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  • 99
    Millipore gst fusion proteins
    HMG2L1 interacts with myocardin in vivo and in vitro . A , HMG2L1 binds to myocardin family proteins in vivo . Myocardin or HA-tagged MRTF-A/B adenovirus were transduced into A7r5 SMCs. Subsequently, nuclear extract was harvested, and proteins were immunoprecipitated with anti-myocardin, HA, HMG2L1, or control IgG antibodies. The immunoprecipitated proteins were detected by Western blotting using anti-myocardin, anti-HA, and anti-HMG2L1 antibodies, as indicated at the right of the blot . 10% total extract was loaded as input. B , HMG2L1 does not bind to <t>SRF.</t> <t>GST</t> fused to SRF or GST alone was expressed in bacteria, conjugated to glutathione-Sepharose beads, and incubated with bacterially expressed T7 tagged amino-terminal-myocardin (amino acids 1–585) or full-length HMG2L1 as indicated. Bound proteins were identified by Western blotting with an anti-T7 antibody ( upper panel ). The lower panel showed the expression of the GST-SRF fusion protein or GST alone (marked by an asterisk to the top left of each protein). The interaction between myocardin and SRF served as a positive control. C , schematic representation of myocardin indicating the GST-fusion proteins analyzed and summary of myocardin domains mapped for interacting with HMG2L1 from D and E. NTD , N-terminal domain; ++, basic domain; Q , poly Q domain; LZ , leuzine zipper domain; TAD , transcriptional activation domain; mut : mutant. D , HMG2L1 binds to the full-length myocardin. Bacterially expressed full-length myocardin was incubated with the GST-HMG2L1 fusion protein. Western blotting was performed to detect the myocardin fusion protein that bound to HMG2L1 ( upper panel ). The lower panel in D indicates the expression of the GST fusion protein of full-length HMG2L1 as detected by a Ponceau S staining. E , HMG2L1 binds to the myocardin N-terminal domain, basic, and poly Q domain. Bacterially expressed HMG2L1 was incubated with the series of GST-myocardin fusion proteins indicated in C . Western blotting was performed to detect the GST-myocardin fusion proteins that bound HMG2L1 ( upper panel ). The lower panel in E indicates the expression of the myocardin GST fusion proteins as detected by a Ponceau S staining. *, GST fusion protein.
    Gst Fusion Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4055 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti gst
    In vivo validation of substrates ubiquitylated on protein microarrays. (A) Ten putative substrates of ubiquitylation identified on the protein microarrays but not reported in the literature were selected for validation of the modification in vivo . Myc- or <t>GST-substrates</t> were co-expressed with HA-ubiquitin in HEK293T cells. HEK293T cell extracts were prepared using denaturing conditions, substrates immunoprecipitated with anti-Myc or anti-GST antibodies, and ubiquitylation detected by immunoblotting with anti-HA antibodies. Empty vector co-expressed with HA-tagged ubiquitin served as control. Substrates indicated in each lane are: 1- ADRBK2, 2- ACVR1B, 3- PIM2, 4- PRKCgamma, 5- KIF2C, 6- RPS6KA5, 7- ITK, 8- EPHA1, 9- TRIM52, and 10- EPHA5. Of 10 substrates 8 were found to be expressed and immunoprecipitated at detectable levels and of these all demonstrated evidence of ubiquitylation in vivo . To best visualize an ubiquitin smear, substrates 1, 2, 3, 4 were separated by 10% SDS-PAGE gels, while larger molecular weight substrates 5, 6, 7, 8 were separated by 6% SDS-PAGE gels. (B) Ubiquitylation of <t>YY1.</t> HEK293T cells were transfected with plasmids that express HA-ubiquitin, endogenous YY1 protein immunoprecipitated from the denatured extracts, and conjugation to ubiquitin determined by Western blot analysis with anti-HA antibodies (left). Immunoprecipitation efficiency was determined by probing blots with anti-YY1 antibodies (right). Immunoprecipitation with IgG antibodies of the same species served as control.
    Anti Gst, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology gst
    The protective effects of CA, QU and AV on UVA-mediated downregulation of Nrf2 target genes. (A) γ-GCL <t>(γ-GCLC</t> and γ-GCLM), (B) <t>GST</t> and (C) NQO1 mRNA expressions were assessed by real-time RT-PCR analysis at 2 h after UVA irradiation in B16F10 cells pretreated with test compounds. The statistical significance of differences between the control and irradiated cells was evaluated by Student’s t test and between UVA-irradiated and compounds-treated cells by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. ## P
    Gst, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1948 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore gst
    Schematic diagram of the GHS-based detoxification pathway in plants. MCB is used as a model substrate for conjugation to <t>GSH</t> by a <t>GST</t> in the cytoplasm and subsequent sequestration in the vacuole by a glutathione S- ).
    Gst, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare anti gst antibody
    Ca Bdf1 BDs are resistant to BETi. ( a ) HTRF assay. A biotinylated tetra-acetylated histone H4 peptide is bound to streptavidin beads coupled to the donor fluorophore. A <t>GST-tagged</t> BD is bound by an anti-GST antibody coupled to the acceptor fluorophor. Peptide binding by the BD results in FRET. The addition of a BETi reduces FRET. ( b ) HTRF assays performed on BDs from Ca Bdf1 and human Brd4 in the presence of the indicated BETi. Inhibition curves are shown as closed (BD1) and open (BD2) circles in green (Brd4) and magenta (Bdf1). IC 50 values are listed below each graph. Data represent the mean and s.d. values from three independent experiments. ( c ) Representative ITC experiments measuring the binding of JQ1 to Ca BDF1 BDs (magenta) and to human Brd4 BD1 (green). The values indicated for K d and N represent the mean and s.d. from three independent experiments. See also Supplementary Table 1 . ( d ) BET inhibitors do not affect C. albicans growth, even when Bdf1 BD1 or BD2 is deleted. Inhibitors were tested at 10 μM concentration. Experiments were performed in the presence of doxycyline to repress expression from the pTetO -BDF1 allele. Data represent the mean and s.d. values from three independent experiments.
    Anti Gst Antibody, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gst
    Identification of the <t>Nedd8</t> binding sequence in Smurf HECT domain. ( A ) Amino acid sequence alignment of Nedd8-binding sites in N-lobe small subdomain and C-lobe domain of Smurf1 and Smurf2. The amino acid highlighted in red indicates the residues conserved in the sites for Nedd8 binding. The conserved residues are found in the sequence L(X7)R(X5)F(X)ALQ. ( B ) Co-IP assay for the interaction between Nedd8 and Smurf1 possessing L(X7)R(X5)F(X)ALQ to A(X7)A(X5)A(X)AAA substitution in the N-lobe small subdomain and C-lobe domain of Smurf1 HECT. Western-blot analysis of whole-cell lysates and immunoprecipitates with Flag antibody. ( C ) <t>GST</t> pull-down assay for the interaction between Nedd8 and Smurf1 possessing L(X7)R(X5)F(X)ALQ to A(X7)A(X5)A(X)AAA substitution of the whole HECT domain. Expressed Myc-tagged Smurf1-HECT (wide type or mutant) proteins in cell lysates were precipitated by GST or GST-Nedd8 from the bacterial lysates. The precipitates were analyzed by western blotting using anti-Myc antibody to detect Smurf-HECT and anti-GST antibody to detect GST or GST-Nedd8. ( D , E ) Co-IP assay for the interaction between Nedd8 and Smurf1/2 possessing L(X7)R(X5)F(X)ALQ to A(X7)A(X5)A(X)AAA substitution in the Smurfs. Western-blot analysis of whole-cell lysates and immunoprecipitates with Flag or Myc antibody.
    Anti Gst, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1077 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore gst tag
    IQGAP1 C terminus aa 1503–1657 is required for binding and suppressing TβRII. ( A ) Top 4 rows: full-length (FL) IQGAP1 and <t>GST-fused</t> truncated IQGAP1 proteins are shown. Bottom, GST fused truncated IQGAP1 proteins extracted from bacteria were incubated with HSC lysates for GST pull-down assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII. Ponceau S staining depicted the purity of the recombinant proteins. ( B ) Top: after the GST tag of GST-TRII was removed by thrombin treatment, <t>detagged</t> TβRII was incubated with GST-fused IQGAP1 proteins for in vitro binding assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII directly in vitro. Ponceau S staining depicted the purity of GST and GST-fused IQGAP1 proteins. Bottom: detagged IQGAP1 aa 746–1657 was incubated with GST or GST-TβRII for in vitro binding assays. GST-TβRII bound to IQGAP1 aa 746-1657 directly in vitro. aa 746–1657 instead of aa 1503–1657 of IQGAP1 was used in this assay because IQGAP1 antibodies could not recognize aa 1503–1657 of IQGAP1. Ponceau S staining depicted the purity of GST and GST fusion proteins. ( C ) HSCs expressing TβRII-HA were transduced with lentiviruses encoding GFP, IQGAP1-FLAG, or IQGAP1 (1-1502)-FLAG, and subjected to WB. In contrast with IQGAP1, IQGAP1 (1-1502) mutant lacking the TβRII binding region failed to repress TβRII protein levels. Densitometric ratios are shown on the bottom. All data shown represent multiple repeats with similar results.
    Gst Tag, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1811 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare gst
    <t>DEK40</t> recognizes and directly binds cox3 , nad2 , and nad5 transcripts. (A) Nucleotide sequence of RNA probes. Edited sites are indicated in red. (B) RNA EMSAs indicated that DEK40 directly binds to cox3 transcripts that surround the cox3 edited site. Unlabeled probe was used as a competitor, and <t>GST</t> was used as a negative control. Black arrows show the shifted bound and free RNA probe. (C) RNA EMSAs indicated that DEK40 directly binds to nad2 transcripts that surround the nad2 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probesw are indicated by black arrows. (D) RNA EMSAs indicated that DEK40 directly binds to nad5 transcripts that surround the nad5 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probes are indicated by black arrows.
    Gst, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 3513 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gst fusion protein
    Bt 2 cAMP does not affect the dephosphorylation of <t>ERK2.</t> A, A <t>GST</t> fusion protein of P-ERK2 (GST-P-ERK2) was incubated with buffer only for 40 min at 4 C or 37 C as indicated. Another sample was incubated for 40 min at 37 C with buffer containing 120 U of
    Gst Fusion Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti glutathione s transferase gst antibody
    Effect of inositol-(1,4,5)triphosphate (IP 3 ) sponge inhibitor on Ca 2+ signaling in MSNs that overexpress HAP1A. (A) Immunoblots of YAC128 MSN cultures that overexpressed the glutathione S -transferase <t>(GST)-tagged</t> IP 3 R1 ligand binding region (226–604 amino acid region) with a point mutation (R441Q: m49) cloned into the pUltra-Chili vector, indicated as p49-GST, and the negative control with a point mutation (K508A: m30), indicated as p30-GST cloned into the pUltra-Chili vector or control (non-transduced) MSNs. MSN cultures on DIV14 from YAC128 mice that overexpressed p49-dTomato and p30-dTomato as an IP 3 sponge and control respectively were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. (B) DHPG-induced Ca 2+ release from the ER in YAC128 MSNs that overexpressed p49-dTomato or p30-dTomato as a control for the IP 3 sponge inhibitor. (C) Protocol to measure DHPG-induced ER Ca 2+ release and DHPG-induced SOCE using the eight-well system. MSN cultures on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP or pLenti-GFP and p49-dTomato or p30-dTomato were loaded with Fura-2AM and incubated in Ca 2 -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (D) Effect of p49-dTomato or p30-dTomato on DHPG-induced ER Ca 2+ release in YAC128 MSNs that overexpressed HAP1A-pLenti-GFP or pLenti-GFP. (E) Effect of p49-dTomato or p30-dTomato on DHPG-induced SOCE in YAC128 MSNs that overexpressed HAP1A-pLenti-GFP or pLenti-GFP. (F) To avoid the firing-effect in MSNs, 1 μM tetrodotoxin (TTX) was added during the Ca 2+ measurement protocol. In the presence of TTX, DHPG-induced ER Ca 2+ release and DHPG-induced SOCE were measured using the eight-well system. MSN cultures on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP or pLenti-GFP and p49-dTomato or p30-dTomato were loaded with Fura-2AM and incubated in Ca 2+ -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. Normalized delta ratio was analyzed as average from three experiments (B,D) . * p
    Anti Glutathione S Transferase Gst Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 685 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc gst
    Phosphorylation of S670 is required for error correction and kinetochore tension. (A) Synchronized HeLa cells depleted of BubR1 by siRNA were injected with various <t>GST/BubR1</t> constructs and treated with MG132 to prevent mitotic exit. Coverslips were chilled on ice for 10 min, extracted, fixed, and stained for GST, ACA, <t>tubulin,</t> and DAPI. Tubulin and ACA signals were deconvolved to visualize microtubule attachments at individual bioriented kinetochores that are shown in the insets. (B) Interkinetochore distances were measured between pairs of ACA foci with end-on attachments (50
    Gst, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 545 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti gst
    <t>TYK2-ΔE8</t> is catalytically active but unable to rescue cytokine signaling. (A) Basal in vitro kinase activity of TYK2 from unstimulated 11,1 cells stably expressing TYK2 WT or TYK2-ΔE8. TYK2 was immunoprecipitated and subjected to an in vitro kinase reaction for 5 min at 30°C in the presence (+) or absence (-) of 30 μM ATP. Phosphorylated TYK2 in the reaction was revealed by immunoblotting with anti-phospho-TYK2. The membrane was reprobed for TYK2. (B) IFN-induced JAK/STAT activation in 11,1 cells stably expressing TYK2 WT or TYK2-ΔE8. Cells were treated with IFN-β for 15 min. The level of tyrosine-phosphorylated TYK2, STAT1, STAT2 and STAT3 was analyzed by with phospho-specific Abs. The membrane was reprobed for TYK2 and total STATs. (C) IFNAR1 level in 11,1 cells (-) and derived clones stably expressing TYK2 WT or TYK2-ΔE8. (D) In vitro interaction of His-TYK2-FERM-SH2 with <t>GST-IFNAR1cyt.</t> His-TYK2-FERM-SH2 WT or ΔE8 were incubated with a GST fusion protein containing the cytoplasmic domain of IFNAR1(IFNAR1 cyt ) or IkB-β. Proteins bound to glutathione-Sepharose beads were separated on SDS-PAGE and visualized with TYK2 Abs. Five % input TYK2 protein shown at the bottom. (E) IL-12-induced JAK/STAT activation in 11,1 cells stably expressing the IL-12 receptor β1 and β2 chains. Cells were transiently transfected with TYK2 WT or TYK2-ΔE8. Twenty-four hrs later, cells were treated with IFN-β (500pM) or IL-12 (20ng/ml) for 15 min. The level of tyrosine-phosphorylated TYK2 and STAT1 was analyzed with phospho-specific Abs. The membrane was reprobed for TYK2 levels. A nonspecific band shown as loading control. (F) 293T cells were transfected with the pRc-CMV empty vector (EV), TYK2-ΔE8 or the triple mutant TYK2-K930R/Y1044F/Y1045F (DN) possessing dominant-negative activity [ 19 ]. Twenty-four hrs later, cells were treated with IFN-α for 15 min. Phosphorylation of STAT1, STAT2 and TYK2 were analyzed with phospho-specific Abs. The membrane strips were reprobed for TYK2 and STAT1 contents. Of note, neither TYK2-ΔE8 nor DN can be inducibly phosphorylated, hence the phospho-TYK2 band corresponds to endogenous TYK2.
    Anti Gst, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 460 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Xenobiotics glutathione s transferases gsts
    Phylogeny of glutathione S-transferases <t>(GSTs)</t> . Branches with dots had > 80% bootstrap support. The tree was rooted with human HsapGSTA1.Aaeg, Aedes aegypti ; Agam, Aedes gambiae ; Amel, Apis mellifera ; Apis, Acyrthosiphon pisum ; Bmor, Bombyx mori ; Cqui, Culex quinquefasciatus ; Dmel, Drosophila melanogaster ; Dpon, Dendroctonus ponderosae ; Hsap; Homo sapiens ; Nvit, Nasonia vitripennis ; Phum, Pediculus humanus ; Tcas, Tribolium castaneum .
    Glutathione S Transferases Gsts, supplied by Xenobiotics, used in various techniques. Bioz Stars score: 91/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare gst beads
    Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing <t>GST–FbxA:F-box/WD40</t> were lysed and the 10,000 × G supernatant was adsorbed to <t>g–Sepharose.</t> The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.
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    91
    Xenobiotics gsts
    Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing <t>GST–FbxA:F-box/WD40</t> were lysed and the 10,000 × G supernatant was adsorbed to <t>g–Sepharose.</t> The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.
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    99
    Millipore glutathione s transferase gst
    Effect of mitochondrial DNA (mtDNA) depletion on the transcripts of the antioxidant defense enzymes. The total RNA from the control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts was prepared. The transcript level of the antioxidant defense enzymes was quantified by RT-PCR (A) and q RT-PCR (B). β-Actin was used as the control. All results represent the mean ± SEM from five independent experiments. GR, glutathione reductase; GPx, glutathione peroxidase; <t>GST,</t> glutathione S-transferase; SOD, superoxide dismutase. *** p
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    GE Healthcare gst sepharose 4b
    Direct physical interaction between GATA-1 and CP2. (A) Purified <t>GST</t> or GST fusion proteins containing full-length CP2 (GST-CP2) preadsorbed to <t>glutathione-Sepharose</t> beads were incubated with 35 S-labeled in vitro-transcribed/translated GATA-1 (lanes 1
    Gst Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gst antibodies
    gcd7 - 201 gcn2∆ rescued Slg + and Gcd + phenotype when transformed with pEG(KG)/ <t>TAN1</t> plasmid. GCD7 gcn2∆ and gcd7 - 201 Gcn2∆ harboring pEG(KG)/ TAN1 or empty vector pEG(KG) were streaked in parallel on SC medium laking uracil, but either containing a raffinose or b galactose. Uracil-based plasmid pEG(KG)/ TAN1 was evicted on SC medium containing, c FOA and further streaked on d SC-Ura medium. e Spotting on SC medium containing 2% galactose and lacking uracil or SC medium containing 2% galactose, 30 mM 3-AT and lacking uracil. Plates were incubated at 30 °C for 2 days. f Western analysis of <t>GST-Tan1p</t> expression in gcd7 - 201 gcn2∆ strain with anti-GST antibody. The whole-cell protein extracts (20 µg) were prepared from uninduced and 2% galactose-induced cultures of the strain harboring pEG(KG)/ TAN1 . Samples were separated on 10% SDS gel followed by western blotting using anti-GST for GST-Tan1 and anti-Gcd6 antibodies (loading control). UI uninduced, I induced
    Anti Gst Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    GE Healthcare gst fusion proteins
    Activation of full-length endophilin-A1 by <t>GST-PRD.</t> A. Negative-stain EM with Folch liposomes. Assays with endophilin-A1 (full-length or N-BAR domain; 10 µM) were carried out as described before. The inset shows a zoomed-in view of the red box area of the image, with scale bar = 100 nm. B. Statistical analysis of vesicle size distribution. Diameters of vesicles produced by endophilin in the presence of GST-PRD were quantified from electron micrographs taken from three independent experiments. C. Liposome co-pelleting assay with Folch liposomes. Liposome binding assays were carried out as described in Fig. 3. The horizontal, dashed lines indicate the lipid-bound fraction of the isolated endophilin-A1 N-BAR (expressed as His 6 -SUMO-fusion protein) domain and isolated full-length endophilin-A1 under similar conditions. Error bars represent standard deviations of a minimum of 3 independent experiments.
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    HMG2L1 interacts with myocardin in vivo and in vitro . A , HMG2L1 binds to myocardin family proteins in vivo . Myocardin or HA-tagged MRTF-A/B adenovirus were transduced into A7r5 SMCs. Subsequently, nuclear extract was harvested, and proteins were immunoprecipitated with anti-myocardin, HA, HMG2L1, or control IgG antibodies. The immunoprecipitated proteins were detected by Western blotting using anti-myocardin, anti-HA, and anti-HMG2L1 antibodies, as indicated at the right of the blot . 10% total extract was loaded as input. B , HMG2L1 does not bind to SRF. GST fused to SRF or GST alone was expressed in bacteria, conjugated to glutathione-Sepharose beads, and incubated with bacterially expressed T7 tagged amino-terminal-myocardin (amino acids 1–585) or full-length HMG2L1 as indicated. Bound proteins were identified by Western blotting with an anti-T7 antibody ( upper panel ). The lower panel showed the expression of the GST-SRF fusion protein or GST alone (marked by an asterisk to the top left of each protein). The interaction between myocardin and SRF served as a positive control. C , schematic representation of myocardin indicating the GST-fusion proteins analyzed and summary of myocardin domains mapped for interacting with HMG2L1 from D and E. NTD , N-terminal domain; ++, basic domain; Q , poly Q domain; LZ , leuzine zipper domain; TAD , transcriptional activation domain; mut : mutant. D , HMG2L1 binds to the full-length myocardin. Bacterially expressed full-length myocardin was incubated with the GST-HMG2L1 fusion protein. Western blotting was performed to detect the myocardin fusion protein that bound to HMG2L1 ( upper panel ). The lower panel in D indicates the expression of the GST fusion protein of full-length HMG2L1 as detected by a Ponceau S staining. E , HMG2L1 binds to the myocardin N-terminal domain, basic, and poly Q domain. Bacterially expressed HMG2L1 was incubated with the series of GST-myocardin fusion proteins indicated in C . Western blotting was performed to detect the GST-myocardin fusion proteins that bound HMG2L1 ( upper panel ). The lower panel in E indicates the expression of the myocardin GST fusion proteins as detected by a Ponceau S staining. *, GST fusion protein.

    Journal: The Journal of Biological Chemistry

    Article Title: Repression of Smooth Muscle Differentiation by a Novel High Mobility Group Box-containing Protein, HMG2L1 *

    doi: 10.1074/jbc.M110.109868

    Figure Lengend Snippet: HMG2L1 interacts with myocardin in vivo and in vitro . A , HMG2L1 binds to myocardin family proteins in vivo . Myocardin or HA-tagged MRTF-A/B adenovirus were transduced into A7r5 SMCs. Subsequently, nuclear extract was harvested, and proteins were immunoprecipitated with anti-myocardin, HA, HMG2L1, or control IgG antibodies. The immunoprecipitated proteins were detected by Western blotting using anti-myocardin, anti-HA, and anti-HMG2L1 antibodies, as indicated at the right of the blot . 10% total extract was loaded as input. B , HMG2L1 does not bind to SRF. GST fused to SRF or GST alone was expressed in bacteria, conjugated to glutathione-Sepharose beads, and incubated with bacterially expressed T7 tagged amino-terminal-myocardin (amino acids 1–585) or full-length HMG2L1 as indicated. Bound proteins were identified by Western blotting with an anti-T7 antibody ( upper panel ). The lower panel showed the expression of the GST-SRF fusion protein or GST alone (marked by an asterisk to the top left of each protein). The interaction between myocardin and SRF served as a positive control. C , schematic representation of myocardin indicating the GST-fusion proteins analyzed and summary of myocardin domains mapped for interacting with HMG2L1 from D and E. NTD , N-terminal domain; ++, basic domain; Q , poly Q domain; LZ , leuzine zipper domain; TAD , transcriptional activation domain; mut : mutant. D , HMG2L1 binds to the full-length myocardin. Bacterially expressed full-length myocardin was incubated with the GST-HMG2L1 fusion protein. Western blotting was performed to detect the myocardin fusion protein that bound to HMG2L1 ( upper panel ). The lower panel in D indicates the expression of the GST fusion protein of full-length HMG2L1 as detected by a Ponceau S staining. E , HMG2L1 binds to the myocardin N-terminal domain, basic, and poly Q domain. Bacterially expressed HMG2L1 was incubated with the series of GST-myocardin fusion proteins indicated in C . Western blotting was performed to detect the GST-myocardin fusion proteins that bound HMG2L1 ( upper panel ). The lower panel in E indicates the expression of the myocardin GST fusion proteins as detected by a Ponceau S staining. *, GST fusion protein.

    Article Snippet: Fragments of mouse myocardin, SRF, and HMG2L1 cDNAs were cloned into pGEX-4T vectors (Stratagene) to generate GST fusion proteins or cloned into pET28 vectors (Novagen) to generate T7 fusion proteins and GST pulldown assays were performed as described in our previous reports ( , ).

    Techniques: In Vivo, In Vitro, Immunoprecipitation, Western Blot, Incubation, Expressing, Positive Control, Activation Assay, Mutagenesis, Staining

    Characterization of HMG2L1 domains that physically and functionally bind to myocardin. A , schematic illustration of the domain structures of HMG2L1 indicating the positions of the truncation mutants used for mapping studies. mut , mutant. B , bacterially expressed HMG2L1 truncation mutants indicated in A were incubated with the N-terminal myocardin (amino acids 1–585) GST fusion protein ( lower panel ). Western blotting was performed to detect the HMG2L1 bound to amino-terminal-myocardin GST fusion protein ( upper panel ). The lower panel in B indicates the expression of the GST or GST-fused amino-terminal-myocardin protein as detected by a Ponceau S staining. Myocardin was found to bind to the HMG2L1 N terminus (amino acids 1–472) as summarized in A. C , HMG box of HMG2L1 is required but not sufficient to abrogate the transactivation of myocardin on the SM22 α promoter. An SM22 α promoter reporter gene was transfected into 10T1/2 cells together with myocardin in the presence of expression plasmids for full-length of HMG2L1 or a variety of HMG2L1 truncation mutants as indicated at the left panel . All data are normalized to the activation produced by myocardin alone ( black bar , set to 100).

    Journal: The Journal of Biological Chemistry

    Article Title: Repression of Smooth Muscle Differentiation by a Novel High Mobility Group Box-containing Protein, HMG2L1 *

    doi: 10.1074/jbc.M110.109868

    Figure Lengend Snippet: Characterization of HMG2L1 domains that physically and functionally bind to myocardin. A , schematic illustration of the domain structures of HMG2L1 indicating the positions of the truncation mutants used for mapping studies. mut , mutant. B , bacterially expressed HMG2L1 truncation mutants indicated in A were incubated with the N-terminal myocardin (amino acids 1–585) GST fusion protein ( lower panel ). Western blotting was performed to detect the HMG2L1 bound to amino-terminal-myocardin GST fusion protein ( upper panel ). The lower panel in B indicates the expression of the GST or GST-fused amino-terminal-myocardin protein as detected by a Ponceau S staining. Myocardin was found to bind to the HMG2L1 N terminus (amino acids 1–472) as summarized in A. C , HMG box of HMG2L1 is required but not sufficient to abrogate the transactivation of myocardin on the SM22 α promoter. An SM22 α promoter reporter gene was transfected into 10T1/2 cells together with myocardin in the presence of expression plasmids for full-length of HMG2L1 or a variety of HMG2L1 truncation mutants as indicated at the left panel . All data are normalized to the activation produced by myocardin alone ( black bar , set to 100).

    Article Snippet: Fragments of mouse myocardin, SRF, and HMG2L1 cDNAs were cloned into pGEX-4T vectors (Stratagene) to generate GST fusion proteins or cloned into pET28 vectors (Novagen) to generate T7 fusion proteins and GST pulldown assays were performed as described in our previous reports ( , ).

    Techniques: Mutagenesis, Incubation, Western Blot, Expressing, Staining, Transfection, Activation Assay, Produced

    NHERF-2 interacts with β-catenin in vitro and in vivo. (A) Schematic of tagged NHERF-2 proteins, indicating the tag, the two PDZ domains, and the ERM-binding domain (ERM BD). (B) An equal amount of each GST-tagged NHERF protein was immobilized

    Journal:

    Article Title:

    doi: 10.1091/mbc.E06-10-0960

    Figure Lengend Snippet: NHERF-2 interacts with β-catenin in vitro and in vivo. (A) Schematic of tagged NHERF-2 proteins, indicating the tag, the two PDZ domains, and the ERM-binding domain (ERM BD). (B) An equal amount of each GST-tagged NHERF protein was immobilized

    Article Snippet: GST-tagged proteins consisting of NHERF-1 and various regions of NHERF-2, including full-length protein, the first PDZ domain, the second PDZ domain including the C terminus, both PDZ domains alone, and the C terminus alone, were purified on a glutathione column (Sigma-Aldrich), washed with Tris-buffered saline (10 mM Tris-HCl, pH 8.0, and 9 g/l NaCl) and eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0.

    Techniques: In Vitro, In Vivo, Binding Assay

    In vivo validation of substrates ubiquitylated on protein microarrays. (A) Ten putative substrates of ubiquitylation identified on the protein microarrays but not reported in the literature were selected for validation of the modification in vivo . Myc- or GST-substrates were co-expressed with HA-ubiquitin in HEK293T cells. HEK293T cell extracts were prepared using denaturing conditions, substrates immunoprecipitated with anti-Myc or anti-GST antibodies, and ubiquitylation detected by immunoblotting with anti-HA antibodies. Empty vector co-expressed with HA-tagged ubiquitin served as control. Substrates indicated in each lane are: 1- ADRBK2, 2- ACVR1B, 3- PIM2, 4- PRKCgamma, 5- KIF2C, 6- RPS6KA5, 7- ITK, 8- EPHA1, 9- TRIM52, and 10- EPHA5. Of 10 substrates 8 were found to be expressed and immunoprecipitated at detectable levels and of these all demonstrated evidence of ubiquitylation in vivo . To best visualize an ubiquitin smear, substrates 1, 2, 3, 4 were separated by 10% SDS-PAGE gels, while larger molecular weight substrates 5, 6, 7, 8 were separated by 6% SDS-PAGE gels. (B) Ubiquitylation of YY1. HEK293T cells were transfected with plasmids that express HA-ubiquitin, endogenous YY1 protein immunoprecipitated from the denatured extracts, and conjugation to ubiquitin determined by Western blot analysis with anti-HA antibodies (left). Immunoprecipitation efficiency was determined by probing blots with anti-YY1 antibodies (right). Immunoprecipitation with IgG antibodies of the same species served as control.

    Journal: PLoS ONE

    Article Title: Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays

    doi: 10.1371/journal.pone.0011332

    Figure Lengend Snippet: In vivo validation of substrates ubiquitylated on protein microarrays. (A) Ten putative substrates of ubiquitylation identified on the protein microarrays but not reported in the literature were selected for validation of the modification in vivo . Myc- or GST-substrates were co-expressed with HA-ubiquitin in HEK293T cells. HEK293T cell extracts were prepared using denaturing conditions, substrates immunoprecipitated with anti-Myc or anti-GST antibodies, and ubiquitylation detected by immunoblotting with anti-HA antibodies. Empty vector co-expressed with HA-tagged ubiquitin served as control. Substrates indicated in each lane are: 1- ADRBK2, 2- ACVR1B, 3- PIM2, 4- PRKCgamma, 5- KIF2C, 6- RPS6KA5, 7- ITK, 8- EPHA1, 9- TRIM52, and 10- EPHA5. Of 10 substrates 8 were found to be expressed and immunoprecipitated at detectable levels and of these all demonstrated evidence of ubiquitylation in vivo . To best visualize an ubiquitin smear, substrates 1, 2, 3, 4 were separated by 10% SDS-PAGE gels, while larger molecular weight substrates 5, 6, 7, 8 were separated by 6% SDS-PAGE gels. (B) Ubiquitylation of YY1. HEK293T cells were transfected with plasmids that express HA-ubiquitin, endogenous YY1 protein immunoprecipitated from the denatured extracts, and conjugation to ubiquitin determined by Western blot analysis with anti-HA antibodies (left). Immunoprecipitation efficiency was determined by probing blots with anti-YY1 antibodies (right). Immunoprecipitation with IgG antibodies of the same species served as control.

    Article Snippet: Antibodies Antibodies used in this study included: anti-ubiquitin (Biomol, PW8805); anti-SUMO1 (Zymed, 33-2400); anti-NEDD8 (Zymed, 34-1400); anti-p27Kip1 (BD pharmingen); anti-c-Src (Biosource); anti-Skp2 (Zymed), anti-YY1 (Santa Cruz Biotechnology); anti-IGF-1R (Zymed); anti-HA (Covance); anti-Flag (Sigma); anti-GST (Santa Cruz Biotechnology); and anti-Myc (9E10, Santa Cruz Biotechnology).

    Techniques: In Vivo, Modification, Immunoprecipitation, Plasmid Preparation, SDS Page, Molecular Weight, Transfection, Conjugation Assay, Western Blot

    Validation of c-Src as a novel SCF SKP2 substrate. (A) SKP2 −/− MEFs were transduced with control (pBABEpuro) or Flag-Skp2-expressing retroviruses and Western blot analysis was used to assess the expression level of known SCF Skp2 substrate p27 Kip1 and putative substrate c-Src. (B) Endogenous c-Src associates with Skp2 in vivo . Anti-Flag antibodies were used to immunoprecipitate Flag-Skp2 from extracts prepared from SKP2 −/− MEFs transduced with control (lanes 1 and 3) or Skp2-expressing retroviruses (lanes 2 and 4). Association of c-Src with Skp2 was determined by Western blot analysis. The same blot was then re-probed with anti-Skp2 antibodies to verify immunoprecipitation. (C) Skp2 promotes c-Src ubiquitylation in vivo . HEK293T cells were co-transfected with plasmids that express GST-c-Src, HA-Ubiquitin, with or without Flag-Skp2. Extracts from cells were denatured, c-Src immunoprecipitated using anti-GST antibodies, and ubiquitylation detected by Western blotting with anti-HA antibodies.

    Journal: PLoS ONE

    Article Title: Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays

    doi: 10.1371/journal.pone.0011332

    Figure Lengend Snippet: Validation of c-Src as a novel SCF SKP2 substrate. (A) SKP2 −/− MEFs were transduced with control (pBABEpuro) or Flag-Skp2-expressing retroviruses and Western blot analysis was used to assess the expression level of known SCF Skp2 substrate p27 Kip1 and putative substrate c-Src. (B) Endogenous c-Src associates with Skp2 in vivo . Anti-Flag antibodies were used to immunoprecipitate Flag-Skp2 from extracts prepared from SKP2 −/− MEFs transduced with control (lanes 1 and 3) or Skp2-expressing retroviruses (lanes 2 and 4). Association of c-Src with Skp2 was determined by Western blot analysis. The same blot was then re-probed with anti-Skp2 antibodies to verify immunoprecipitation. (C) Skp2 promotes c-Src ubiquitylation in vivo . HEK293T cells were co-transfected with plasmids that express GST-c-Src, HA-Ubiquitin, with or without Flag-Skp2. Extracts from cells were denatured, c-Src immunoprecipitated using anti-GST antibodies, and ubiquitylation detected by Western blotting with anti-HA antibodies.

    Article Snippet: Antibodies Antibodies used in this study included: anti-ubiquitin (Biomol, PW8805); anti-SUMO1 (Zymed, 33-2400); anti-NEDD8 (Zymed, 34-1400); anti-p27Kip1 (BD pharmingen); anti-c-Src (Biosource); anti-Skp2 (Zymed), anti-YY1 (Santa Cruz Biotechnology); anti-IGF-1R (Zymed); anti-HA (Covance); anti-Flag (Sigma); anti-GST (Santa Cruz Biotechnology); and anti-Myc (9E10, Santa Cruz Biotechnology).

    Techniques: Transduction, Expressing, Western Blot, In Vivo, Immunoprecipitation, Transfection

    The casein kinase Yck2p binds to the autoinhibitory region of Sec2p and phosphorylates Sec2p in vitro. (A) Top, domain organization of Sec2p. The N-terminal region contains a coiled-coil domain (CC), which catalyzes the exchange of GDP for GTP on Sec4p. Downstream are overlapping binding sites for Sec15p and Ypt32p-GTP, which compete against each other to bind to Sec2p. The choice of Sec2p between these two binding partners is, in part, regulated by phosphorylation of the phosphoregion at aa 181–188. Three positively charged patches allow Sec2p to interact with the phosphoinositide PI(4)P. Finally, the region of aa 450–508, named the autoinhibitory region, negatively regulates Sec15p binding to Sec2p by an autoinhibitory mechanism. Bottom, in vivo phosphorylation results of different Sec2p constructs ( Elkind et al. , 2000 ). (B) Yck2p binds to the autoinhibitory region of Sec2p. Yeast lysate overexpressing GFP-Yck2p (NY3138) was incubated with different GST-Sec2p constructs purified from bacteria and immobilized on glutathione beads. Bound GFP-Yck2p protein was detected with anti-GFP antibody. Because GST–Sec2p 1-508 protein runs at the same molecular weight as GFP-Yck2p (which explains the distortion of the GFP signal), we checked that the GFP signal is specific to GFP-Yck2p and does not cross-react with GST–Sec2p 1-508. GST-Sec2p was detected with anti-GST antibody. The intensity of the bands was quantified using ImageJ. The percentage of Yck2p bound to Sec2p was calculated and is indicated on a logarithmic scale. Mean and SD of at least three different experiments. * p

    Journal: Molecular Biology of the Cell

    Article Title: The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

    doi: 10.1091/mbc.E15-09-0651

    Figure Lengend Snippet: The casein kinase Yck2p binds to the autoinhibitory region of Sec2p and phosphorylates Sec2p in vitro. (A) Top, domain organization of Sec2p. The N-terminal region contains a coiled-coil domain (CC), which catalyzes the exchange of GDP for GTP on Sec4p. Downstream are overlapping binding sites for Sec15p and Ypt32p-GTP, which compete against each other to bind to Sec2p. The choice of Sec2p between these two binding partners is, in part, regulated by phosphorylation of the phosphoregion at aa 181–188. Three positively charged patches allow Sec2p to interact with the phosphoinositide PI(4)P. Finally, the region of aa 450–508, named the autoinhibitory region, negatively regulates Sec15p binding to Sec2p by an autoinhibitory mechanism. Bottom, in vivo phosphorylation results of different Sec2p constructs ( Elkind et al. , 2000 ). (B) Yck2p binds to the autoinhibitory region of Sec2p. Yeast lysate overexpressing GFP-Yck2p (NY3138) was incubated with different GST-Sec2p constructs purified from bacteria and immobilized on glutathione beads. Bound GFP-Yck2p protein was detected with anti-GFP antibody. Because GST–Sec2p 1-508 protein runs at the same molecular weight as GFP-Yck2p (which explains the distortion of the GFP signal), we checked that the GFP signal is specific to GFP-Yck2p and does not cross-react with GST–Sec2p 1-508. GST-Sec2p was detected with anti-GST antibody. The intensity of the bands was quantified using ImageJ. The percentage of Yck2p bound to Sec2p was calculated and is indicated on a logarithmic scale. Mean and SD of at least three different experiments. * p

    Article Snippet: The amount of GST-Sec2p in the reaction was verified by Western blotting with anti-GST antibody (1:1000 dilution; sc-459; Santa Cruz Biotechnology).

    Techniques: In Vitro, Binding Assay, In Vivo, Construct, Incubation, Purification, Molecular Weight

    The casein kinases Yck1p and Yck2p phosphorylate Sec2p in vivo and target phosphosites within the Sec15p/Ypt32p binding region. (A) Control yeast cells or cells deleted for YCK3 , YCK1 , or YCK2 genes and overexpressing Sec2p 1-508 on a CEN vector (NY3099, NY3100, NY3101, and NY3102, respectively) were grown overnight at 25°C and quickly lysed by NaOH treatment. The mobility of Sec2p 1-508 protein was visualized after SDS–PAGE gel with anti-Sec2p antibody. (B) GST–Sec2p 1-508 protein, overexpressed on a CEN vector in control yeast cells or cells deleted for YCK3 , YCK1 , or YCK2 genes (NY3103, NY3104, NY3105, and NY3106, respectively), was purified, treated or not with the phosphatase CIP, and visualized after SDS–PAGE with anti-Sec2p antibody. (C) Control yeast cells, deleted for YCK1 , expressing yck2-1 or the yck-ts mutant, all overexpressing GST–Sec2p 1-508 on a CEN vector (NY3111, NY3112, NY3113, and NY3114, respectively), were grown overnight at 25°C and then shifted to 37°C for 2 h. The mobility of GST–Sec2p 1-508 protein was analyzed as in A. (D) Control yeast strain or the yck-ts mutant, overexpressing GST–Sec2p full length (FL) on a CEN vector (NY3115 and NY3118, respectively), were grown overnight at 25°C and then shifted to 37°C for 1 h. GST–Sec2p FL protein was purified, treated or not with the phosphatase CIP ± EDTA and visualized after SDS–PAGE by Coomassie brilliant blue staining. (E) Top, control yeast strain or the yck-ts mutant, overexpressing GST–Sec2p FL WT, S181-8A, or S181-8D/E on a CEN vector (NY3115, NY3118, NY3116, NY3119, NY3117, and NY3120), were grown overnight at 25°C and then shifted to 37°C for 1 h. GST Sec2p FL protein was purified and visualized after SDS–PAGE by Coomassie brilliant blue staining. Bottom, control yeast strain or the yck-ts mutant, overexpressing GST–Sec2p 1-508 WT, S181-8A, or S181-8D/E on a CEN vector (NY3111, NY3114, NY3121, NY3123, NY3122, and NY3124), were grown overnight at 25°C and quickly lysed by NaOH treatment. The mobility of Sec2p 1-508 protein was visualized after SDS–PAGE with anti-Sec2p antibody. (F) The Sec2p–Sec15p interaction is impaired in the yck-ts mutant. Sec2p-3xGFP was immunoprecipitated with GFP antibody in the control yeast strain or in the yck-ts mutant coexpressing Sec15p-13xmyc (NY3136 and NY3137, respectively). Yeast strains expressing untagged Sec2p (NY3134 and NY3135) were used as negative control. Coprecipitated Sec15p-13xmyc and the amount of Sec15p-13xmyc in 0.05% of lysates were detected with anti-myc antibody. The intensity of the bands was quantified using ImageJ (National Institutes of Health, Bethesda, MD). The percentage of Sec15p bound to Sec2p was calculated and indicated. Mean and SD of three different experiments. * p

    Journal: Molecular Biology of the Cell

    Article Title: The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

    doi: 10.1091/mbc.E15-09-0651

    Figure Lengend Snippet: The casein kinases Yck1p and Yck2p phosphorylate Sec2p in vivo and target phosphosites within the Sec15p/Ypt32p binding region. (A) Control yeast cells or cells deleted for YCK3 , YCK1 , or YCK2 genes and overexpressing Sec2p 1-508 on a CEN vector (NY3099, NY3100, NY3101, and NY3102, respectively) were grown overnight at 25°C and quickly lysed by NaOH treatment. The mobility of Sec2p 1-508 protein was visualized after SDS–PAGE gel with anti-Sec2p antibody. (B) GST–Sec2p 1-508 protein, overexpressed on a CEN vector in control yeast cells or cells deleted for YCK3 , YCK1 , or YCK2 genes (NY3103, NY3104, NY3105, and NY3106, respectively), was purified, treated or not with the phosphatase CIP, and visualized after SDS–PAGE with anti-Sec2p antibody. (C) Control yeast cells, deleted for YCK1 , expressing yck2-1 or the yck-ts mutant, all overexpressing GST–Sec2p 1-508 on a CEN vector (NY3111, NY3112, NY3113, and NY3114, respectively), were grown overnight at 25°C and then shifted to 37°C for 2 h. The mobility of GST–Sec2p 1-508 protein was analyzed as in A. (D) Control yeast strain or the yck-ts mutant, overexpressing GST–Sec2p full length (FL) on a CEN vector (NY3115 and NY3118, respectively), were grown overnight at 25°C and then shifted to 37°C for 1 h. GST–Sec2p FL protein was purified, treated or not with the phosphatase CIP ± EDTA and visualized after SDS–PAGE by Coomassie brilliant blue staining. (E) Top, control yeast strain or the yck-ts mutant, overexpressing GST–Sec2p FL WT, S181-8A, or S181-8D/E on a CEN vector (NY3115, NY3118, NY3116, NY3119, NY3117, and NY3120), were grown overnight at 25°C and then shifted to 37°C for 1 h. GST Sec2p FL protein was purified and visualized after SDS–PAGE by Coomassie brilliant blue staining. Bottom, control yeast strain or the yck-ts mutant, overexpressing GST–Sec2p 1-508 WT, S181-8A, or S181-8D/E on a CEN vector (NY3111, NY3114, NY3121, NY3123, NY3122, and NY3124), were grown overnight at 25°C and quickly lysed by NaOH treatment. The mobility of Sec2p 1-508 protein was visualized after SDS–PAGE with anti-Sec2p antibody. (F) The Sec2p–Sec15p interaction is impaired in the yck-ts mutant. Sec2p-3xGFP was immunoprecipitated with GFP antibody in the control yeast strain or in the yck-ts mutant coexpressing Sec15p-13xmyc (NY3136 and NY3137, respectively). Yeast strains expressing untagged Sec2p (NY3134 and NY3135) were used as negative control. Coprecipitated Sec15p-13xmyc and the amount of Sec15p-13xmyc in 0.05% of lysates were detected with anti-myc antibody. The intensity of the bands was quantified using ImageJ (National Institutes of Health, Bethesda, MD). The percentage of Sec15p bound to Sec2p was calculated and indicated. Mean and SD of three different experiments. * p

    Article Snippet: The amount of GST-Sec2p in the reaction was verified by Western blotting with anti-GST antibody (1:1000 dilution; sc-459; Santa Cruz Biotechnology).

    Techniques: In Vivo, Binding Assay, Plasmid Preparation, SDS Page, Purification, Expressing, Mutagenesis, Staining, Immunoprecipitation, Negative Control

    Modulation of Sec2p phosphorylation. (A) Sec2p in vitro phosphorylation by Yck2p is inhibited by the phosphoinositide PI(4)P. GFP-Yck2p was immunoprecipitated from a yeast lysate with protein A/G beads (NY3138) and incubated with bacterial eluted GST–Sec2p 1-508 and with or without ATP and liposomes containing different amounts of PI(4)P (liposomes 1–4, which contain, respectively, 0, 2, 5, and 10% PI(4)P). GST–Sec2p 1-508 was detected with anti-GST antibody, and the phosphorylated form is indicated with an arrow. GFP-Yck2p protein was detected with anti-GFP antibody. The intensity of the phosphorylated form of GST–Sec2p 1-508 was quantified using ImageJ and normalized and is indicated. Mean and SD of three different experiments. * p

    Journal: Molecular Biology of the Cell

    Article Title: The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

    doi: 10.1091/mbc.E15-09-0651

    Figure Lengend Snippet: Modulation of Sec2p phosphorylation. (A) Sec2p in vitro phosphorylation by Yck2p is inhibited by the phosphoinositide PI(4)P. GFP-Yck2p was immunoprecipitated from a yeast lysate with protein A/G beads (NY3138) and incubated with bacterial eluted GST–Sec2p 1-508 and with or without ATP and liposomes containing different amounts of PI(4)P (liposomes 1–4, which contain, respectively, 0, 2, 5, and 10% PI(4)P). GST–Sec2p 1-508 was detected with anti-GST antibody, and the phosphorylated form is indicated with an arrow. GFP-Yck2p protein was detected with anti-GFP antibody. The intensity of the phosphorylated form of GST–Sec2p 1-508 was quantified using ImageJ and normalized and is indicated. Mean and SD of three different experiments. * p

    Article Snippet: The amount of GST-Sec2p in the reaction was verified by Western blotting with anti-GST antibody (1:1000 dilution; sc-459; Santa Cruz Biotechnology).

    Techniques: In Vitro, Immunoprecipitation, Incubation

    The protective effects of CA, QU and AV on UVA-mediated downregulation of Nrf2 target genes. (A) γ-GCL (γ-GCLC and γ-GCLM), (B) GST and (C) NQO1 mRNA expressions were assessed by real-time RT-PCR analysis at 2 h after UVA irradiation in B16F10 cells pretreated with test compounds. The statistical significance of differences between the control and irradiated cells was evaluated by Student’s t test and between UVA-irradiated and compounds-treated cells by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. ## P

    Journal: Redox Biology

    Article Title: Photoprotection by dietary phenolics against melanogenesis induced by UVA through Nrf2-dependent antioxidant responses

    doi: 10.1016/j.redox.2015.12.006

    Figure Lengend Snippet: The protective effects of CA, QU and AV on UVA-mediated downregulation of Nrf2 target genes. (A) γ-GCL (γ-GCLC and γ-GCLM), (B) GST and (C) NQO1 mRNA expressions were assessed by real-time RT-PCR analysis at 2 h after UVA irradiation in B16F10 cells pretreated with test compounds. The statistical significance of differences between the control and irradiated cells was evaluated by Student’s t test and between UVA-irradiated and compounds-treated cells by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. ## P

    Article Snippet: The membranes were blocked with 5% skim milk (Tris-buffer saline containing 0.1% (v /v ) Tween 20 and 5% (w/v) skim milk) for 1.5 h and then incubated overnight at 4 °C with the primary antibody against tyrosinase (ab178676; Abcam, Cambridge, MA, USA) (1:10000), Nrf2 (sc-722; Santa Cruz Biotechnology, Santa Cruz, CA) (1:2000), GCLC (ab53179; Abcam, Cambridge, MA, USA) (1:2000), GST (sc-459; Santa Cruz Biotechnology, Santa Cruz, CA) (1:1000) and NQO1 (ab34173; Abcam, Cambridge, MA, USA) (1:2000) in 5% skim milk.

    Techniques: Quantitative RT-PCR, Irradiation

    Schematic diagram of the GHS-based detoxification pathway in plants. MCB is used as a model substrate for conjugation to GSH by a GST in the cytoplasm and subsequent sequestration in the vacuole by a glutathione S- ).

    Journal: Plant Physiology

    Article Title: Gene Expression and Microscopic Analysis of Arabidopsis Exposed to Chloroacetanilide Herbicides and Explosive Compounds. A Phytoremediation Approach 1

    doi: 10.1104/pp.104.056168

    Figure Lengend Snippet: Schematic diagram of the GHS-based detoxification pathway in plants. MCB is used as a model substrate for conjugation to GSH by a GST in the cytoplasm and subsequent sequestration in the vacuole by a glutathione S- ).

    Article Snippet: The calibration standards were prepared using a 10 m m fresh stock solution of GSB, which was made from 10 m m MCB and 100 m m GSH (G 1404; Sigma-Aldrich) in the presence of GST (Equine Liver GST, G 6511; Sigma-Aldrich).

    Techniques: Conjugation Assay

    Ca Bdf1 BDs are resistant to BETi. ( a ) HTRF assay. A biotinylated tetra-acetylated histone H4 peptide is bound to streptavidin beads coupled to the donor fluorophore. A GST-tagged BD is bound by an anti-GST antibody coupled to the acceptor fluorophor. Peptide binding by the BD results in FRET. The addition of a BETi reduces FRET. ( b ) HTRF assays performed on BDs from Ca Bdf1 and human Brd4 in the presence of the indicated BETi. Inhibition curves are shown as closed (BD1) and open (BD2) circles in green (Brd4) and magenta (Bdf1). IC 50 values are listed below each graph. Data represent the mean and s.d. values from three independent experiments. ( c ) Representative ITC experiments measuring the binding of JQ1 to Ca BDF1 BDs (magenta) and to human Brd4 BD1 (green). The values indicated for K d and N represent the mean and s.d. from three independent experiments. See also Supplementary Table 1 . ( d ) BET inhibitors do not affect C. albicans growth, even when Bdf1 BD1 or BD2 is deleted. Inhibitors were tested at 10 μM concentration. Experiments were performed in the presence of doxycyline to repress expression from the pTetO -BDF1 allele. Data represent the mean and s.d. values from three independent experiments.

    Journal: Nature Communications

    Article Title: Selective BET bromodomain inhibition as an antifungal therapeutic strategy

    doi: 10.1038/ncomms15482

    Figure Lengend Snippet: Ca Bdf1 BDs are resistant to BETi. ( a ) HTRF assay. A biotinylated tetra-acetylated histone H4 peptide is bound to streptavidin beads coupled to the donor fluorophore. A GST-tagged BD is bound by an anti-GST antibody coupled to the acceptor fluorophor. Peptide binding by the BD results in FRET. The addition of a BETi reduces FRET. ( b ) HTRF assays performed on BDs from Ca Bdf1 and human Brd4 in the presence of the indicated BETi. Inhibition curves are shown as closed (BD1) and open (BD2) circles in green (Brd4) and magenta (Bdf1). IC 50 values are listed below each graph. Data represent the mean and s.d. values from three independent experiments. ( c ) Representative ITC experiments measuring the binding of JQ1 to Ca BDF1 BDs (magenta) and to human Brd4 BD1 (green). The values indicated for K d and N represent the mean and s.d. from three independent experiments. See also Supplementary Table 1 . ( d ) BET inhibitors do not affect C. albicans growth, even when Bdf1 BD1 or BD2 is deleted. Inhibitors were tested at 10 μM concentration. Experiments were performed in the presence of doxycyline to repress expression from the pTetO -BDF1 allele. Data represent the mean and s.d. values from three independent experiments.

    Article Snippet: Specifically, the assay used a terbium(III) cryptate donor reagent conjugated to an anti-GST antibody (MAb anti-GST-Tb; ref. 61GSTTLA), a streptavidin-conjugated acceptor reagent (streptavidin-d2; ref. 610SADLA) and Cisbio proprietary buffers (EPIgeneous Binding Domain Diluent and Detection buffer; refs. 62DLBDDF and 62DB2FDG, respectively).

    Techniques: HTRF Assay, Binding Assay, Inhibition, Concentration Assay, Expressing

    Ubiquitination but not activity of duck 2CARD is lost in double K-R mutant. A. GST pulldown and immunoblotting with anti-GST and anti-HA of samples from cells transfected with different GST-d2CARD mutants (Q170K, K167R, K193R, K167R/K193R) and HA-Ub plasmids showing a loss of ubiquitination only in the double mutant. B. GST pulldown and immunoblotting of K167/K193R mutant showing interaction with duck TRIM25-V5. C. GST-d2CARD and mutants Q170K, K167R, K193R, K167/193R activate the chIFN-β promoter when co-transfected into chicken DF-1 cells. Data represent the mean ± SD (n = 3). All mutants tested significantly activate the chIFN-β promoter compared with the GST control (P

    Journal: PLoS ONE

    Article Title: Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

    doi: 10.1371/journal.pone.0086968

    Figure Lengend Snippet: Ubiquitination but not activity of duck 2CARD is lost in double K-R mutant. A. GST pulldown and immunoblotting with anti-GST and anti-HA of samples from cells transfected with different GST-d2CARD mutants (Q170K, K167R, K193R, K167R/K193R) and HA-Ub plasmids showing a loss of ubiquitination only in the double mutant. B. GST pulldown and immunoblotting of K167/K193R mutant showing interaction with duck TRIM25-V5. C. GST-d2CARD and mutants Q170K, K167R, K193R, K167/193R activate the chIFN-β promoter when co-transfected into chicken DF-1 cells. Data represent the mean ± SD (n = 3). All mutants tested significantly activate the chIFN-β promoter compared with the GST control (P

    Article Snippet: Immunodetection was achieved with anti-V5 (1∶5,000) (Invitrogen), anti-Flag (1∶2,000) (Sigma), anti-HA (1∶1000) (Sigma-Aldrich) or anti-GST (1∶1000) (Dana-Farber Monoclonal Core Facility (DG-122) antibodies and proteins were visualized by chemiluminescence using the ECL kit (GE- Healthcare).

    Techniques: Activity Assay, Mutagenesis, Transfection

    RIG-I splicing variant is not ubiquitinated and cannot activate innate immune signaling. A. Reverse transcription PCR showing amplification across exon 2 of duck RIG-I in lung tissue of ducks that were mock challenged, or infected with influenza A virus strains, BC500 or VN1203. B. Alignment of sequences of duck and human RIG-I CARD domains. Exon 2 sequence missing in splicing variant is overlined and Lys residues ubiquitinated in human RIG-I are marked with an asterisk. C. GST pulldown followed by immunoblotting indicates that GST-d2CARD interacts with human TRIM25-V5, while this interaction is greatly reduced for the splicing variant SVCARD. D. GST pulldown and immunoblotting showing no interaction between duck RIG-I CARD splice variant (SVCARD) and duck TRIM25-V5. E. GST-d2CARD is ubiquitinated while splicing variant is not. F. GST-2CARD activates the chIFN-β promoter in DF-1 cells, while splicing variant does not. Human TRIM25 significantly increased the chIFN-β promoter activity of d2CARD, but not SVCARD, compared to d2CARD alone (* indicates P

    Journal: PLoS ONE

    Article Title: Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

    doi: 10.1371/journal.pone.0086968

    Figure Lengend Snippet: RIG-I splicing variant is not ubiquitinated and cannot activate innate immune signaling. A. Reverse transcription PCR showing amplification across exon 2 of duck RIG-I in lung tissue of ducks that were mock challenged, or infected with influenza A virus strains, BC500 or VN1203. B. Alignment of sequences of duck and human RIG-I CARD domains. Exon 2 sequence missing in splicing variant is overlined and Lys residues ubiquitinated in human RIG-I are marked with an asterisk. C. GST pulldown followed by immunoblotting indicates that GST-d2CARD interacts with human TRIM25-V5, while this interaction is greatly reduced for the splicing variant SVCARD. D. GST pulldown and immunoblotting showing no interaction between duck RIG-I CARD splice variant (SVCARD) and duck TRIM25-V5. E. GST-d2CARD is ubiquitinated while splicing variant is not. F. GST-2CARD activates the chIFN-β promoter in DF-1 cells, while splicing variant does not. Human TRIM25 significantly increased the chIFN-β promoter activity of d2CARD, but not SVCARD, compared to d2CARD alone (* indicates P

    Article Snippet: Immunodetection was achieved with anti-V5 (1∶5,000) (Invitrogen), anti-Flag (1∶2,000) (Sigma), anti-HA (1∶1000) (Sigma-Aldrich) or anti-GST (1∶1000) (Dana-Farber Monoclonal Core Facility (DG-122) antibodies and proteins were visualized by chemiluminescence using the ECL kit (GE- Healthcare).

    Techniques: Variant Assay, Polymerase Chain Reaction, Amplification, Infection, Sequencing, Activity Assay

    Identification of the Nedd8 binding sequence in Smurf HECT domain. ( A ) Amino acid sequence alignment of Nedd8-binding sites in N-lobe small subdomain and C-lobe domain of Smurf1 and Smurf2. The amino acid highlighted in red indicates the residues conserved in the sites for Nedd8 binding. The conserved residues are found in the sequence L(X7)R(X5)F(X)ALQ. ( B ) Co-IP assay for the interaction between Nedd8 and Smurf1 possessing L(X7)R(X5)F(X)ALQ to A(X7)A(X5)A(X)AAA substitution in the N-lobe small subdomain and C-lobe domain of Smurf1 HECT. Western-blot analysis of whole-cell lysates and immunoprecipitates with Flag antibody. ( C ) GST pull-down assay for the interaction between Nedd8 and Smurf1 possessing L(X7)R(X5)F(X)ALQ to A(X7)A(X5)A(X)AAA substitution of the whole HECT domain. Expressed Myc-tagged Smurf1-HECT (wide type or mutant) proteins in cell lysates were precipitated by GST or GST-Nedd8 from the bacterial lysates. The precipitates were analyzed by western blotting using anti-Myc antibody to detect Smurf-HECT and anti-GST antibody to detect GST or GST-Nedd8. ( D , E ) Co-IP assay for the interaction between Nedd8 and Smurf1/2 possessing L(X7)R(X5)F(X)ALQ to A(X7)A(X5)A(X)AAA substitution in the Smurfs. Western-blot analysis of whole-cell lysates and immunoprecipitates with Flag or Myc antibody.

    Journal: Scientific Reports

    Article Title: The Nedd8 Non-covalent Binding Region in the Smurf HECT Domain is Critical to its Ubiquitn Ligase Function

    doi: 10.1038/srep41364

    Figure Lengend Snippet: Identification of the Nedd8 binding sequence in Smurf HECT domain. ( A ) Amino acid sequence alignment of Nedd8-binding sites in N-lobe small subdomain and C-lobe domain of Smurf1 and Smurf2. The amino acid highlighted in red indicates the residues conserved in the sites for Nedd8 binding. The conserved residues are found in the sequence L(X7)R(X5)F(X)ALQ. ( B ) Co-IP assay for the interaction between Nedd8 and Smurf1 possessing L(X7)R(X5)F(X)ALQ to A(X7)A(X5)A(X)AAA substitution in the N-lobe small subdomain and C-lobe domain of Smurf1 HECT. Western-blot analysis of whole-cell lysates and immunoprecipitates with Flag antibody. ( C ) GST pull-down assay for the interaction between Nedd8 and Smurf1 possessing L(X7)R(X5)F(X)ALQ to A(X7)A(X5)A(X)AAA substitution of the whole HECT domain. Expressed Myc-tagged Smurf1-HECT (wide type or mutant) proteins in cell lysates were precipitated by GST or GST-Nedd8 from the bacterial lysates. The precipitates were analyzed by western blotting using anti-Myc antibody to detect Smurf-HECT and anti-GST antibody to detect GST or GST-Nedd8. ( D , E ) Co-IP assay for the interaction between Nedd8 and Smurf1/2 possessing L(X7)R(X5)F(X)ALQ to A(X7)A(X5)A(X)AAA substitution in the Smurfs. Western-blot analysis of whole-cell lysates and immunoprecipitates with Flag or Myc antibody.

    Article Snippet: Antibodies All antibodies were purchased as follows: anti-Smurf1 (ab117552, Abcam), anti-Smurf2 (sc-25511, Santa Cruz), anti-p-Smad1/5 (cst9246S, Cell Signaling Technology), anti-Smad1/5 (sc6031, Sant Cruz), anti-p-Smad3 (cst9520S, Cell Signaling Technology), anti-Smad3 (cst9523S, Cell Signaling Technology), anti-ubiquitin (sc-166553, Santa Cruz), anti-Nedd8 (ALX-210-194-R200, Alexis Biochemicals), anti-Myc, anti-GST, anti-His, anti-GAPDH, anti-HA (MBL), anti-Flag (Sigma-Aldrih), and mouse/rabbit IgG (Santa Cruz).

    Techniques: Binding Assay, Sequencing, Co-Immunoprecipitation Assay, Western Blot, Pull Down Assay, Mutagenesis

    The Nedd8 binding sequence is required for autoubiquitylation of Smurf1. ( A ) Analysis of direct binding between the wide type, Y439A and 10 A mutants of Smurf1-HECT and GST-ubiquitin in GST pull-down experiments. ( B ) In vitro autoubiquitylation of Smurf Nedd8 binding mutant. In vitro autoubiquitylation assays were performed using purified Smurf1-HECT wild-type or C669A, Y439A, 10 A mutants and ubiquitin. Autoubiquitylated Smurf1 was analyzed by immunoblotting using anti-ubiquitin antibody. ( C ) In vitro autoubiquitylation assays were carried out using Smurf1 WT and 10 A mutants in the presence of lysine-null ubiquitin (Ubiquitin-K0) or Ubiquitin-K48 only or wide type ubiquitin and analyzed by immunoblotting with an anti-ubiquitin antibody. ( D ) Analysis of ubiquitin thioester intermediates formed by Smurf1-HECT WT, C699A and 10 A mutants with ubiquitin. The Smurf1 truncates were incubated for 10 min at room temperature and analyzed using immunoblotting under non-reducing (−DTT) or reducing conditions (+DTT) using a ubiquitin antibody.

    Journal: Scientific Reports

    Article Title: The Nedd8 Non-covalent Binding Region in the Smurf HECT Domain is Critical to its Ubiquitn Ligase Function

    doi: 10.1038/srep41364

    Figure Lengend Snippet: The Nedd8 binding sequence is required for autoubiquitylation of Smurf1. ( A ) Analysis of direct binding between the wide type, Y439A and 10 A mutants of Smurf1-HECT and GST-ubiquitin in GST pull-down experiments. ( B ) In vitro autoubiquitylation of Smurf Nedd8 binding mutant. In vitro autoubiquitylation assays were performed using purified Smurf1-HECT wild-type or C669A, Y439A, 10 A mutants and ubiquitin. Autoubiquitylated Smurf1 was analyzed by immunoblotting using anti-ubiquitin antibody. ( C ) In vitro autoubiquitylation assays were carried out using Smurf1 WT and 10 A mutants in the presence of lysine-null ubiquitin (Ubiquitin-K0) or Ubiquitin-K48 only or wide type ubiquitin and analyzed by immunoblotting with an anti-ubiquitin antibody. ( D ) Analysis of ubiquitin thioester intermediates formed by Smurf1-HECT WT, C699A and 10 A mutants with ubiquitin. The Smurf1 truncates were incubated for 10 min at room temperature and analyzed using immunoblotting under non-reducing (−DTT) or reducing conditions (+DTT) using a ubiquitin antibody.

    Article Snippet: Antibodies All antibodies were purchased as follows: anti-Smurf1 (ab117552, Abcam), anti-Smurf2 (sc-25511, Santa Cruz), anti-p-Smad1/5 (cst9246S, Cell Signaling Technology), anti-Smad1/5 (sc6031, Sant Cruz), anti-p-Smad3 (cst9520S, Cell Signaling Technology), anti-Smad3 (cst9523S, Cell Signaling Technology), anti-ubiquitin (sc-166553, Santa Cruz), anti-Nedd8 (ALX-210-194-R200, Alexis Biochemicals), anti-Myc, anti-GST, anti-His, anti-GAPDH, anti-HA (MBL), anti-Flag (Sigma-Aldrih), and mouse/rabbit IgG (Santa Cruz).

    Techniques: Binding Assay, Sequencing, In Vitro, Mutagenesis, Purification, Incubation

    Smurf2 interacts with Nedd8 both in vitro and in vivo . ( A ) Direct interaction between Nedd8 and Smurf2 was revealed by GST pull-down assays. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies. Input represents 20% of that used for pull down. ( B ) Co-immunoprecipitation (Co-IP) of endogenous Nedd8 and endogenous Smurf2 from HEK293 cells. Western-blot analysis of whole-cell lysates and immunoprecipitates with Smurf2 antibody or control IgG. To avoid Smurf1 degradation, proteasome inhibitor, MG132 was added and incubated for 12 h before cells were harvested. IP, immunoprecipitate; IB, immunoblotting; IgG, immunoglobulin; IgG HC, IgG heavy chain. IgG LC, IgG light chain. ( C ) Smurf2 was co-immunoprecipitated with both wild type and ΔGG mutant forms of Nedd8 but did not interact with the mutant of I44A. HEK293T cells transfected with Myc-Nedd8 or the mutants and Flag-Smurf2 were immunoprecipitated with anti-Flag, followed by immunoblotting analysis. ( D ) Mapping of interacting regions of Smurf2 with Nedd8. Flag-Nedd8 and the indicated Smurf2 truncates were coexpressed in HEK293T cells. Cell lysates were incubated with anti-Flag antibody to precipitate Smurf2 deletion mutants. Both the lysates and the immunoprecipitates were analyzed using western blot analysis with the indicated antibodies.

    Journal: Scientific Reports

    Article Title: The Nedd8 Non-covalent Binding Region in the Smurf HECT Domain is Critical to its Ubiquitn Ligase Function

    doi: 10.1038/srep41364

    Figure Lengend Snippet: Smurf2 interacts with Nedd8 both in vitro and in vivo . ( A ) Direct interaction between Nedd8 and Smurf2 was revealed by GST pull-down assays. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies. Input represents 20% of that used for pull down. ( B ) Co-immunoprecipitation (Co-IP) of endogenous Nedd8 and endogenous Smurf2 from HEK293 cells. Western-blot analysis of whole-cell lysates and immunoprecipitates with Smurf2 antibody or control IgG. To avoid Smurf1 degradation, proteasome inhibitor, MG132 was added and incubated for 12 h before cells were harvested. IP, immunoprecipitate; IB, immunoblotting; IgG, immunoglobulin; IgG HC, IgG heavy chain. IgG LC, IgG light chain. ( C ) Smurf2 was co-immunoprecipitated with both wild type and ΔGG mutant forms of Nedd8 but did not interact with the mutant of I44A. HEK293T cells transfected with Myc-Nedd8 or the mutants and Flag-Smurf2 were immunoprecipitated with anti-Flag, followed by immunoblotting analysis. ( D ) Mapping of interacting regions of Smurf2 with Nedd8. Flag-Nedd8 and the indicated Smurf2 truncates were coexpressed in HEK293T cells. Cell lysates were incubated with anti-Flag antibody to precipitate Smurf2 deletion mutants. Both the lysates and the immunoprecipitates were analyzed using western blot analysis with the indicated antibodies.

    Article Snippet: Antibodies All antibodies were purchased as follows: anti-Smurf1 (ab117552, Abcam), anti-Smurf2 (sc-25511, Santa Cruz), anti-p-Smad1/5 (cst9246S, Cell Signaling Technology), anti-Smad1/5 (sc6031, Sant Cruz), anti-p-Smad3 (cst9520S, Cell Signaling Technology), anti-Smad3 (cst9523S, Cell Signaling Technology), anti-ubiquitin (sc-166553, Santa Cruz), anti-Nedd8 (ALX-210-194-R200, Alexis Biochemicals), anti-Myc, anti-GST, anti-His, anti-GAPDH, anti-HA (MBL), anti-Flag (Sigma-Aldrih), and mouse/rabbit IgG (Santa Cruz).

    Techniques: In Vitro, In Vivo, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Incubation, Mutagenesis, Transfection

    HECTD3 interacts with caspase-8 through the DOC and DED domains. ( a ) Schematic representation of the HECTD3 and caspase-8 proteins and their mutants. ( b ) WT HECTD3 interacts with endogenous caspase-8, but not caspase-3 and -7. Flag-HECTD3, Flag-HΔ1–511 and an empty vector were transfected into HEK293T for 2 days. Immunoprecipitation was performed using the anti-FLAG M2 beads. ( c ) Caspase-8 interacts with HECTD3. Flag-caspase-8 or an empty vector was co-transfected with HECTD3 into HEK293T for 2 days. Immunoprecipitation was performed using the anti-FLAG M2 beads. ( d ) HECTD3 directly binds to caspase-8 but not caspase-3 in vitro . The recombinant GST-HECTD3 and GST were purified from E.coli ( Supplementary Figure S1A ) and were incubated with in vitro -translated caspase-8 and caspase-3 proteins, respectively, with Glutathione-Sepharose 4B slurry beads overnight at 4 °C. The beads were washed three times with 1 ml of 1 × cell lysis buffer. The proteins were resuspended with 20–50 μ l of SDS sample buffer and analyzed by WB. ( e ) The endogenous HECTD3 protein forms a complex with the endogenous caspase-8 protein in HeLa. The endogenous HECTD3 was immunoprecipitated by the anti-HECTD3 Ab. Rabbit IgG was used as a negative control. ( f ) Both HECTD3 and caspase-8 are predominantly localized in the cytoplasm. The localization of Flag-HECTD3 and caspase-8 proteins was examined by immunofluorescence staining in HEK293T cells after co-transfection. DAPI was used to stain cell nuclei. ( g ) HΔ1–511 and HΔ109–393 significantly decrease the binding affinity with caspase-8. Flag-HECTD3 and its mutants were co-transfected with caspase-8 into HEK293T for 2 days. Immunoprecipitation was performed using the anti-FLAG M2 beads. ( h ) GST-fused H109–393 is sufficient to bind to caspase-8 as determined by performing the GST pull-down assay. ( i ) Flag-HECTD3 binds to both DED1 and DED2 of caspase-8 as determined by the GST pull-down assay

    Journal: Cell Death & Disease

    Article Title: The HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by promoting K63-linked polyubiquitination of caspase-8

    doi: 10.1038/cddis.2013.464

    Figure Lengend Snippet: HECTD3 interacts with caspase-8 through the DOC and DED domains. ( a ) Schematic representation of the HECTD3 and caspase-8 proteins and their mutants. ( b ) WT HECTD3 interacts with endogenous caspase-8, but not caspase-3 and -7. Flag-HECTD3, Flag-HΔ1–511 and an empty vector were transfected into HEK293T for 2 days. Immunoprecipitation was performed using the anti-FLAG M2 beads. ( c ) Caspase-8 interacts with HECTD3. Flag-caspase-8 or an empty vector was co-transfected with HECTD3 into HEK293T for 2 days. Immunoprecipitation was performed using the anti-FLAG M2 beads. ( d ) HECTD3 directly binds to caspase-8 but not caspase-3 in vitro . The recombinant GST-HECTD3 and GST were purified from E.coli ( Supplementary Figure S1A ) and were incubated with in vitro -translated caspase-8 and caspase-3 proteins, respectively, with Glutathione-Sepharose 4B slurry beads overnight at 4 °C. The beads were washed three times with 1 ml of 1 × cell lysis buffer. The proteins were resuspended with 20–50 μ l of SDS sample buffer and analyzed by WB. ( e ) The endogenous HECTD3 protein forms a complex with the endogenous caspase-8 protein in HeLa. The endogenous HECTD3 was immunoprecipitated by the anti-HECTD3 Ab. Rabbit IgG was used as a negative control. ( f ) Both HECTD3 and caspase-8 are predominantly localized in the cytoplasm. The localization of Flag-HECTD3 and caspase-8 proteins was examined by immunofluorescence staining in HEK293T cells after co-transfection. DAPI was used to stain cell nuclei. ( g ) HΔ1–511 and HΔ109–393 significantly decrease the binding affinity with caspase-8. Flag-HECTD3 and its mutants were co-transfected with caspase-8 into HEK293T for 2 days. Immunoprecipitation was performed using the anti-FLAG M2 beads. ( h ) GST-fused H109–393 is sufficient to bind to caspase-8 as determined by performing the GST pull-down assay. ( i ) Flag-HECTD3 binds to both DED1 and DED2 of caspase-8 as determined by the GST pull-down assay

    Article Snippet: The anti-GST antibody and anti-Flag M2 monoclonal antibody are from Sigma (St. Louis, MO, USA).

    Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, In Vitro, Recombinant, Purification, Incubation, Lysis, Western Blot, Negative Control, Immunofluorescence, Staining, Cotransfection, Binding Assay, Pull Down Assay

    IQGAP1 C terminus aa 1503–1657 is required for binding and suppressing TβRII. ( A ) Top 4 rows: full-length (FL) IQGAP1 and GST-fused truncated IQGAP1 proteins are shown. Bottom, GST fused truncated IQGAP1 proteins extracted from bacteria were incubated with HSC lysates for GST pull-down assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII. Ponceau S staining depicted the purity of the recombinant proteins. ( B ) Top: after the GST tag of GST-TRII was removed by thrombin treatment, detagged TβRII was incubated with GST-fused IQGAP1 proteins for in vitro binding assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII directly in vitro. Ponceau S staining depicted the purity of GST and GST-fused IQGAP1 proteins. Bottom: detagged IQGAP1 aa 746–1657 was incubated with GST or GST-TβRII for in vitro binding assays. GST-TβRII bound to IQGAP1 aa 746-1657 directly in vitro. aa 746–1657 instead of aa 1503–1657 of IQGAP1 was used in this assay because IQGAP1 antibodies could not recognize aa 1503–1657 of IQGAP1. Ponceau S staining depicted the purity of GST and GST fusion proteins. ( C ) HSCs expressing TβRII-HA were transduced with lentiviruses encoding GFP, IQGAP1-FLAG, or IQGAP1 (1-1502)-FLAG, and subjected to WB. In contrast with IQGAP1, IQGAP1 (1-1502) mutant lacking the TβRII binding region failed to repress TβRII protein levels. Densitometric ratios are shown on the bottom. All data shown represent multiple repeats with similar results.

    Journal: The Journal of Clinical Investigation

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

    doi: 10.1172/JCI63836

    Figure Lengend Snippet: IQGAP1 C terminus aa 1503–1657 is required for binding and suppressing TβRII. ( A ) Top 4 rows: full-length (FL) IQGAP1 and GST-fused truncated IQGAP1 proteins are shown. Bottom, GST fused truncated IQGAP1 proteins extracted from bacteria were incubated with HSC lysates for GST pull-down assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII. Ponceau S staining depicted the purity of the recombinant proteins. ( B ) Top: after the GST tag of GST-TRII was removed by thrombin treatment, detagged TβRII was incubated with GST-fused IQGAP1 proteins for in vitro binding assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII directly in vitro. Ponceau S staining depicted the purity of GST and GST-fused IQGAP1 proteins. Bottom: detagged IQGAP1 aa 746–1657 was incubated with GST or GST-TβRII for in vitro binding assays. GST-TβRII bound to IQGAP1 aa 746-1657 directly in vitro. aa 746–1657 instead of aa 1503–1657 of IQGAP1 was used in this assay because IQGAP1 antibodies could not recognize aa 1503–1657 of IQGAP1. Ponceau S staining depicted the purity of GST and GST fusion proteins. ( C ) HSCs expressing TβRII-HA were transduced with lentiviruses encoding GFP, IQGAP1-FLAG, or IQGAP1 (1-1502)-FLAG, and subjected to WB. In contrast with IQGAP1, IQGAP1 (1-1502) mutant lacking the TβRII binding region failed to repress TβRII protein levels. Densitometric ratios are shown on the bottom. All data shown represent multiple repeats with similar results.

    Article Snippet: For in vitro binding assays, the GST tag was first removed by thrombin, and detagged proteins were purified and recovered by the Thrombin Cleavage Capture Kit (Novagen, 69022).

    Techniques: Binding Assay, Incubation, Staining, Recombinant, In Vitro, Expressing, Transduction, Western Blot, Mutagenesis

    DEK40 recognizes and directly binds cox3 , nad2 , and nad5 transcripts. (A) Nucleotide sequence of RNA probes. Edited sites are indicated in red. (B) RNA EMSAs indicated that DEK40 directly binds to cox3 transcripts that surround the cox3 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. Black arrows show the shifted bound and free RNA probe. (C) RNA EMSAs indicated that DEK40 directly binds to nad2 transcripts that surround the nad2 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probesw are indicated by black arrows. (D) RNA EMSAs indicated that DEK40 directly binds to nad5 transcripts that surround the nad5 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probes are indicated by black arrows.

    Journal: Journal of Experimental Botany

    Article Title: Pentatricopeptide repeat protein DEK40 is required for mitochondrial function and kernel development in maize

    doi: 10.1093/jxb/erz391

    Figure Lengend Snippet: DEK40 recognizes and directly binds cox3 , nad2 , and nad5 transcripts. (A) Nucleotide sequence of RNA probes. Edited sites are indicated in red. (B) RNA EMSAs indicated that DEK40 directly binds to cox3 transcripts that surround the cox3 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. Black arrows show the shifted bound and free RNA probe. (C) RNA EMSAs indicated that DEK40 directly binds to nad2 transcripts that surround the nad2 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probesw are indicated by black arrows. (D) RNA EMSAs indicated that DEK40 directly binds to nad5 transcripts that surround the nad5 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probes are indicated by black arrows.

    Article Snippet: DEK40 tagged with GST was purified using glutathione Sepharose 4B (GE Healthcare).

    Techniques: Sequencing, Negative Control

    Bt 2 cAMP does not affect the dephosphorylation of ERK2. A, A GST fusion protein of P-ERK2 (GST-P-ERK2) was incubated with buffer only for 40 min at 4 C or 37 C as indicated. Another sample was incubated for 40 min at 37 C with buffer containing 120 U of

    Journal: Molecular Endocrinology

    Article Title: Reactive Oxygen Species (ROS) Play a Critical Role in the cAMP-Induced Activation of Ras and the Phosphorylation of ERK1/2 in Leydig Cells

    doi: 10.1210/me.2010-0489

    Figure Lengend Snippet: Bt 2 cAMP does not affect the dephosphorylation of ERK2. A, A GST fusion protein of P-ERK2 (GST-P-ERK2) was incubated with buffer only for 40 min at 4 C or 37 C as indicated. Another sample was incubated for 40 min at 37 C with buffer containing 120 U of

    Article Snippet: Seventy five microliters of these lysates were mixed with 225 μl of a buffer containing 100 m m MgCl2 , 100 m m HEPES (pH 7.4), 10 μ m UO126, and 30 ng of a GST fusion protein of phosphorylated ERK2 (Sigma Chemical Co., St. Louis, MO; catalog no. E-1283).

    Techniques: De-Phosphorylation Assay, Incubation

    Effect of inositol-(1,4,5)triphosphate (IP 3 ) sponge inhibitor on Ca 2+ signaling in MSNs that overexpress HAP1A. (A) Immunoblots of YAC128 MSN cultures that overexpressed the glutathione S -transferase (GST)-tagged IP 3 R1 ligand binding region (226–604 amino acid region) with a point mutation (R441Q: m49) cloned into the pUltra-Chili vector, indicated as p49-GST, and the negative control with a point mutation (K508A: m30), indicated as p30-GST cloned into the pUltra-Chili vector or control (non-transduced) MSNs. MSN cultures on DIV14 from YAC128 mice that overexpressed p49-dTomato and p30-dTomato as an IP 3 sponge and control respectively were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. (B) DHPG-induced Ca 2+ release from the ER in YAC128 MSNs that overexpressed p49-dTomato or p30-dTomato as a control for the IP 3 sponge inhibitor. (C) Protocol to measure DHPG-induced ER Ca 2+ release and DHPG-induced SOCE using the eight-well system. MSN cultures on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP or pLenti-GFP and p49-dTomato or p30-dTomato were loaded with Fura-2AM and incubated in Ca 2 -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (D) Effect of p49-dTomato or p30-dTomato on DHPG-induced ER Ca 2+ release in YAC128 MSNs that overexpressed HAP1A-pLenti-GFP or pLenti-GFP. (E) Effect of p49-dTomato or p30-dTomato on DHPG-induced SOCE in YAC128 MSNs that overexpressed HAP1A-pLenti-GFP or pLenti-GFP. (F) To avoid the firing-effect in MSNs, 1 μM tetrodotoxin (TTX) was added during the Ca 2+ measurement protocol. In the presence of TTX, DHPG-induced ER Ca 2+ release and DHPG-induced SOCE were measured using the eight-well system. MSN cultures on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP or pLenti-GFP and p49-dTomato or p30-dTomato were loaded with Fura-2AM and incubated in Ca 2+ -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. Normalized delta ratio was analyzed as average from three experiments (B,D) . * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

    doi: 10.3389/fncel.2018.00381

    Figure Lengend Snippet: Effect of inositol-(1,4,5)triphosphate (IP 3 ) sponge inhibitor on Ca 2+ signaling in MSNs that overexpress HAP1A. (A) Immunoblots of YAC128 MSN cultures that overexpressed the glutathione S -transferase (GST)-tagged IP 3 R1 ligand binding region (226–604 amino acid region) with a point mutation (R441Q: m49) cloned into the pUltra-Chili vector, indicated as p49-GST, and the negative control with a point mutation (K508A: m30), indicated as p30-GST cloned into the pUltra-Chili vector or control (non-transduced) MSNs. MSN cultures on DIV14 from YAC128 mice that overexpressed p49-dTomato and p30-dTomato as an IP 3 sponge and control respectively were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. (B) DHPG-induced Ca 2+ release from the ER in YAC128 MSNs that overexpressed p49-dTomato or p30-dTomato as a control for the IP 3 sponge inhibitor. (C) Protocol to measure DHPG-induced ER Ca 2+ release and DHPG-induced SOCE using the eight-well system. MSN cultures on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP or pLenti-GFP and p49-dTomato or p30-dTomato were loaded with Fura-2AM and incubated in Ca 2 -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (D) Effect of p49-dTomato or p30-dTomato on DHPG-induced ER Ca 2+ release in YAC128 MSNs that overexpressed HAP1A-pLenti-GFP or pLenti-GFP. (E) Effect of p49-dTomato or p30-dTomato on DHPG-induced SOCE in YAC128 MSNs that overexpressed HAP1A-pLenti-GFP or pLenti-GFP. (F) To avoid the firing-effect in MSNs, 1 μM tetrodotoxin (TTX) was added during the Ca 2+ measurement protocol. In the presence of TTX, DHPG-induced ER Ca 2+ release and DHPG-induced SOCE were measured using the eight-well system. MSN cultures on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP or pLenti-GFP and p49-dTomato or p30-dTomato were loaded with Fura-2AM and incubated in Ca 2+ -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. Normalized delta ratio was analyzed as average from three experiments (B,D) . * p

    Article Snippet: The nitrocellulose sheets were then incubated at 4°C overnight in blocking solution with primary polyclonal antibodies against GST (1:5,000; catalog no. G7781, Sigma) and monoclonal antibodies against HAP1 protein (1:300; catalog no. 611302, BD Transduction Laboratories), CacyBP/SIP (1:1,000; catalog no. ab51288, Abcam), or GFP (1:1,000; catalog no. 11814460001, Roche).

    Techniques: Western Blot, Ligand Binding Assay, Mutagenesis, Clone Assay, Plasmid Preparation, Negative Control, Mouse Assay, Incubation

    Phosphorylation of S670 is required for error correction and kinetochore tension. (A) Synchronized HeLa cells depleted of BubR1 by siRNA were injected with various GST/BubR1 constructs and treated with MG132 to prevent mitotic exit. Coverslips were chilled on ice for 10 min, extracted, fixed, and stained for GST, ACA, tubulin, and DAPI. Tubulin and ACA signals were deconvolved to visualize microtubule attachments at individual bioriented kinetochores that are shown in the insets. (B) Interkinetochore distances were measured between pairs of ACA foci with end-on attachments (50

    Journal: The Journal of Cell Biology

    Article Title: Phosphorylation sites in BubR1 that regulate kinetochore attachment, tension, and mitotic exit

    doi: 10.1083/jcb.200805163

    Figure Lengend Snippet: Phosphorylation of S670 is required for error correction and kinetochore tension. (A) Synchronized HeLa cells depleted of BubR1 by siRNA were injected with various GST/BubR1 constructs and treated with MG132 to prevent mitotic exit. Coverslips were chilled on ice for 10 min, extracted, fixed, and stained for GST, ACA, tubulin, and DAPI. Tubulin and ACA signals were deconvolved to visualize microtubule attachments at individual bioriented kinetochores that are shown in the insets. (B) Interkinetochore distances were measured between pairs of ACA foci with end-on attachments (50

    Article Snippet: Commercial antibodies to tubulin (Sigma-Aldrich), GST (Cell Signaling Technology), Flag (Sigma-Aldrich), and Plk1 (Santa Cruz Biotechnology, Inc.) were used.

    Techniques: Injection, Construct, Staining

    TYK2-ΔE8 is catalytically active but unable to rescue cytokine signaling. (A) Basal in vitro kinase activity of TYK2 from unstimulated 11,1 cells stably expressing TYK2 WT or TYK2-ΔE8. TYK2 was immunoprecipitated and subjected to an in vitro kinase reaction for 5 min at 30°C in the presence (+) or absence (-) of 30 μM ATP. Phosphorylated TYK2 in the reaction was revealed by immunoblotting with anti-phospho-TYK2. The membrane was reprobed for TYK2. (B) IFN-induced JAK/STAT activation in 11,1 cells stably expressing TYK2 WT or TYK2-ΔE8. Cells were treated with IFN-β for 15 min. The level of tyrosine-phosphorylated TYK2, STAT1, STAT2 and STAT3 was analyzed by with phospho-specific Abs. The membrane was reprobed for TYK2 and total STATs. (C) IFNAR1 level in 11,1 cells (-) and derived clones stably expressing TYK2 WT or TYK2-ΔE8. (D) In vitro interaction of His-TYK2-FERM-SH2 with GST-IFNAR1cyt. His-TYK2-FERM-SH2 WT or ΔE8 were incubated with a GST fusion protein containing the cytoplasmic domain of IFNAR1(IFNAR1 cyt ) or IkB-β. Proteins bound to glutathione-Sepharose beads were separated on SDS-PAGE and visualized with TYK2 Abs. Five % input TYK2 protein shown at the bottom. (E) IL-12-induced JAK/STAT activation in 11,1 cells stably expressing the IL-12 receptor β1 and β2 chains. Cells were transiently transfected with TYK2 WT or TYK2-ΔE8. Twenty-four hrs later, cells were treated with IFN-β (500pM) or IL-12 (20ng/ml) for 15 min. The level of tyrosine-phosphorylated TYK2 and STAT1 was analyzed with phospho-specific Abs. The membrane was reprobed for TYK2 levels. A nonspecific band shown as loading control. (F) 293T cells were transfected with the pRc-CMV empty vector (EV), TYK2-ΔE8 or the triple mutant TYK2-K930R/Y1044F/Y1045F (DN) possessing dominant-negative activity [ 19 ]. Twenty-four hrs later, cells were treated with IFN-α for 15 min. Phosphorylation of STAT1, STAT2 and TYK2 were analyzed with phospho-specific Abs. The membrane strips were reprobed for TYK2 and STAT1 contents. Of note, neither TYK2-ΔE8 nor DN can be inducibly phosphorylated, hence the phospho-TYK2 band corresponds to endogenous TYK2.

    Journal: PLoS ONE

    Article Title: Two common disease-associated TYK2 variants impact exon splicing and TYK2 dosage

    doi: 10.1371/journal.pone.0225289

    Figure Lengend Snippet: TYK2-ΔE8 is catalytically active but unable to rescue cytokine signaling. (A) Basal in vitro kinase activity of TYK2 from unstimulated 11,1 cells stably expressing TYK2 WT or TYK2-ΔE8. TYK2 was immunoprecipitated and subjected to an in vitro kinase reaction for 5 min at 30°C in the presence (+) or absence (-) of 30 μM ATP. Phosphorylated TYK2 in the reaction was revealed by immunoblotting with anti-phospho-TYK2. The membrane was reprobed for TYK2. (B) IFN-induced JAK/STAT activation in 11,1 cells stably expressing TYK2 WT or TYK2-ΔE8. Cells were treated with IFN-β for 15 min. The level of tyrosine-phosphorylated TYK2, STAT1, STAT2 and STAT3 was analyzed by with phospho-specific Abs. The membrane was reprobed for TYK2 and total STATs. (C) IFNAR1 level in 11,1 cells (-) and derived clones stably expressing TYK2 WT or TYK2-ΔE8. (D) In vitro interaction of His-TYK2-FERM-SH2 with GST-IFNAR1cyt. His-TYK2-FERM-SH2 WT or ΔE8 were incubated with a GST fusion protein containing the cytoplasmic domain of IFNAR1(IFNAR1 cyt ) or IkB-β. Proteins bound to glutathione-Sepharose beads were separated on SDS-PAGE and visualized with TYK2 Abs. Five % input TYK2 protein shown at the bottom. (E) IL-12-induced JAK/STAT activation in 11,1 cells stably expressing the IL-12 receptor β1 and β2 chains. Cells were transiently transfected with TYK2 WT or TYK2-ΔE8. Twenty-four hrs later, cells were treated with IFN-β (500pM) or IL-12 (20ng/ml) for 15 min. The level of tyrosine-phosphorylated TYK2 and STAT1 was analyzed with phospho-specific Abs. The membrane was reprobed for TYK2 levels. A nonspecific band shown as loading control. (F) 293T cells were transfected with the pRc-CMV empty vector (EV), TYK2-ΔE8 or the triple mutant TYK2-K930R/Y1044F/Y1045F (DN) possessing dominant-negative activity [ 19 ]. Twenty-four hrs later, cells were treated with IFN-α for 15 min. Phosphorylation of STAT1, STAT2 and TYK2 were analyzed with phospho-specific Abs. The membrane strips were reprobed for TYK2 and STAT1 contents. Of note, neither TYK2-ΔE8 nor DN can be inducibly phosphorylated, hence the phospho-TYK2 band corresponds to endogenous TYK2.

    Article Snippet: The following Abs were used: TYK2 mAb T10-2 and anti-GST (Hybridolab, Institut Pasteur); anti-IFNAR1 (64G12 mAbs) [ ]; anti-STAT2-phospho-Y689 (R & D); Abs to STAT1, STAT2, STAT3, and STAT1-P-Y701, STAT3-P-Y705, and TYK2–P-YY1054/55 (Cell Signaling Technology, Beverly, MA).

    Techniques: In Vitro, Activity Assay, Stable Transfection, Expressing, Immunoprecipitation, Activation Assay, Derivative Assay, Clone Assay, Incubation, SDS Page, Transfection, Plasmid Preparation, Mutagenesis, Dominant Negative Mutation

    Phylogeny of glutathione S-transferases (GSTs) . Branches with dots had > 80% bootstrap support. The tree was rooted with human HsapGSTA1.Aaeg, Aedes aegypti ; Agam, Aedes gambiae ; Amel, Apis mellifera ; Apis, Acyrthosiphon pisum ; Bmor, Bombyx mori ; Cqui, Culex quinquefasciatus ; Dmel, Drosophila melanogaster ; Dpon, Dendroctonus ponderosae ; Hsap; Homo sapiens ; Nvit, Nasonia vitripennis ; Phum, Pediculus humanus ; Tcas, Tribolium castaneum .

    Journal: Genome Biology

    Article Title: Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest

    doi: 10.1186/gb-2013-14-3-r27

    Figure Lengend Snippet: Phylogeny of glutathione S-transferases (GSTs) . Branches with dots had > 80% bootstrap support. The tree was rooted with human HsapGSTA1.Aaeg, Aedes aegypti ; Agam, Aedes gambiae ; Amel, Apis mellifera ; Apis, Acyrthosiphon pisum ; Bmor, Bombyx mori ; Cqui, Culex quinquefasciatus ; Dmel, Drosophila melanogaster ; Dpon, Dendroctonus ponderosae ; Hsap; Homo sapiens ; Nvit, Nasonia vitripennis ; Phum, Pediculus humanus ; Tcas, Tribolium castaneum .

    Article Snippet: We examined two gene families, the P450 cytochromes, and the glutathione S-transferases (GSTs), which are commonly involved in detoxification of plant chemicals, and from which some members are likely to be involved in the sequential pathway of metabolizing xenobiotics by making them more polar and excretable.

    Techniques:

    Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing GST–FbxA:F-box/WD40 were lysed and the 10,000 × G supernatant was adsorbed to g–Sepharose. The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.

    Journal: Genes & Development

    Article Title: Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium

    doi: 10.1101/gad.871101

    Figure Lengend Snippet: Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing GST–FbxA:F-box/WD40 were lysed and the 10,000 × G supernatant was adsorbed to g–Sepharose. The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.

    Article Snippet: Samples were centrifuged at 15,000 rpm for 10 min. We incubated the supernatant with 80 μL of GST beads (glutathione–Sepharose 4B, purchased from Amersham Pharmacia Biotech) for 1 h at 4°C with gentle rocking.

    Techniques: Western Blot, Mutagenesis, Expressing, Transformation Assay

    Effect of mitochondrial DNA (mtDNA) depletion on the transcripts of the antioxidant defense enzymes. The total RNA from the control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts was prepared. The transcript level of the antioxidant defense enzymes was quantified by RT-PCR (A) and q RT-PCR (B). β-Actin was used as the control. All results represent the mean ± SEM from five independent experiments. GR, glutathione reductase; GPx, glutathione peroxidase; GST, glutathione S-transferase; SOD, superoxide dismutase. *** p

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Alteration of mitochondrial DNA content modulates antioxidant enzyme expressions and oxidative stress in myoblasts

    doi: 10.4196/kjpp.2019.23.6.519

    Figure Lengend Snippet: Effect of mitochondrial DNA (mtDNA) depletion on the transcripts of the antioxidant defense enzymes. The total RNA from the control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts was prepared. The transcript level of the antioxidant defense enzymes was quantified by RT-PCR (A) and q RT-PCR (B). β-Actin was used as the control. All results represent the mean ± SEM from five independent experiments. GR, glutathione reductase; GPx, glutathione peroxidase; GST, glutathione S-transferase; SOD, superoxide dismutase. *** p

    Article Snippet: The kits for measuring the glutathione S-transferase (GST) and glucose-6 phosphate dehydrogenase (G6PD) activities were obtained from Sigma-Aldrich.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Effect of mitochondrial DNA (mtDNA) depletion on the reduced glutathione (GSH) or oxidized glutathione (GSSG) contents and glutathione S-transferase (GST) activity. (A, B) The cellular GSH and GSSG contents were measured in the control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts. (C) The cellular redox status is presented as the GSH/GSSG ratio. (D) The GST activity was assayed in the supernatant of the cell lysates and was calculated using a known extinction coefficient. The values are expressed as the mean ± SEM from three independent experiments. ** p

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Alteration of mitochondrial DNA content modulates antioxidant enzyme expressions and oxidative stress in myoblasts

    doi: 10.4196/kjpp.2019.23.6.519

    Figure Lengend Snippet: Effect of mitochondrial DNA (mtDNA) depletion on the reduced glutathione (GSH) or oxidized glutathione (GSSG) contents and glutathione S-transferase (GST) activity. (A, B) The cellular GSH and GSSG contents were measured in the control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts. (C) The cellular redox status is presented as the GSH/GSSG ratio. (D) The GST activity was assayed in the supernatant of the cell lysates and was calculated using a known extinction coefficient. The values are expressed as the mean ± SEM from three independent experiments. ** p

    Article Snippet: The kits for measuring the glutathione S-transferase (GST) and glucose-6 phosphate dehydrogenase (G6PD) activities were obtained from Sigma-Aldrich.

    Techniques: Activity Assay

    Direct physical interaction between GATA-1 and CP2. (A) Purified GST or GST fusion proteins containing full-length CP2 (GST-CP2) preadsorbed to glutathione-Sepharose beads were incubated with 35 S-labeled in vitro-transcribed/translated GATA-1 (lanes 1

    Journal:

    Article Title: Functional Interaction of CP2 with GATA-1 in the Regulation of Erythroid Promoters

    doi: 10.1128/MCB.26.10.3942-3954.2006

    Figure Lengend Snippet: Direct physical interaction between GATA-1 and CP2. (A) Purified GST or GST fusion proteins containing full-length CP2 (GST-CP2) preadsorbed to glutathione-Sepharose beads were incubated with 35 S-labeled in vitro-transcribed/translated GATA-1 (lanes 1

    Article Snippet: GST-CP2 and GST-GATA-1 proteins present in the soluble fraction were bound to GST-Sepharose 4B (Amersham Bioscience) and purified according to the manufacturer's instructions.

    Techniques: Purification, Incubation, Labeling, In Vitro

    Binding of PBP to ER and potentiation of estrogen dependent transcription from ER. ( A ) Binding of PBP to ER. [ 35 S] methionine-labeled PBP generated by in vitro translation was incubated with glutathione-Sepharose beads containing GST, or GST-ER, in the presence of estrogen (E) or tamoxifen (T) or absence of the ligand (-). The bound protein was eluted and analyzed by SDS/PAGE and was autoradiographed. PBP binds to ER in the absence of estrogen and in the presence of tamoxifen, but estrogen increases the interaction of ER with PBP. No binding was seen for GST alone, indicating that the interaction is specific for ER. ( B ) PBP potentiates the transcriptional activity of ER. CV-1 cell were transiently cotransfected with 1.5 μg of reporter construct ERE-TK-luc, 0.25 μg of pCMV-ER, 0.5 μg of pCMX-PBP or pCMX, and 0.5 μg of pCMVβ in the presence of β-estradiol or absence of ligand. PBP increased the transcription of luciferase gene ≈2- fold in the presence of β-estradiol. Results are the mean of four independent transfections and are normalized to the internal control β-galactosidase expression.

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

    Article Title: Amplification and overexpression of peroxisome proliferatoractivated receptor binding protein (PBP/PPARBP) gene in breast cancer

    doi:

    Figure Lengend Snippet: Binding of PBP to ER and potentiation of estrogen dependent transcription from ER. ( A ) Binding of PBP to ER. [ 35 S] methionine-labeled PBP generated by in vitro translation was incubated with glutathione-Sepharose beads containing GST, or GST-ER, in the presence of estrogen (E) or tamoxifen (T) or absence of the ligand (-). The bound protein was eluted and analyzed by SDS/PAGE and was autoradiographed. PBP binds to ER in the absence of estrogen and in the presence of tamoxifen, but estrogen increases the interaction of ER with PBP. No binding was seen for GST alone, indicating that the interaction is specific for ER. ( B ) PBP potentiates the transcriptional activity of ER. CV-1 cell were transiently cotransfected with 1.5 μg of reporter construct ERE-TK-luc, 0.25 μg of pCMV-ER, 0.5 μg of pCMX-PBP or pCMX, and 0.5 μg of pCMVβ in the presence of β-estradiol or absence of ligand. PBP increased the transcription of luciferase gene ≈2- fold in the presence of β-estradiol. Results are the mean of four independent transfections and are normalized to the internal control β-galactosidase expression.

    Article Snippet: GST and GST-ER were produced in Escherichia coli and were bound to GST-Sepharose beads (Amersham Pharmacia).

    Techniques: Binding Assay, Labeling, Generated, In Vitro, Incubation, SDS Page, Activity Assay, Construct, Luciferase, Transfection, Expressing

    gcd7 - 201 gcn2∆ rescued Slg + and Gcd + phenotype when transformed with pEG(KG)/ TAN1 plasmid. GCD7 gcn2∆ and gcd7 - 201 Gcn2∆ harboring pEG(KG)/ TAN1 or empty vector pEG(KG) were streaked in parallel on SC medium laking uracil, but either containing a raffinose or b galactose. Uracil-based plasmid pEG(KG)/ TAN1 was evicted on SC medium containing, c FOA and further streaked on d SC-Ura medium. e Spotting on SC medium containing 2% galactose and lacking uracil or SC medium containing 2% galactose, 30 mM 3-AT and lacking uracil. Plates were incubated at 30 °C for 2 days. f Western analysis of GST-Tan1p expression in gcd7 - 201 gcn2∆ strain with anti-GST antibody. The whole-cell protein extracts (20 µg) were prepared from uninduced and 2% galactose-induced cultures of the strain harboring pEG(KG)/ TAN1 . Samples were separated on 10% SDS gel followed by western blotting using anti-GST for GST-Tan1 and anti-Gcd6 antibodies (loading control). UI uninduced, I induced

    Journal: 3 Biotech

    Article Title: Role of Saccharomyces cerevisiae TAN1 (tRNA acetyltransferase) in eukaryotic initiation factor 2B (eIF2B)-mediated translation control and stress response

    doi: 10.1007/s13205-017-0857-8

    Figure Lengend Snippet: gcd7 - 201 gcn2∆ rescued Slg + and Gcd + phenotype when transformed with pEG(KG)/ TAN1 plasmid. GCD7 gcn2∆ and gcd7 - 201 Gcn2∆ harboring pEG(KG)/ TAN1 or empty vector pEG(KG) were streaked in parallel on SC medium laking uracil, but either containing a raffinose or b galactose. Uracil-based plasmid pEG(KG)/ TAN1 was evicted on SC medium containing, c FOA and further streaked on d SC-Ura medium. e Spotting on SC medium containing 2% galactose and lacking uracil or SC medium containing 2% galactose, 30 mM 3-AT and lacking uracil. Plates were incubated at 30 °C for 2 days. f Western analysis of GST-Tan1p expression in gcd7 - 201 gcn2∆ strain with anti-GST antibody. The whole-cell protein extracts (20 µg) were prepared from uninduced and 2% galactose-induced cultures of the strain harboring pEG(KG)/ TAN1 . Samples were separated on 10% SDS gel followed by western blotting using anti-GST for GST-Tan1 and anti-Gcd6 antibodies (loading control). UI uninduced, I induced

    Article Snippet: Whole-cell extracts (WCE) of gcd7 - 201 gcn2∆ mutant expressing GST-Tan1 or GST alone were analyzed by western blotting using anti-GST antibodies.

    Techniques: Transformation Assay, Plasmid Preparation, Incubation, Western Blot, Expressing, SDS-Gel

    A. PDZ-domain protein arrays were overlaid with purified GST-MapC50, GST-NleH1C150 or GST followed by detection with anti-GST antibodies. MapC50 interacts with PDZ1 and 2 domains of NHERF1 and NHERF2 whereas NleH1C150 interacts only with the PDZ2 domain of NHERF2.B. Purified His-NHERF2, MBP-PDZ1, MBP-PDZ2, MBP-EBD and MBP were transferred onto PVDF membrane and overlaid with purified MBP-Map followed by detection with anti-Map, His-EspI followed by detection with anti-EspI or GST-NleH1 followed by detection with anti-NleH. Equivalent protein loading was confirmed by Coomassie staining. Map, EspI and NleH1 interact with His-NHERF2, but only Map and EspI interacted with MBP-PDZ2 (black arrows).C and D. Bacterial extracts of EPEC Δ map overexpressing Map and MapΔC3 (C) or EPEC Δ espI overexpressing EspI and EspIΔC7 (D) were transferred onto PVDF membrane and overlaid with purified His-NHERF2 followed by detection with anti-NHERF2 or immunoblotted with an anti-Map or anti-EspI antibody. NHERF2 was able to interact with Map and EspI but not MapΔC3 or EspIΔC7.E. Purified His-NHERF2, GST, GST-NleH1 and His-EspI were transferred onto PVDF membrane and overlaid with purified His-NHERF2 followed by detection with anti-NHERF2 or immunoblotted with anti-GST. NHERF2 interacted with His-EspI and GST-NleH1 (black arrow).

    Journal: Cellular Microbiology

    Article Title: Binding to Na+/H+ exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map, EspI and NleH

    doi: 10.1111/j.1462-5822.2010.01503.x

    Figure Lengend Snippet: A. PDZ-domain protein arrays were overlaid with purified GST-MapC50, GST-NleH1C150 or GST followed by detection with anti-GST antibodies. MapC50 interacts with PDZ1 and 2 domains of NHERF1 and NHERF2 whereas NleH1C150 interacts only with the PDZ2 domain of NHERF2.B. Purified His-NHERF2, MBP-PDZ1, MBP-PDZ2, MBP-EBD and MBP were transferred onto PVDF membrane and overlaid with purified MBP-Map followed by detection with anti-Map, His-EspI followed by detection with anti-EspI or GST-NleH1 followed by detection with anti-NleH. Equivalent protein loading was confirmed by Coomassie staining. Map, EspI and NleH1 interact with His-NHERF2, but only Map and EspI interacted with MBP-PDZ2 (black arrows).C and D. Bacterial extracts of EPEC Δ map overexpressing Map and MapΔC3 (C) or EPEC Δ espI overexpressing EspI and EspIΔC7 (D) were transferred onto PVDF membrane and overlaid with purified His-NHERF2 followed by detection with anti-NHERF2 or immunoblotted with an anti-Map or anti-EspI antibody. NHERF2 was able to interact with Map and EspI but not MapΔC3 or EspIΔC7.E. Purified His-NHERF2, GST, GST-NleH1 and His-EspI were transferred onto PVDF membrane and overlaid with purified His-NHERF2 followed by detection with anti-NHERF2 or immunoblotted with anti-GST. NHERF2 interacted with His-EspI and GST-NleH1 (black arrow).

    Article Snippet: Western blots were performed following standard methods using rabbit anti-NHERF2 (raised against purified human His-NHERF2, Covalab), anti-EspI , anti-NleH (raised against purified His-NleH1, Covalab), mouse anti-Map (Immune Systems limited), anti-poly-Histidine (Sigma), anti-GST (AbCam) and anti-HA HRP-conjugated (Roche) antibodies.

    Techniques: Purification, Staining

    Activation of full-length endophilin-A1 by GST-PRD. A. Negative-stain EM with Folch liposomes. Assays with endophilin-A1 (full-length or N-BAR domain; 10 µM) were carried out as described before. The inset shows a zoomed-in view of the red box area of the image, with scale bar = 100 nm. B. Statistical analysis of vesicle size distribution. Diameters of vesicles produced by endophilin in the presence of GST-PRD were quantified from electron micrographs taken from three independent experiments. C. Liposome co-pelleting assay with Folch liposomes. Liposome binding assays were carried out as described in Fig. 3. The horizontal, dashed lines indicate the lipid-bound fraction of the isolated endophilin-A1 N-BAR (expressed as His 6 -SUMO-fusion protein) domain and isolated full-length endophilin-A1 under similar conditions. Error bars represent standard deviations of a minimum of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Versatile Membrane Deformation Potential of Activated Pacsin

    doi: 10.1371/journal.pone.0051628

    Figure Lengend Snippet: Activation of full-length endophilin-A1 by GST-PRD. A. Negative-stain EM with Folch liposomes. Assays with endophilin-A1 (full-length or N-BAR domain; 10 µM) were carried out as described before. The inset shows a zoomed-in view of the red box area of the image, with scale bar = 100 nm. B. Statistical analysis of vesicle size distribution. Diameters of vesicles produced by endophilin in the presence of GST-PRD were quantified from electron micrographs taken from three independent experiments. C. Liposome co-pelleting assay with Folch liposomes. Liposome binding assays were carried out as described in Fig. 3. The horizontal, dashed lines indicate the lipid-bound fraction of the isolated endophilin-A1 N-BAR (expressed as His 6 -SUMO-fusion protein) domain and isolated full-length endophilin-A1 under similar conditions. Error bars represent standard deviations of a minimum of 3 independent experiments.

    Article Snippet: All PRD constructs were expressed and purified as GST fusion proteins (resin: GSTrap, GE Healthcare), following the manufacturer's instructions.

    Techniques: Activation Assay, Staining, Produced, Binding Assay, Isolation

    The role of arginine residues within the dynamin-1 PRD in pacsin-1 ' s membrane deformation potential. A. Membrane deformation of Folch liposomes. The positions of Arg-to-Ala mutations (GST-PRD ArgKO1 , GST-PRD ArgKO2 and GST-PRD ArgKO3 ) in the mouse dynamin-1 PRD protein sequence are shown (top panel). Negative-stain EM images are shown after incubation of liposomes with the indicated protein complexes. B. Liposome co-pelleting assay. Liposome binding assays were carried out as described in Fig. 3. Error bars represent standard deviations of a minimum of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Versatile Membrane Deformation Potential of Activated Pacsin

    doi: 10.1371/journal.pone.0051628

    Figure Lengend Snippet: The role of arginine residues within the dynamin-1 PRD in pacsin-1 ' s membrane deformation potential. A. Membrane deformation of Folch liposomes. The positions of Arg-to-Ala mutations (GST-PRD ArgKO1 , GST-PRD ArgKO2 and GST-PRD ArgKO3 ) in the mouse dynamin-1 PRD protein sequence are shown (top panel). Negative-stain EM images are shown after incubation of liposomes with the indicated protein complexes. B. Liposome co-pelleting assay. Liposome binding assays were carried out as described in Fig. 3. Error bars represent standard deviations of a minimum of 3 independent experiments.

    Article Snippet: All PRD constructs were expressed and purified as GST fusion proteins (resin: GSTrap, GE Healthcare), following the manufacturer's instructions.

    Techniques: Sequencing, Staining, Incubation, Binding Assay

    Activation of pacsin-1 by the proline-rich domain (PRD) of dynamin-1. A. Sequence of the mouse dynamin-1 PRD. A regulatory sequence (phospho-box), the core pacsin-1 binding region (orange) and arginine residues are highlighted. The sequence is 100% identical to the human dynamin-1 PRD. The mouse PRD was expressed as GST-fusion protein. B. Negative-stain EM with Folch liposomes. Liposomes were imaged as described before following incubations with the indicated proteins and protein complexes (top panel). The histogram (middle panel) shows the size distribution of the vesicles produced by pacsin-1 in the presence of GST-PRD. Vesicle diameters were quantified from electron micrographs taken from three independent experiments. Liposome-protein co-pelleting assays (bottom panel) were used to assess the amount of protein bound to lipid vesicles. The horizontal, dashed line indicates the lipid-bound fraction of the isolated pacsin-1 F-BAR domain under similar conditions. Two-tailed unpaired t-tests for both pacsin-1 and GST-PRD were p

    Journal: PLoS ONE

    Article Title: Versatile Membrane Deformation Potential of Activated Pacsin

    doi: 10.1371/journal.pone.0051628

    Figure Lengend Snippet: Activation of pacsin-1 by the proline-rich domain (PRD) of dynamin-1. A. Sequence of the mouse dynamin-1 PRD. A regulatory sequence (phospho-box), the core pacsin-1 binding region (orange) and arginine residues are highlighted. The sequence is 100% identical to the human dynamin-1 PRD. The mouse PRD was expressed as GST-fusion protein. B. Negative-stain EM with Folch liposomes. Liposomes were imaged as described before following incubations with the indicated proteins and protein complexes (top panel). The histogram (middle panel) shows the size distribution of the vesicles produced by pacsin-1 in the presence of GST-PRD. Vesicle diameters were quantified from electron micrographs taken from three independent experiments. Liposome-protein co-pelleting assays (bottom panel) were used to assess the amount of protein bound to lipid vesicles. The horizontal, dashed line indicates the lipid-bound fraction of the isolated pacsin-1 F-BAR domain under similar conditions. Two-tailed unpaired t-tests for both pacsin-1 and GST-PRD were p

    Article Snippet: All PRD constructs were expressed and purified as GST fusion proteins (resin: GSTrap, GE Healthcare), following the manufacturer's instructions.

    Techniques: Activation Assay, Sequencing, Binding Assay, Staining, Produced, Isolation, Two Tailed Test

    Pacsin-PRD complex formation. GST pull-down experiments were carried out by using wild-type and mutant forms of GST-PRD to examine their interactions with pacsin-1. Complexes were eluted and analyzed by SDS-PAGE and Coomassie-staining. Bait proteins: wild-type GST-PRD (PRD wt ), GST-PRD trunc1 (tr1), GST-PRD trunc2 (tr2), GST-PRD ArgKO1 (KO1), GST-PRD ArgKO2 (KO2), GST-PRD ArgKO3 (KO3) and GST (negative control).

    Journal: PLoS ONE

    Article Title: Versatile Membrane Deformation Potential of Activated Pacsin

    doi: 10.1371/journal.pone.0051628

    Figure Lengend Snippet: Pacsin-PRD complex formation. GST pull-down experiments were carried out by using wild-type and mutant forms of GST-PRD to examine their interactions with pacsin-1. Complexes were eluted and analyzed by SDS-PAGE and Coomassie-staining. Bait proteins: wild-type GST-PRD (PRD wt ), GST-PRD trunc1 (tr1), GST-PRD trunc2 (tr2), GST-PRD ArgKO1 (KO1), GST-PRD ArgKO2 (KO2), GST-PRD ArgKO3 (KO3) and GST (negative control).

    Article Snippet: All PRD constructs were expressed and purified as GST fusion proteins (resin: GSTrap, GE Healthcare), following the manufacturer's instructions.

    Techniques: Mutagenesis, SDS Page, Staining, Negative Control

    Effect of GST-PRD truncation mutants on the membrane deformation activity of pacsin-1. A. Membrane deformation of Folch liposomes. The sequences of mouse dynamin-1 PRD truncation mutants GST-PRD trunc1 and GST-PRD trunc2 are shown (top panel). Negative-stain EM images are shown after incubation of liposomes with the indicated protein complexes. Either wild-type human pacsin-1 or a corresponding protein with a single-point mutation in the SH3 domain (pacsin-1 P437L ) was used. B. Liposome co-pelleting assay. Liposome binding assays were carried out with the complexes used in (A). The horizontal, dashed lines indicate the lipid-bound fraction of the isolated pacsin-1 F-BAR domain and isolated full-length pacsin-1 under similar conditions. Error bars represent standard deviations of a minimum of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Versatile Membrane Deformation Potential of Activated Pacsin

    doi: 10.1371/journal.pone.0051628

    Figure Lengend Snippet: Effect of GST-PRD truncation mutants on the membrane deformation activity of pacsin-1. A. Membrane deformation of Folch liposomes. The sequences of mouse dynamin-1 PRD truncation mutants GST-PRD trunc1 and GST-PRD trunc2 are shown (top panel). Negative-stain EM images are shown after incubation of liposomes with the indicated protein complexes. Either wild-type human pacsin-1 or a corresponding protein with a single-point mutation in the SH3 domain (pacsin-1 P437L ) was used. B. Liposome co-pelleting assay. Liposome binding assays were carried out with the complexes used in (A). The horizontal, dashed lines indicate the lipid-bound fraction of the isolated pacsin-1 F-BAR domain and isolated full-length pacsin-1 under similar conditions. Error bars represent standard deviations of a minimum of 3 independent experiments.

    Article Snippet: All PRD constructs were expressed and purified as GST fusion proteins (resin: GSTrap, GE Healthcare), following the manufacturer's instructions.

    Techniques: Activity Assay, Staining, Incubation, Mutagenesis, Binding Assay, Isolation