011 ). The RNA sequence was obtained from Protein Data Bank (PDB) ID 2ZKO (Cheng et al ., 2009 ). C. Structural simulation on 2ZKO structure employing DUET (Pires et al ., 2014b ) was predicted as destabilizing (−0.17 Kcal/mol) resulting in an unfavourable RNA binding ability. The figure shows the stereo‐chemical effect of E49 compared with T49. The RNA helix is shown in yellow, while the two monomers of the NS1 dimer are shown in green and blue. NS1 residues T49 and E49 are shown in red and purple respectively. Molecular graphic simulation was performed using Bioblender (Andrei et al ., 2012 ). D. For analysis of NS1‐RIG‐I interaction, HEK293 cells transiently expressing FLAG‐tagged RIG‐I and the indicated NS1 proteins were subjected to crosslinking with DSP after 48 h, followed by quenching with glycine. Control cells were mock transfected. FLAG‐tagged RIG‐I was immunoprecipitated with anti‐FLAG M2 antibody. Detection of FLAG‐tagged RIG‐I and co‐precipitated NS1 protein was performed by Western blotting. Detection of FLAG‐tagged RIG‐I and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels. E. For analysis of NS1‐TRIM25 interaction HEK293 cells were infected with PR8/NS1‐T49, PR8/NS1‐T49A or PR8/NS1‐T49E (MOI of 1, 5 or 10 respectively) or were mock infected (control).Eighteen h p.i. cells were lysed and lysates subjected to immunoprecipitation with mouse anti‐TRIM25 antibody. TRIM25 and the co‐precipitated NS1 were detected by Western blotting using mouse anti‐TRIM25 antibody or mouse anti‐NS1 antibody. Detection of TRIM25 and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels.
    Immunoprecipitations, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 1741 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    AhSLF-S 2 Interacts with ASK1- and CUL1-Like Proteins. (A) Extracts of styles of S 1 S 4 or S 2 S 5 lines (50 μg each) and pollen of an S 2 S 5 line (50 μg) were coimmunoprecipitated with anti-AhSLF-S 2 -C antibody (3 μL) (lanes 1 and 3) or control preimmune serum (3 μL) (lanes 2 and 4), respectively. As additional controls, the extracts of styles of S 1 S 4 (lane 5) and S 2 S 5 (lane 6) lines or pollen of S 1 S 4 (lane 7) and S 2 S 5 (lane 8) lines also were coimmunoprecipitated with anti-AhSLF-S 2 -C antibody (3 μL). After immunoprecipitation, the proteins were subjected to 12% SDS-PAGE followed by protein gel blotting with specific antibodies against AtASK1 of Arabidopsis (top panel). Total proteins of Antirrhinum pollen (lane 9) and Arabidopsis leaf (lane 10) also were used as positive controls. The bottom panel represents the Coomassie blue–stained gel before protein gel blotting for equal loading of proteins. (B) Immunoblot detection of CUL1-like protein from total pollen proteins of Antirrhinum pollen (lane 1) and Arabidopsis (lane 2). (C) A pull-down assay for an interaction between AhSLF-S 2 and CUL1-like proteins. His-NAT resin or the purified fusion proteins of His-AhSLF-S 2 -C were incubated with the extracts of Antirrhinum styles from S 2 S 5 and pollen of S 2 S 5 or S 1 S 4 (lanes 5 and 10), respectively. The extracts of the Antirrhinum styles from S 2 S 5 and pollen of S 2 S 5 or S 1 S 4 were incubated without His-AhSLF-S 2 -C as negative controls (lanes 4 and 9), and style and pollen extracts were used as positive controls (lanes 1 and 6). As additional controls, the extracts of the styles of S 2 S 5 (lane 2) and S 1 S 4 (lane 7) lines or pollen of S 2 S 5 (lane 3) and S 1 S 4 (lane 8) lines were incubated with anti-AhSLF-S 2 -C antibody (3 μL). Proteins bound were pulled down with His-NAT resin, eluted with lysis buffer, separated by 12% SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with Arabidopsis anti-CUL1 antibody.

    Journal: The Plant Cell

    Article Title: The F-Box Protein AhSLF-S2 Physically Interacts with S-RNases That May Be Inhibited by the Ubiquitin/26S Proteasome Pathway of Protein Degradation during Compatible Pollination in Antirrhinum

    doi: 10.1105/tpc.017673

    Figure Lengend Snippet: AhSLF-S 2 Interacts with ASK1- and CUL1-Like Proteins. (A) Extracts of styles of S 1 S 4 or S 2 S 5 lines (50 μg each) and pollen of an S 2 S 5 line (50 μg) were coimmunoprecipitated with anti-AhSLF-S 2 -C antibody (3 μL) (lanes 1 and 3) or control preimmune serum (3 μL) (lanes 2 and 4), respectively. As additional controls, the extracts of styles of S 1 S 4 (lane 5) and S 2 S 5 (lane 6) lines or pollen of S 1 S 4 (lane 7) and S 2 S 5 (lane 8) lines also were coimmunoprecipitated with anti-AhSLF-S 2 -C antibody (3 μL). After immunoprecipitation, the proteins were subjected to 12% SDS-PAGE followed by protein gel blotting with specific antibodies against AtASK1 of Arabidopsis (top panel). Total proteins of Antirrhinum pollen (lane 9) and Arabidopsis leaf (lane 10) also were used as positive controls. The bottom panel represents the Coomassie blue–stained gel before protein gel blotting for equal loading of proteins. (B) Immunoblot detection of CUL1-like protein from total pollen proteins of Antirrhinum pollen (lane 1) and Arabidopsis (lane 2). (C) A pull-down assay for an interaction between AhSLF-S 2 and CUL1-like proteins. His-NAT resin or the purified fusion proteins of His-AhSLF-S 2 -C were incubated with the extracts of Antirrhinum styles from S 2 S 5 and pollen of S 2 S 5 or S 1 S 4 (lanes 5 and 10), respectively. The extracts of the Antirrhinum styles from S 2 S 5 and pollen of S 2 S 5 or S 1 S 4 were incubated without His-AhSLF-S 2 -C as negative controls (lanes 4 and 9), and style and pollen extracts were used as positive controls (lanes 1 and 6). As additional controls, the extracts of the styles of S 2 S 5 (lane 2) and S 1 S 4 (lane 7) lines or pollen of S 2 S 5 (lane 3) and S 1 S 4 (lane 8) lines were incubated with anti-AhSLF-S 2 -C antibody (3 μL). Proteins bound were pulled down with His-NAT resin, eluted with lysis buffer, separated by 12% SDS-PAGE, transferred to membranes, and analyzed by immunoblotting with Arabidopsis anti-CUL1 antibody.

    Article Snippet: Immunoprecipitation was performed following the manufacturer's recommendation of an immunoprecipitation kit (IP-50; Sigma-Aldrich).

    Techniques: Immunoprecipitation, SDS Page, Staining, Pull Down Assay, Purification, Incubation, Lysis

    Coimmunoprecipitation of AhSLF-S 2 and S-RNases. (A) Detection of AhSLF-S 2 in several tissues and E. coli expressing AhSLF-S 2 C by a polyclonal antibody against the C-terminal region of AhSLF-S 2 . Bottom panel: Coomassie blue–stained gel before protein gel blot analysis showing control loading of proteins. Lanes 1 to 4 represent protein extracts from sepal (Sp), petal (Pe), style (St), and anther (An) from an S 2 S 5 line. AhSLF-S 2 C indicates the detection of AhSLF-S 2 C in E. coli expressing AhSLF-S 2 C . (B) Immunoblot analysis of style proteins from several lines of an S -allele–segregating family using allele-specific antibodies against peptides derived from S 2 - and S 4 -RNases. Genotypes are indicated at the top. (C) Extracts of pollinated styles of S 1 S 4 (lane 1) or S 2 S 5 (lane 3) lines (50 μg) and pollen of an S 2 S 5 line (50 μg) were immunoprecipitated with anti-AhSLF-S 2 -C antibody (3 μL) (lanes 1 and 3) or control preimmune serum (3 μL) (lanes 2 and 4), respectively. After immunoprecipitation, the proteins were subjected to 12% SDS-PAGE followed by protein gel blotting with specific antibodies against S 2 - and S 4 -RNases, respectively (top panels). S-RNases detected are indicated. Bottom panels: Coomassie blue–stained gels before protein gel blot analysis showing equal loading of proteins. The strongest bands represent immunoglobulin heavy chains. Molecular mass markers are indicated in kilodaltons.

    Journal: The Plant Cell

    Article Title: The F-Box Protein AhSLF-S2 Physically Interacts with S-RNases That May Be Inhibited by the Ubiquitin/26S Proteasome Pathway of Protein Degradation during Compatible Pollination in Antirrhinum

    doi: 10.1105/tpc.017673

    Figure Lengend Snippet: Coimmunoprecipitation of AhSLF-S 2 and S-RNases. (A) Detection of AhSLF-S 2 in several tissues and E. coli expressing AhSLF-S 2 C by a polyclonal antibody against the C-terminal region of AhSLF-S 2 . Bottom panel: Coomassie blue–stained gel before protein gel blot analysis showing control loading of proteins. Lanes 1 to 4 represent protein extracts from sepal (Sp), petal (Pe), style (St), and anther (An) from an S 2 S 5 line. AhSLF-S 2 C indicates the detection of AhSLF-S 2 C in E. coli expressing AhSLF-S 2 C . (B) Immunoblot analysis of style proteins from several lines of an S -allele–segregating family using allele-specific antibodies against peptides derived from S 2 - and S 4 -RNases. Genotypes are indicated at the top. (C) Extracts of pollinated styles of S 1 S 4 (lane 1) or S 2 S 5 (lane 3) lines (50 μg) and pollen of an S 2 S 5 line (50 μg) were immunoprecipitated with anti-AhSLF-S 2 -C antibody (3 μL) (lanes 1 and 3) or control preimmune serum (3 μL) (lanes 2 and 4), respectively. After immunoprecipitation, the proteins were subjected to 12% SDS-PAGE followed by protein gel blotting with specific antibodies against S 2 - and S 4 -RNases, respectively (top panels). S-RNases detected are indicated. Bottom panels: Coomassie blue–stained gels before protein gel blot analysis showing equal loading of proteins. The strongest bands represent immunoglobulin heavy chains. Molecular mass markers are indicated in kilodaltons.

    Article Snippet: Immunoprecipitation was performed following the manufacturer's recommendation of an immunoprecipitation kit (IP-50; Sigma-Aldrich).

    Techniques: Expressing, Staining, Western Blot, Derivative Assay, Immunoprecipitation, SDS Page

    Infusion of HSV-RGS4 into the dSTR results in over-expression of RGS4 protein as well as binding of recombinant RGS4 to mGluR5 receptors Animals received bilateral intra-dSTR infusion of HSV-RGS4- FLAG or a control vector HSV-LacZ (2μl/side) and brains were analyzed 3 days later. A , Upper panel: Outline of the rat brain coronal section depicting a representative placement of HSV infusions; adapted from Paxinos and Watson (2005). Lower panels: Immunohistochemical detection of RGS4 and β-galactosidase over-expression in the dSTR near the infusion site (black box represents a tip of the injector). Representative image of Nissl-stained striatal section showing no necrosis or abnormal cytoarchitecture in the proximity of the infusion site. B , Protein levels of β-galactosidase, RGS4, RGS4- FLAG and calnexin as measured by immunoblotting in tissue lysates prepared from the dSTR. C , FLAG -tagged RGS4 co-immunoprecipitates with mGluR5, but not with, dopamine D1 or D2 receptors in the dSTR ( D ). Immunoprecipitates (IP) were analyzed by immunoblotting (IB) using antibodies against FLAG -tag, mGluR5 or D1 and D2 receptors. T - total lysate used as an immunoprecipitation input. co-IP analysis included negative controls in which lysate, FLAG IP beads or RGS4- FLAG over-expression was omitted.

    Journal: Psychopharmacology

    Article Title: RGS4 over-expression in the rat dorsal striatum modulates mGluR5- and amphetamine-mediated behavior and signaling

    doi: 10.1007/s00213-011-2606-8

    Figure Lengend Snippet: Infusion of HSV-RGS4 into the dSTR results in over-expression of RGS4 protein as well as binding of recombinant RGS4 to mGluR5 receptors Animals received bilateral intra-dSTR infusion of HSV-RGS4- FLAG or a control vector HSV-LacZ (2μl/side) and brains were analyzed 3 days later. A , Upper panel: Outline of the rat brain coronal section depicting a representative placement of HSV infusions; adapted from Paxinos and Watson (2005). Lower panels: Immunohistochemical detection of RGS4 and β-galactosidase over-expression in the dSTR near the infusion site (black box represents a tip of the injector). Representative image of Nissl-stained striatal section showing no necrosis or abnormal cytoarchitecture in the proximity of the infusion site. B , Protein levels of β-galactosidase, RGS4, RGS4- FLAG and calnexin as measured by immunoblotting in tissue lysates prepared from the dSTR. C , FLAG -tagged RGS4 co-immunoprecipitates with mGluR5, but not with, dopamine D1 or D2 receptors in the dSTR ( D ). Immunoprecipitates (IP) were analyzed by immunoblotting (IB) using antibodies against FLAG -tag, mGluR5 or D1 and D2 receptors. T - total lysate used as an immunoprecipitation input. co-IP analysis included negative controls in which lysate, FLAG IP beads or RGS4- FLAG over-expression was omitted.

    Article Snippet: Co-immunoprecipitation experiments included a positive control (immunoprecipitation of FLAG -BAP fusion protein; Sigma-Aldrich) as well as negative controls (omission of lysate, FLAG M2 beads or no over-expression of FLAG -tagged RGS4).

    Techniques: Over Expression, Binding Assay, Recombinant, Plasmid Preparation, Immunohistochemistry, Staining, FLAG-tag, Immunoprecipitation, Co-Immunoprecipitation Assay

    EZH2 interacts with DNMT3A in imatinib-resistant EOL-1 cells. Western blot analysis. ( a ) EOL-1 cells were cultured with imatinib (1 n) for 3 or 4 months. Cells were harvested and subjected to western blot analysis to monitor the levels of tri-methyl-histone H3 (Lys27), histone H3, EZH2 and DNMT3A. Each lane was loaded with 20 μg of nuclear protein lysates. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured as a loading control. Band intensities were quantified with ImageJ software (Wayne Rasband, NIH). Immunoprecipitation. ( b ) EOL-1 cells were cultured with imatinib (1 n) for 3 or 4 months. Cells were harvested and proteins were extracted. The EZH2 proteins were immunoprecipitated and subjected to western blot analysis. The membrane was probed sequentially with anti-DNMT3A (top) and anti-EZH2 antibodies (bottom). Binding of EZH2 and DNMT3A in PTEN promoter region. Chromatin immunoprecipitation assay. ( c ) Binding of DNMT3A and EZH2 in the PTEN promoter was analyzed by ChIP assay. To quantify acetylated DNA precisely, we employed real-time PCR. The amplified sequences were normalized to those from input (the cross-linked DNA/protein complexes, which were not immunoprecipitated with anti-DNMT3A and -EZH2 antibodies). Results represent the mean±s.d. of two experiments performed in duplicate. The statistical significance was assessed by a paired t -test. * P

    Journal: Blood Cancer Journal

    Article Title: Imatinib causes epigenetic alterations of PTEN gene via upregulation of DNA methyltransferases and polycomb group proteins

    doi: 10.1038/bcj.2011.33

    Figure Lengend Snippet: EZH2 interacts with DNMT3A in imatinib-resistant EOL-1 cells. Western blot analysis. ( a ) EOL-1 cells were cultured with imatinib (1 n) for 3 or 4 months. Cells were harvested and subjected to western blot analysis to monitor the levels of tri-methyl-histone H3 (Lys27), histone H3, EZH2 and DNMT3A. Each lane was loaded with 20 μg of nuclear protein lysates. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured as a loading control. Band intensities were quantified with ImageJ software (Wayne Rasband, NIH). Immunoprecipitation. ( b ) EOL-1 cells were cultured with imatinib (1 n) for 3 or 4 months. Cells were harvested and proteins were extracted. The EZH2 proteins were immunoprecipitated and subjected to western blot analysis. The membrane was probed sequentially with anti-DNMT3A (top) and anti-EZH2 antibodies (bottom). Binding of EZH2 and DNMT3A in PTEN promoter region. Chromatin immunoprecipitation assay. ( c ) Binding of DNMT3A and EZH2 in the PTEN promoter was analyzed by ChIP assay. To quantify acetylated DNA precisely, we employed real-time PCR. The amplified sequences were normalized to those from input (the cross-linked DNA/protein complexes, which were not immunoprecipitated with anti-DNMT3A and -EZH2 antibodies). Results represent the mean±s.d. of two experiments performed in duplicate. The statistical significance was assessed by a paired t -test. * P

    Article Snippet: The cells were collected and subjected to chromatin immunoprecipitation kit (Millipore, Temecula, CA, USA) according to the manufacturer's protocol.

    Techniques: Western Blot, Cell Culture, Software, Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification

    p65 binds directly to the K8 promoter. (a) The luciferase activity of the plasmid containing the pGL3-K8 promoter was greatly increased compared with that of control. (b) Schematic of the putative K8 promoter with two potential p65 response elements. (c) Luciferase assays in HEK293A cells. p65 significantly increased the luciferase activity of the vector containing the pGL3-K8 promoter. (d) Chromatin immunoprecipitation assays revealed that p65 interacts with the K8 promoter. Data are presented as mean ± SD. ∗ p

    Journal: International Journal of Endocrinology

    Article Title: Resistin Activates p65 Pathway and Reduces Glycogen Content through Keratin 8

    doi: 10.1155/2020/9767926

    Figure Lengend Snippet: p65 binds directly to the K8 promoter. (a) The luciferase activity of the plasmid containing the pGL3-K8 promoter was greatly increased compared with that of control. (b) Schematic of the putative K8 promoter with two potential p65 response elements. (c) Luciferase assays in HEK293A cells. p65 significantly increased the luciferase activity of the vector containing the pGL3-K8 promoter. (d) Chromatin immunoprecipitation assays revealed that p65 interacts with the K8 promoter. Data are presented as mean ± SD. ∗ p

    Article Snippet: Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) assays were performed using Chromatin Immunoprecipitation Kits (Millipore) according to the manufacturer's instructions.

    Techniques: Luciferase, Activity Assay, Plasmid Preparation, Chromatin Immunoprecipitation

    HDAC1 repression enhances miR-200b promoter activities and up-regulates the histone H3-acetylation level at the promoters through the Sp1-dependent pathway. (A) Western blotting detection of HDAC1 protein expression in CSCs from docetaxel-resistant LAD cells or parental docetaxel-resistant LAD cells. GAPDH was used as an internal control. (B) Western blotting detection of HDAC1 protein expression in CSCs from SPC-A1/DTX cells transfected with either HDAC1-shRNAs (HDAC1-shRNA#1, #2 or #3) or shRNA-control vectors, which indicated that the inhibitory effect of HDAC1-shRNA#2 was the most effective shRNA vector. GAPDH was used as an internal control. (C) Luciferase activities after transfection of the sh-HDAC1#2 or the control vectors into CSCs (SPC-A1/DTX) that had been previously cotransfected with Renilla and either wild-type, Sp1 mutant or PGL3-basic vector-firefly-promoter reporter constructs. Data was normalized to Renilla luciferase activity and determined relative to PGL3-basic-vector promoter activity. (D) The co-immunoprecipitation assay indicated that HDAC1 co-immunoprecipitated with Sp1 in vivo in CSCs (SPC-A1/DTX). HDAC1 and Sp1 were visualized by Western blotting (WB). (E) Chromatin immunoprecipitation (ChIP) assay indicated that HDAC1 and Sp1 bound to the miR-200b promoters in vivo . ChIP assays were performed in CSCs (SPC-A1/DTX) with antibodies directly against Sp1, HDAC1 or IgG control. ChIP-derived DNA was amplified after immunoprecipitation by qRT-PCR with specific primers that were designed to amplify the sequences containing the putative Sp1-binding sites. Data were shown relative to qRT-PCR products amplified with input DNA before immunoprecipitation. p21 was used as a positive control. (F) The ChIP assays indicated that HDAC1 decreased the histone H3-acetylation level at the miR-200b promoters through the Sp1-dependent pathway. ChIP assays were performed with antibody directly against acetyl-Histone H3 (AcH3) in CSCs (SPC-A1/DTX) that were transfected with shRNA-control or shRNA-HDAC1#2. ChIP-derived DNA was amplified after immunoprecipitation by qRT-PCR with primers designed to amplify the sequences containing the putative Sp1-binding sites at the miR-200b promoters. Data was normalized to qRT-PCR products that were amplified with input DNA before immunoprecipitation. (G) The mRNA level of miR-200b as measured by qRT-PCR in CSCs from docetaxel-resistant LAD cells after transfection with the sh-HDAC1#2 or the control vectors or co-transfection with siRNA-Sp1 or siRNA-control vector, which indicated that HDAC1 repression upregulated the miR-200b expression at least partially through the Sp1-dependent pathway. Data were normalized to U6 RNA. Data were presented as mean ± SD of at least three independent experiments. * p

    Journal: PLoS ONE

    Article Title: Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

    doi: 10.1371/journal.pone.0109578

    Figure Lengend Snippet: HDAC1 repression enhances miR-200b promoter activities and up-regulates the histone H3-acetylation level at the promoters through the Sp1-dependent pathway. (A) Western blotting detection of HDAC1 protein expression in CSCs from docetaxel-resistant LAD cells or parental docetaxel-resistant LAD cells. GAPDH was used as an internal control. (B) Western blotting detection of HDAC1 protein expression in CSCs from SPC-A1/DTX cells transfected with either HDAC1-shRNAs (HDAC1-shRNA#1, #2 or #3) or shRNA-control vectors, which indicated that the inhibitory effect of HDAC1-shRNA#2 was the most effective shRNA vector. GAPDH was used as an internal control. (C) Luciferase activities after transfection of the sh-HDAC1#2 or the control vectors into CSCs (SPC-A1/DTX) that had been previously cotransfected with Renilla and either wild-type, Sp1 mutant or PGL3-basic vector-firefly-promoter reporter constructs. Data was normalized to Renilla luciferase activity and determined relative to PGL3-basic-vector promoter activity. (D) The co-immunoprecipitation assay indicated that HDAC1 co-immunoprecipitated with Sp1 in vivo in CSCs (SPC-A1/DTX). HDAC1 and Sp1 were visualized by Western blotting (WB). (E) Chromatin immunoprecipitation (ChIP) assay indicated that HDAC1 and Sp1 bound to the miR-200b promoters in vivo . ChIP assays were performed in CSCs (SPC-A1/DTX) with antibodies directly against Sp1, HDAC1 or IgG control. ChIP-derived DNA was amplified after immunoprecipitation by qRT-PCR with specific primers that were designed to amplify the sequences containing the putative Sp1-binding sites. Data were shown relative to qRT-PCR products amplified with input DNA before immunoprecipitation. p21 was used as a positive control. (F) The ChIP assays indicated that HDAC1 decreased the histone H3-acetylation level at the miR-200b promoters through the Sp1-dependent pathway. ChIP assays were performed with antibody directly against acetyl-Histone H3 (AcH3) in CSCs (SPC-A1/DTX) that were transfected with shRNA-control or shRNA-HDAC1#2. ChIP-derived DNA was amplified after immunoprecipitation by qRT-PCR with primers designed to amplify the sequences containing the putative Sp1-binding sites at the miR-200b promoters. Data was normalized to qRT-PCR products that were amplified with input DNA before immunoprecipitation. (G) The mRNA level of miR-200b as measured by qRT-PCR in CSCs from docetaxel-resistant LAD cells after transfection with the sh-HDAC1#2 or the control vectors or co-transfection with siRNA-Sp1 or siRNA-control vector, which indicated that HDAC1 repression upregulated the miR-200b expression at least partially through the Sp1-dependent pathway. Data were normalized to U6 RNA. Data were presented as mean ± SD of at least three independent experiments. * p

    Article Snippet: Chromatin immunoprecipitation (ChIP) assay ChIP assay was performed with Immunoprecipitation Assay Kits (Millipore) according to the manufacturer's instructions.

    Techniques: Western Blot, Expressing, Transfection, shRNA, Plasmid Preparation, Luciferase, Mutagenesis, Construct, Activity Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, In Vivo, Chromatin Immunoprecipitation, Derivative Assay, Amplification, Quantitative RT-PCR, Binding Assay, Positive Control, Cotransfection

    Reciprocal immunoprecipitation experiments confirm an interaction between Slo and CDK5. CDK5 was immunoprecipitated from lysates of a stable cell line expressing FLAG-cSlo-YFP (yellow fluorescent protein) and CDK5 using the anti-CDK5 C-8 antibody. Slo

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: CDK5 interacts with Slo and affects its surface expression and kinetics through direct phosphorylation

    doi: 10.1152/ajpcell.00339.2011

    Figure Lengend Snippet: Reciprocal immunoprecipitation experiments confirm an interaction between Slo and CDK5. CDK5 was immunoprecipitated from lysates of a stable cell line expressing FLAG-cSlo-YFP (yellow fluorescent protein) and CDK5 using the anti-CDK5 C-8 antibody. Slo

    Article Snippet: Reciprocal immunoprecipitation was done with anti-Flag (FLAG-cSlo) antibody and anti-CDK5 (CDK5) antibody using immunoprecipitation kits purchased from Sigma (St. Louis, MO).

    Techniques: Immunoprecipitation, Stable Transfection, Expressing

    UBE2K modulates ubiquitination and proteasomal degradation of histone H3. a In vitro ubiquitination assay of 6xHis-tagged H3F3A with UBE2K and FLAG::RNF2 or GFP::RNF138 ubiquitin ligases followed by immunoblotting with antibodies to 6xHis, UBE2K, FLAG and GFP. The images are representative of two independent experiments. b In vitro ubiquitination of recombinant p53 followed by immunoblotting with antibodies to p53, UBE2K, and GFP. The images are representative of two independent experiments. c In vitro ubiquitination of 6xHis::H1 followed by immunoblotting with antibodies to 6xHis, UBE2K, and GFP. The images are representative of two independent experiments. d Western blot of UBE2K overexpressing (OE) HEK293 with antibodies to H3, H3K9me3 and β-actin. The graphs represent the relative percentage of H3/β-actin and H3K9me3/H3 to DMSO-empty vector cells (mean ± s.e.m. of four independent experiments). When indicated in the figure, cells were treated with 5 µM MG-132 for 16 h. e Knockdown levels of proteasome activators in HEK293 cells. The graph (relative expression to non-targeting (NT) shRNA HEK293 cells) represents the mean ± s.e.m. (PSMD11 shRNA (n = 8), PSME4 shRNA ( n = 5), PSME3 shRNA ( n = 6)). f Percentage of chymotrypsin-like proteasome activity relative to NT shRNA HEK293 cells (mean ± s.e.m. of three independent experiments). MG-132 treatment: 5 µM MG-132 for 16 h. g Western blot of HEK293 cells with antibodies to H3 and UBE2K. The graph represents the relative percentage of H3/β-actin to NT shRNA cells (mean ± s.e.m. of three independent experiments). h Western blot of HEK293 cells with antibodies to H3, PSMD11 and UBE2K. The graph represents the relative percentage of H3/β-actin to NT shRNA + empty vector cells (mean ± s.e.m. of three independent experiments). i After immunoprecipitation with anti-H3 and anti-FLAG antibodies in HEK293 cells, we performed a re-immunoprecipitation (Re-IP) with the same antibodies. Re-IP was followed by western blot with antibodies against H3 and polyubiquitinated proteins (polyUb) to detect immunoprecipitated H3 protein and polyUb-H3, respectively. The images are representative of two independent experiments. Prior to immunoprecipitation, cells were treated with 5 µM MG-132 (16 h). All the statistical comparisons were made by two-tailed Student’s t -test for unpaired samples. P value: * P

    Journal: Communications Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells

    doi: 10.1038/s42003-020-0984-3

    Figure Lengend Snippet: UBE2K modulates ubiquitination and proteasomal degradation of histone H3. a In vitro ubiquitination assay of 6xHis-tagged H3F3A with UBE2K and FLAG::RNF2 or GFP::RNF138 ubiquitin ligases followed by immunoblotting with antibodies to 6xHis, UBE2K, FLAG and GFP. The images are representative of two independent experiments. b In vitro ubiquitination of recombinant p53 followed by immunoblotting with antibodies to p53, UBE2K, and GFP. The images are representative of two independent experiments. c In vitro ubiquitination of 6xHis::H1 followed by immunoblotting with antibodies to 6xHis, UBE2K, and GFP. The images are representative of two independent experiments. d Western blot of UBE2K overexpressing (OE) HEK293 with antibodies to H3, H3K9me3 and β-actin. The graphs represent the relative percentage of H3/β-actin and H3K9me3/H3 to DMSO-empty vector cells (mean ± s.e.m. of four independent experiments). When indicated in the figure, cells were treated with 5 µM MG-132 for 16 h. e Knockdown levels of proteasome activators in HEK293 cells. The graph (relative expression to non-targeting (NT) shRNA HEK293 cells) represents the mean ± s.e.m. (PSMD11 shRNA (n = 8), PSME4 shRNA ( n = 5), PSME3 shRNA ( n = 6)). f Percentage of chymotrypsin-like proteasome activity relative to NT shRNA HEK293 cells (mean ± s.e.m. of three independent experiments). MG-132 treatment: 5 µM MG-132 for 16 h. g Western blot of HEK293 cells with antibodies to H3 and UBE2K. The graph represents the relative percentage of H3/β-actin to NT shRNA cells (mean ± s.e.m. of three independent experiments). h Western blot of HEK293 cells with antibodies to H3, PSMD11 and UBE2K. The graph represents the relative percentage of H3/β-actin to NT shRNA + empty vector cells (mean ± s.e.m. of three independent experiments). i After immunoprecipitation with anti-H3 and anti-FLAG antibodies in HEK293 cells, we performed a re-immunoprecipitation (Re-IP) with the same antibodies. Re-IP was followed by western blot with antibodies against H3 and polyubiquitinated proteins (polyUb) to detect immunoprecipitated H3 protein and polyUb-H3, respectively. The images are representative of two independent experiments. Prior to immunoprecipitation, cells were treated with 5 µM MG-132 (16 h). All the statistical comparisons were made by two-tailed Student’s t -test for unpaired samples. P value: * P

    Article Snippet: Protein immunoprecipitation for interactome analysis Protein immunoprecipitation for interactome assays were performed as we reported in ref. .

    Techniques: In Vitro, Ubiquitin Assay, Recombinant, Western Blot, Plasmid Preparation, Expressing, shRNA, Activity Assay, Immunoprecipitation, Two Tailed Test

    Knockdown of UBE2K increases SETDB1 levels in hESCs. a Western blot analysis of H9 hESCs with antibodies to H3K9me1, H3K9me2, total H3 and β-actin. Graph represents the H3K9me1/H3 and H3K9me2/H3 relative percentage values to NT shRNA hESCs (mean ± s.e.m., five independent experiments). b Western blot analysis of H9 hESCs with antibodies to H3K9me3, H3K4me3, H3K27me3, H3K27ac, total H3 and β-actin. Graph represents the mean ± s.e.m of H3K9me3/H3 ( n = 5 independent experiments), H3K4me3/H3 ( n = 3), H3K27me3/H3 ( n = 5) and H3K27ac/H3 ( n = 3) relative percentage values to NT shRNA hESCs. c Immunoprecipitation with UBE2K and control FLAG antibodies in H9 hESCs followed by western blot with H3K9me3, H3K4me3, H3K27me3, H3K27ac, H3 and UBE2K antibodies. The images are representative of three independent experiments. d Immunoprecipitation with UBE2K and control FLAG antibodies in H9 hESCs followed by western blot with HTT, SETDB1 and UBE2K antibodies. The images are representative of three independent experiments. e Western blot analysis of H9 hESCs with antibodies to HTT and SETDB1. The graph represents the relative percentage values (corrected for β-actin loading control) to NT shRNA control hESCs (mean ± s.e.m., three independent experiments). f Immunoprecipitation with H3 and control FLAG antibodies in H9 hESCs followed by western blot with H3 and SETDB1 antibodies. The images are representative of two independent experiments. All the statistical comparisons were made by two-tailed Student’s t test for unpaired samples. P value: * P

    Journal: Communications Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells

    doi: 10.1038/s42003-020-0984-3

    Figure Lengend Snippet: Knockdown of UBE2K increases SETDB1 levels in hESCs. a Western blot analysis of H9 hESCs with antibodies to H3K9me1, H3K9me2, total H3 and β-actin. Graph represents the H3K9me1/H3 and H3K9me2/H3 relative percentage values to NT shRNA hESCs (mean ± s.e.m., five independent experiments). b Western blot analysis of H9 hESCs with antibodies to H3K9me3, H3K4me3, H3K27me3, H3K27ac, total H3 and β-actin. Graph represents the mean ± s.e.m of H3K9me3/H3 ( n = 5 independent experiments), H3K4me3/H3 ( n = 3), H3K27me3/H3 ( n = 5) and H3K27ac/H3 ( n = 3) relative percentage values to NT shRNA hESCs. c Immunoprecipitation with UBE2K and control FLAG antibodies in H9 hESCs followed by western blot with H3K9me3, H3K4me3, H3K27me3, H3K27ac, H3 and UBE2K antibodies. The images are representative of three independent experiments. d Immunoprecipitation with UBE2K and control FLAG antibodies in H9 hESCs followed by western blot with HTT, SETDB1 and UBE2K antibodies. The images are representative of three independent experiments. e Western blot analysis of H9 hESCs with antibodies to HTT and SETDB1. The graph represents the relative percentage values (corrected for β-actin loading control) to NT shRNA control hESCs (mean ± s.e.m., three independent experiments). f Immunoprecipitation with H3 and control FLAG antibodies in H9 hESCs followed by western blot with H3 and SETDB1 antibodies. The images are representative of two independent experiments. All the statistical comparisons were made by two-tailed Student’s t test for unpaired samples. P value: * P

    Article Snippet: Protein immunoprecipitation for interactome analysis Protein immunoprecipitation for interactome assays were performed as we reported in ref. .

    Techniques: Western Blot, shRNA, Immunoprecipitation, Two Tailed Test

    Loss of UBE2K increases total H3 levels and H3K9 trimethylation in hESCs. a Immunoprecipitation with UBE2K and control FLAG antibodies in H9 hESCs followed by western blot with H3, H1 and UBE2K antibodies. The images are representative of three independent experiments. b Western blot analysis of H9 hESCs with antibodies to total histone H3 and H1. Relative percentage values of H3/β-actin (mean ± s.e.m., eight independent experiments) and H1/β-actin (mean ± s.e.m., three independent experiments) to NT shRNA control hESCs are presented. c qPCR analysis of histone H3 variants in H9 hESCs. Graph (relative expression to NT shRNA control hESCs) represents the mean ± s.e.m. of five independent experiments. d Western blot analysis of H9 hESCs with antibodies to H3K9me3 and total H3. Graph represents the relative percentage of H3K9me3/H3 ratio to NT shRNA control hESCs (mean ± s.e.m., eight independent experiments). e Immunocytochemistry of H9 hESCs with antibody to H3K9me3. Hoechst staining was used as a marker of nuclei. Scale bar represents 20 μm. The images are representative of three independent experiments. All the statistical comparisons were made by two-tailed Student’s t test for unpaired samples. P value: ** P

    Journal: Communications Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells

    doi: 10.1038/s42003-020-0984-3

    Figure Lengend Snippet: Loss of UBE2K increases total H3 levels and H3K9 trimethylation in hESCs. a Immunoprecipitation with UBE2K and control FLAG antibodies in H9 hESCs followed by western blot with H3, H1 and UBE2K antibodies. The images are representative of three independent experiments. b Western blot analysis of H9 hESCs with antibodies to total histone H3 and H1. Relative percentage values of H3/β-actin (mean ± s.e.m., eight independent experiments) and H1/β-actin (mean ± s.e.m., three independent experiments) to NT shRNA control hESCs are presented. c qPCR analysis of histone H3 variants in H9 hESCs. Graph (relative expression to NT shRNA control hESCs) represents the mean ± s.e.m. of five independent experiments. d Western blot analysis of H9 hESCs with antibodies to H3K9me3 and total H3. Graph represents the relative percentage of H3K9me3/H3 ratio to NT shRNA control hESCs (mean ± s.e.m., eight independent experiments). e Immunocytochemistry of H9 hESCs with antibody to H3K9me3. Hoechst staining was used as a marker of nuclei. Scale bar represents 20 μm. The images are representative of three independent experiments. All the statistical comparisons were made by two-tailed Student’s t test for unpaired samples. P value: ** P

    Article Snippet: Protein immunoprecipitation for interactome analysis Protein immunoprecipitation for interactome assays were performed as we reported in ref. .

    Techniques: Immunoprecipitation, Western Blot, shRNA, Real-time Polymerase Chain Reaction, Expressing, Immunocytochemistry, Staining, Marker, Two Tailed Test

    NME2 interacts with telomere binding factor TRF2. ( A ) Co-immunoprecipitation of NME2 with TRF2. HT-1080 nuclear lysate immunoprecipitated with anti-TRF2 antibody, followed by immunoblotting with anti-NME2 or anti-TRF2 antibody. ( B ) Reverse co-immunoprecipitation TRF2 by NME2. HT-1080 nuclear lysate immunoprecipitated with anti-NME2 or specific isotype and immunoblotted with anti-TRF2 or anti-NME2 antibody. ( C ) Interaction of NME2 with TRF2 in vitro . Ni-NTA only or Ni-NTA NME2 (purified his-tagged) beads were incubated with cell extracts from HT-1080 following by detection of bound TRF2 by immunoblot using TRF2-specific antibody. ( D ) Association of TRF2 and NME2 in HT-1080 cells was not affected after treatment with DNase I, ethidiumbromide (EtBr) or RNase A. Quantification is shown for IP with anti-TRF2 and anti-NME2 antibodies with respect to respective input fractions; average of three independent pull-down experiments is shown.

    Journal: Nucleic Acids Research

    Article Title: Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells

    doi: 10.1093/nar/gkr1109

    Figure Lengend Snippet: NME2 interacts with telomere binding factor TRF2. ( A ) Co-immunoprecipitation of NME2 with TRF2. HT-1080 nuclear lysate immunoprecipitated with anti-TRF2 antibody, followed by immunoblotting with anti-NME2 or anti-TRF2 antibody. ( B ) Reverse co-immunoprecipitation TRF2 by NME2. HT-1080 nuclear lysate immunoprecipitated with anti-NME2 or specific isotype and immunoblotted with anti-TRF2 or anti-NME2 antibody. ( C ) Interaction of NME2 with TRF2 in vitro . Ni-NTA only or Ni-NTA NME2 (purified his-tagged) beads were incubated with cell extracts from HT-1080 following by detection of bound TRF2 by immunoblot using TRF2-specific antibody. ( D ) Association of TRF2 and NME2 in HT-1080 cells was not affected after treatment with DNase I, ethidiumbromide (EtBr) or RNase A. Quantification is shown for IP with anti-TRF2 and anti-NME2 antibodies with respect to respective input fractions; average of three independent pull-down experiments is shown.

    Article Snippet: Immunoprecipitation For immunoprecipitation experiments, 1 mg of nuclear extract was incubated for 2 h at 4°C with 6 μg of anti-NME2 antibody (MC-412 Kamiya Biomedical Company, USA), anti-telomerase reverse transcriptase antibody (Y182; ab32020 Abcam) or anti-TRF2 (NB110-57130 Novus Biologicals), and immunoprecipitation was performed using Catch and Release co-immunoprecipitation kit (Millipore, USA) as per manufacturer's protocol.

    Techniques: Binding Assay, Immunoprecipitation, In Vitro, Purification, Incubation

    NME2 directly interacts with hTERT ( A–C ). Co-immunoprecipitation of NME2 and hTERT. HT-1080 nuclear lysate was subjected to immunoprecipitation with anti-NME2 (A), -hTERT (B), -HA-hTERT (C) antibody, followed by immunoblotting with either anti-hTERT, anti-NME2 or anti-HA-tag antibody (A, B, C, respectively). ( D ) Interaction of NME2 with hTERT in vitro . Ni-NTA only or Ni-NTA NME2 (His-tagged) beads were incubated with cell extracts from HT-1080 following by detection of bound hTERT by immunoblot using anti-hTERT antibody. ( E ) Association of endogenous hTERT with NME2 in HT-1080 cells was not changed after treatment with DNase I, ethidiumbromide (EtBr) or RNase A. Quantification is shown for IP with anti-NME2 and anti-hTERT antibodies with respect to respective input fractions; average of three independent pull-down experiments is shown.

    Journal: Nucleic Acids Research

    Article Title: Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells

    doi: 10.1093/nar/gkr1109

    Figure Lengend Snippet: NME2 directly interacts with hTERT ( A–C ). Co-immunoprecipitation of NME2 and hTERT. HT-1080 nuclear lysate was subjected to immunoprecipitation with anti-NME2 (A), -hTERT (B), -HA-hTERT (C) antibody, followed by immunoblotting with either anti-hTERT, anti-NME2 or anti-HA-tag antibody (A, B, C, respectively). ( D ) Interaction of NME2 with hTERT in vitro . Ni-NTA only or Ni-NTA NME2 (His-tagged) beads were incubated with cell extracts from HT-1080 following by detection of bound hTERT by immunoblot using anti-hTERT antibody. ( E ) Association of endogenous hTERT with NME2 in HT-1080 cells was not changed after treatment with DNase I, ethidiumbromide (EtBr) or RNase A. Quantification is shown for IP with anti-NME2 and anti-hTERT antibodies with respect to respective input fractions; average of three independent pull-down experiments is shown.

    Article Snippet: Immunoprecipitation For immunoprecipitation experiments, 1 mg of nuclear extract was incubated for 2 h at 4°C with 6 μg of anti-NME2 antibody (MC-412 Kamiya Biomedical Company, USA), anti-telomerase reverse transcriptase antibody (Y182; ab32020 Abcam) or anti-TRF2 (NB110-57130 Novus Biologicals), and immunoprecipitation was performed using Catch and Release co-immunoprecipitation kit (Millipore, USA) as per manufacturer's protocol.

    Techniques: Immunoprecipitation, In Vitro, Incubation

    RagC complexes isolated by immunoprecipitation from Huh7 cells. ( A ) Huh7 cultured with Si-OH, Si-NH2, or PS-NH2 NPs (all 50 µg/ml) bearing BSA or RNase (both 50 µM) as hard protein corona or bare NPs for 4 h. After 4 h post NP treatment, cells were lysed with lysis buffer from immunoprecipitation kit (Abcam). RagC-mTOR complexes were co-immunoprecipitated from the precleared cell lysates with appropriate Ab as described in the manufacturer’s instruction. The resulting protein complex was eluted from the beads with Laemmli protein sample buffer for SDS-PAGE (Bio-Rad) and resolved on SDS-PAGE with specific antibody against mTOR (Cell Signaling) (full blots are presented in Supporting Information). ( B ) Scheme of district biochemical signaling activation in cells after stimulation with amino-functionalized non-biodegradable PS and biodegradable silica NPs. ∆mΦ – mitochondrial membrane potential; LMP – lysosomal membrane permeabilization.

    Journal: Scientific Reports

    Article Title: Nanoparticle core stability and surface functionalization drive the mTOR signaling pathway in hepatocellular cell lines

    doi: 10.1038/s41598-017-16447-6

    Figure Lengend Snippet: RagC complexes isolated by immunoprecipitation from Huh7 cells. ( A ) Huh7 cultured with Si-OH, Si-NH2, or PS-NH2 NPs (all 50 µg/ml) bearing BSA or RNase (both 50 µM) as hard protein corona or bare NPs for 4 h. After 4 h post NP treatment, cells were lysed with lysis buffer from immunoprecipitation kit (Abcam). RagC-mTOR complexes were co-immunoprecipitated from the precleared cell lysates with appropriate Ab as described in the manufacturer’s instruction. The resulting protein complex was eluted from the beads with Laemmli protein sample buffer for SDS-PAGE (Bio-Rad) and resolved on SDS-PAGE with specific antibody against mTOR (Cell Signaling) (full blots are presented in Supporting Information). ( B ) Scheme of district biochemical signaling activation in cells after stimulation with amino-functionalized non-biodegradable PS and biodegradable silica NPs. ∆mΦ – mitochondrial membrane potential; LMP – lysosomal membrane permeabilization.

    Article Snippet: Co-immunoprecipitation After 4 h post NP treatment, cells were lysed with lysis buffer from immunoprecipitation kit (Abcam, catalogue no. ab206996).

    Techniques: Isolation, Immunoprecipitation, Cell Culture, Lysis, SDS Page, Activation Assay

    Validation of interaction between Shisa9 and its putative interactors by means of co-immunoprecipitation from HEK293T cells. A. Schematic view of Shisa9-constructs used in co-immunoprecipitations. SP, signal sequence; TM, transmembrane domain; HA, HA-tag; EVTV, PDZ-ligand motif. B. Co-immunoprecipitation of Shisa9-interactor complexes from HEK293T cells. HA-Shisa9WT and HA-Shisa9ΔEVTV were overexpressed in HEK293T cells in combination with interacting proteins (one at a time). Anti-HA-tag antibody was added to immunoprecipitate HA-Shisa9-interactor complexes. Obtained samples were resolved on SDS-PAGE, western blotted and immunostained with anti-V5 antibody against V5-tagged interactors. Shisa9WT co-immunoprecipitates with PSD95, PSD93, GRIP1, PICK1 and Lin7b proteins, whereas Shisa9ΔEVTV lost the possibility to establish the interaction with named proteins (left panel). The right panel shows the same membranes as in the left panel stained with the anti-HA antibody in order to visualize the presence of Shisa9 in the immunoprecipitated samples. The 50 kDa band is indicated.

    Journal: PLoS ONE

    Article Title: C-Terminal Interactors of the AMPA Receptor Auxiliary Subunit Shisa9

    doi: 10.1371/journal.pone.0087360

    Figure Lengend Snippet: Validation of interaction between Shisa9 and its putative interactors by means of co-immunoprecipitation from HEK293T cells. A. Schematic view of Shisa9-constructs used in co-immunoprecipitations. SP, signal sequence; TM, transmembrane domain; HA, HA-tag; EVTV, PDZ-ligand motif. B. Co-immunoprecipitation of Shisa9-interactor complexes from HEK293T cells. HA-Shisa9WT and HA-Shisa9ΔEVTV were overexpressed in HEK293T cells in combination with interacting proteins (one at a time). Anti-HA-tag antibody was added to immunoprecipitate HA-Shisa9-interactor complexes. Obtained samples were resolved on SDS-PAGE, western blotted and immunostained with anti-V5 antibody against V5-tagged interactors. Shisa9WT co-immunoprecipitates with PSD95, PSD93, GRIP1, PICK1 and Lin7b proteins, whereas Shisa9ΔEVTV lost the possibility to establish the interaction with named proteins (left panel). The right panel shows the same membranes as in the left panel stained with the anti-HA antibody in order to visualize the presence of Shisa9 in the immunoprecipitated samples. The 50 kDa band is indicated.

    Article Snippet: For immunostaining of co-immunoprecipitation samples from HEK293T cells the following antibodies were used: anti-V5 (Abcam, 1∶1,000), anti-HA (3F10, Roche, 1∶1,000) in 5% milk TBST, incubation was done overnight at 4°C on a shaking platform.

    Techniques: Immunoprecipitation, Construct, Sequencing, SDS Page, Western Blot, Staining

    KIAA1199 connects Semaphorin 3A signalling to EGFR phosphorylation. ( a ) Semaphorin 3A-mediated EGFR phosphorylation requires KIAA1199. Control or KIAA1199-depleted CaSki cells were untreated or stimulated with Semaphorin 3A (100 ng ml −1 ) and WB analyses were carried out on the resulting cell extracts (lysis in SDS 1%). ( b ) KIAA1199 deficiency does not have an impact on EGFR mRNA levels in cervical cancer cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR, to assess EGFR mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from two independent experiments (means±s.d.) are shown. ( c ) Plexin A2 deficiency potentiates Semaphorin 3A-mediated EGFR phosphorylation. Control or Plexin A2-depleted CaSki cells were left untreated or stimulated with Semaphorin 3A for the indicated periods of time. The resulting cell extracts (lysis in SDS 1%) were subjected to WBs using the indicated antibodies. ( d ) Plexin A2 deficiency prolongs the binding of KIAA1199 to EGFR on EGF stimulation. Control or Plexin A2-deficient cells were untreated or stimulated with EGF for the indicated periods of time. Cell extracts were subjected to anti-FLAG (negative control) or -EGFR immunoprecipitations followed by anti- KIAA1199 or -EGFR WBs (top panels). Crude cell extracts were subjected to anti-pEGFR (Y845 and Y1068) (to validate the triggering of the EGF-dependent pathway), -EGFR, -KIAA1199, -Plexin A2 and -HSP90 WBs, as indicated. ( e ) KIAA1199 binds EGFR through its N-terminal domain. Cells were transfected with the indicated expression plasmids and protein extracts were subjected to anti-HA (negative control) or -FLAG IPs followed by an anti-EGFR WB (top panel). Crude cell extracts were also subjected to anti-EGFR and -FLAG WB analyses (bottom panels).

    Journal: Nature Communications

    Article Title: NF-κB-induced KIAA1199 promotes survival through EGFR signalling

    doi: 10.1038/ncomms6232

    Figure Lengend Snippet: KIAA1199 connects Semaphorin 3A signalling to EGFR phosphorylation. ( a ) Semaphorin 3A-mediated EGFR phosphorylation requires KIAA1199. Control or KIAA1199-depleted CaSki cells were untreated or stimulated with Semaphorin 3A (100 ng ml −1 ) and WB analyses were carried out on the resulting cell extracts (lysis in SDS 1%). ( b ) KIAA1199 deficiency does not have an impact on EGFR mRNA levels in cervical cancer cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR, to assess EGFR mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from two independent experiments (means±s.d.) are shown. ( c ) Plexin A2 deficiency potentiates Semaphorin 3A-mediated EGFR phosphorylation. Control or Plexin A2-depleted CaSki cells were left untreated or stimulated with Semaphorin 3A for the indicated periods of time. The resulting cell extracts (lysis in SDS 1%) were subjected to WBs using the indicated antibodies. ( d ) Plexin A2 deficiency prolongs the binding of KIAA1199 to EGFR on EGF stimulation. Control or Plexin A2-deficient cells were untreated or stimulated with EGF for the indicated periods of time. Cell extracts were subjected to anti-FLAG (negative control) or -EGFR immunoprecipitations followed by anti- KIAA1199 or -EGFR WBs (top panels). Crude cell extracts were subjected to anti-pEGFR (Y845 and Y1068) (to validate the triggering of the EGF-dependent pathway), -EGFR, -KIAA1199, -Plexin A2 and -HSP90 WBs, as indicated. ( e ) KIAA1199 binds EGFR through its N-terminal domain. Cells were transfected with the indicated expression plasmids and protein extracts were subjected to anti-HA (negative control) or -FLAG IPs followed by an anti-EGFR WB (top panel). Crude cell extracts were also subjected to anti-EGFR and -FLAG WB analyses (bottom panels).

    Article Snippet: The anti-EGFR antibody used for immunoprecipitations was from GeneTex (Irvine, CA, USA).

    Techniques: Western Blot, Lysis, shRNA, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control, Transfection, Expressing

    Occupancy of Sp1 and κB Sites in Bright and Off States. (A) Flow cytometry histograms of expanded populations of Off- and Bright-sorted Jurkats infected with LGIT and each LGIT mutant (as in Figure 3A , panels 5–6). 10 6 cells were initially sorted from Off and Bright regions, and seven days of expansion was conducted to achieve 5×10 7 cells necessary for this ChIP protocol. We observed a moderate extent of Bright→Off and Off→Bright dynamic switching over this one-week expansion. (B) RelA ChIP results for Off- and Bright-sorted populations of LGIT , mutI Sp1 , mutII Sp1 , mutIII Sp1 , mutI NF-κB , and mutII NF-κB . Immunoprecipitations were performed using RelA antibody, and immunoprecipitated DNA was quantified using QPCR with primers against the HIV LTR. For analysis of input DNA and RelA immunoprecipitation, all LTR QPCR measurements were normalized by with ChIP-QPCR measurements for the endogenous TAP1/LMP2 regulatory domain [86] , which contains single κB and Sp1 sites that recruit RelA and p50 (refer to Figure S4A and S4B for non-normalized results). Primer sequences and QPCR conditions for HIV LTR and TAP1/LMP2 are supplied in Materials and Methods and Table S2 . The QPCR measurements for LTR and control TAP1/LMP2 were performed in triplicate, and error bars are standard deviations. Statistically significant differences from WT LGIT are denoted by black single asterisks (*, p

    Journal: PLoS Pathogens

    Article Title: Control of Stochastic Gene Expression by Host Factors at the HIV Promoter

    doi: 10.1371/journal.ppat.1000260

    Figure Lengend Snippet: Occupancy of Sp1 and κB Sites in Bright and Off States. (A) Flow cytometry histograms of expanded populations of Off- and Bright-sorted Jurkats infected with LGIT and each LGIT mutant (as in Figure 3A , panels 5–6). 10 6 cells were initially sorted from Off and Bright regions, and seven days of expansion was conducted to achieve 5×10 7 cells necessary for this ChIP protocol. We observed a moderate extent of Bright→Off and Off→Bright dynamic switching over this one-week expansion. (B) RelA ChIP results for Off- and Bright-sorted populations of LGIT , mutI Sp1 , mutII Sp1 , mutIII Sp1 , mutI NF-κB , and mutII NF-κB . Immunoprecipitations were performed using RelA antibody, and immunoprecipitated DNA was quantified using QPCR with primers against the HIV LTR. For analysis of input DNA and RelA immunoprecipitation, all LTR QPCR measurements were normalized by with ChIP-QPCR measurements for the endogenous TAP1/LMP2 regulatory domain [86] , which contains single κB and Sp1 sites that recruit RelA and p50 (refer to Figure S4A and S4B for non-normalized results). Primer sequences and QPCR conditions for HIV LTR and TAP1/LMP2 are supplied in Materials and Methods and Table S2 . The QPCR measurements for LTR and control TAP1/LMP2 were performed in triplicate, and error bars are standard deviations. Statistically significant differences from WT LGIT are denoted by black single asterisks (*, p

    Article Snippet: Immunoprecipitations were performed with Upstate polyclonal antibodies anti-p50 (06–886), anti-p65 (06–418), anti-p300 (05–257), anti-Sp1 (07–645), and anti-AcH3H9/14 (06–599) and Abcam polyclonal antibodies anti-HDAC1 (ab7028), anti-H3 (ab1791), and anti-TriMetH3K9 (ab8898).

    Techniques: Flow Cytometry, Cytometry, Infection, Mutagenesis, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

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

    Journal: Cellular Microbiology

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

    doi: 10.1111/cmi.12559

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

    Article Snippet: Twenty‐four hours later, the cells were lysed in RIPA buffer and cell lysates subjected to immunoprecipitations using mouse anti‐NS1 antibody (clone NS1‐23‐1, IMV Münster) and protein G agarose beads (Roche) for 4 h at 4°C.

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