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  • 94
    Thermo Fisher gst
    A small region within the DNA-binding domain of GRHL2, aa 425–437, inhibits <t>p300.</t> (A) Schematic of GRHL2 domains. DBD, DNA-binding domain; DD, dimerization domain; TAD, transactivation domain. (B) HAT assays using the indicated GRHL2 fragments, assayed as <t>GST-fusion</t> proteins. (C) GRHL2 aa 425–437 inhibits p300 HAT activity. The indicated fragments derived from GRHL2 were assayed as GST-fusions (left) or recombinant peptides (right) for inhibition of HAT activity. (D) GRHL2 aa 425–437 are required for inhibition of an AP-1 reporter, GAL4 reporter in conjunction with GAL4-p300, MMP1 reporter, or MMP14 reporter. Values represent relative luciferase activity normalized to TK-β-galactosidase control.
    Gst, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore control gst protein
    Tr-kit does not stably associate with <t>PLCγ1.</t> ( A ) COS cells were transfected with no DNA ( mock ), or 20 μg/dish pCMV5-c-kit ( c-kit ), or 20 μg/dish pCMV5-tr-kit ( tr-kit ). C-kit–transfected cells were incubated for the final 10 min with or without 100 ng/ml SCF. Cell extracts were either analyzed immediately in Western blot (50 μg in each lane) with an anti-kit antibody ( right side of the panel ), or incubated for 2 h with a <t>GST-PLCγ1-SH2SH2SH3</t> fusion protein linked to glutathione–agarose beads. Proteins bound to the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody ( left side of the panel ). ( B ) Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were immunoprecipitated using an anti-kit antibody preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to the one expected for tr-kit is present in all the samples, regardless of tr-kit presence, indicating that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. ( C ) Cell extracts (50 μg) from the same samples shown in B were analyzed in Western blot using an anti-kit antibody. All panels are representative of at least three separate experiments.
    Control Gst Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 33 article reviews
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    gst  (Bio-Rad)
    93
    Bio-Rad gst
    Effects of kinase inhibitors on Claspin and <t>Chk1</t> phosphorylation The extract was incubated with radiolabelled Claspin 679–1332 , radiolabelled <t>GST-CKBD,</t> OA+poly(dA/dT) 70 and the following additions: staurosporine (staur), UCN01, roscovitine (rosco) and caffeine (upper panels); Gö6976, Ro318220, Bim1, SB203580, rapamycin (Rapa), PD98059 and H89 (lower panels). Proteins were detected by immunoblotting with antibodies raised against Chk1 (anti-Chk1) or specific phosphorylation sites on Chk1 (α-p345, α-p317 and α-p296). Claspin 679–1332 and GST–CKBD proteins were detected by autoradiography.
    Gst, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 85 article reviews
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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 520 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gst/product/Cell Signaling Technology Inc
    Average 93 stars, based on 520 article reviews
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    91
    WuXi AppTec gst
    IFT25 interacts physically with IFT27. Purified <t>GST-tagged</t> IFT27 and <t>MBP-tagged</t> IFT25 were used for in vitro binding assay. The left panel shows that immobilized GST-IFT27 protein on the beads could retain MBP-IFT25 protein but not the control protein, MBP. The right panel shows that immobilized MBP-IFT25 protein on beads could retain GST-IFT27 protein but not the control protein, GST. The molecular marker is labeled on the left of each figure. From top to bottom for both panels, the first figure is the Coomassie blue-stained gel. The second and third figures represent the immunoblots probed with antibodies against IFT25 and IFT27, respectively. The fourth one is the immunoblots probed with antibodies against either MBP (left panel) or GST (right panel). The loading materials for each lane of the gels are shown in the tables at the top of each panel. S stands for supernatant and P for bead pellet.
    Gst, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gst  (Abcam)
    93
    Abcam gst
    IRGMd binds to cardiolipin and causes loss of mitochondrial membrane potential a . Splice variants of <t>IRGM.</t> G1-G5, GTPase motifs. The S47N mutation is indicated in blue. b. HeLa cells were transfected with IRGMb, IRGMd-WT (wild type IRGMd, unaltered) or IRGM-S47N mutant (SN) for 48 h, labeled with MTR and imaged by live microscopy. c. Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative. d . Quantification of GFP + MTR + cells. e,f . Analysis of <t>GST-IRGMd</t> binding to lipids by lipid protein binding dot blots (e) and cardiolipin bound agarose bead pull down assay (f; details in Methods). g. IRGMd, IRGMdS47 mutant, and IRGMb isoform concentration-dependent analysis of binding to lipids on strip blots (numbers identifying lipids correspond to the legend in panel e). GST control is in Suppl. Fig. S1d . Membranes in e-g were probed with anti-GST antibody.
    Gst, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa gst purification
    IRGMd binds to cardiolipin and causes loss of mitochondrial membrane potential a . Splice variants of <t>IRGM.</t> G1-G5, GTPase motifs. The S47N mutation is indicated in blue. b. HeLa cells were transfected with IRGMb, IRGMd-WT (wild type IRGMd, unaltered) or IRGM-S47N mutant (SN) for 48 h, labeled with MTR and imaged by live microscopy. c. Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative. d . Quantification of GFP + MTR + cells. e,f . Analysis of <t>GST-IRGMd</t> binding to lipids by lipid protein binding dot blots (e) and cardiolipin bound agarose bead pull down assay (f; details in Methods). g. IRGMd, IRGMdS47 mutant, and IRGMb isoform concentration-dependent analysis of binding to lipids on strip blots (numbers identifying lipids correspond to the legend in panel e). GST control is in Suppl. Fig. S1d . Membranes in e-g were probed with anti-GST antibody.
    Gst Purification, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam anti gst
    TMEM74 associates with ATG16L1 via its C-terminal and influences the interaction between ATG5 and ATG16L1. ( a ) Schematic representations of WT ATG16L1 and its mutants: ATG16L1(1–320), and ATG16L1 △(1–320) . ( b ) HeLa cells were co-transfected with <t>GFP-ATG16L1</t> and mCherry-TMEM74 for 24 h, Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( c ) HeLa cells were co-transfected with GFP-TMEM74 and mCherry-ATG16L1 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, ATG16L1 was detected in the washed beads using an anti-ATG16L1 IgG by western blotting. ( d ) <t>GST</t> and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG16L1, GFP-ATG16L1 was detected in the washed beads by western blotting. ( e , f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG16L1(1–320), or GFP-ATG16L1 △(1–320) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 were detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG16L1 for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, ATG5-ATG12 complex pulled down was detected in the immunoprecipitates using anti-ATG5 by western blotting. Quantification of ATG5-ATG12 pulled down relative to GFP-ATG16L1 was shown as column. Data are means±S.D. of three experiments. * P
    Anti Gst, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A small region within the DNA-binding domain of GRHL2, aa 425–437, inhibits p300. (A) Schematic of GRHL2 domains. DBD, DNA-binding domain; DD, dimerization domain; TAD, transactivation domain. (B) HAT assays using the indicated GRHL2 fragments, assayed as GST-fusion proteins. (C) GRHL2 aa 425–437 inhibits p300 HAT activity. The indicated fragments derived from GRHL2 were assayed as GST-fusions (left) or recombinant peptides (right) for inhibition of HAT activity. (D) GRHL2 aa 425–437 are required for inhibition of an AP-1 reporter, GAL4 reporter in conjunction with GAL4-p300, MMP1 reporter, or MMP14 reporter. Values represent relative luciferase activity normalized to TK-β-galactosidase control.

    Journal: Molecular Biology of the Cell

    Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

    doi: 10.1091/mbc.E16-04-0249

    Figure Lengend Snippet: A small region within the DNA-binding domain of GRHL2, aa 425–437, inhibits p300. (A) Schematic of GRHL2 domains. DBD, DNA-binding domain; DD, dimerization domain; TAD, transactivation domain. (B) HAT assays using the indicated GRHL2 fragments, assayed as GST-fusion proteins. (C) GRHL2 aa 425–437 inhibits p300 HAT activity. The indicated fragments derived from GRHL2 were assayed as GST-fusions (left) or recombinant peptides (right) for inhibition of HAT activity. (D) GRHL2 aa 425–437 are required for inhibition of an AP-1 reporter, GAL4 reporter in conjunction with GAL4-p300, MMP1 reporter, or MMP14 reporter. Values represent relative luciferase activity normalized to TK-β-galactosidase control.

    Article Snippet: Primary antibodies used were as follows: GRHL2, rabbit (rb; Sigma-Aldrich, St. Louis, MO); β-actin, mouse (ms; Thermo Pierce); fibronectin, ms (BD Biosciences, Franklin Lakes, NJ); vimentin, ms (Santa Cruz Biotechnology, Dallas, TX); β-actin, ms (Sigma-Aldrich); E-cadherin, ms (BD Biosciences); β-catenin, ms (BD Biosciences); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ms (OriGene, Rockville, MD); acetyl-lysine, rb (Millipore); H3K18AC, rb (Cell Signaling, Danvers, MA); H3K27AC, rb (Cell Signaling); total H3, rb (Cell Signaling); GST, ms (Thermo Pierce, Waltham, MA); p300, rb (sc-584; Santa Cruz Biotechnology); p300, rb (sc-585; Santa Cruz Biotechnology); FLAG, ms (Sigma-Aldrich); and GAL4, rb (Santa Cruz Biotechnology).

    Techniques: Binding Assay, HAT Assay, Activity Assay, Derivative Assay, Recombinant, Inhibition, Luciferase

    Tr-kit does not stably associate with PLCγ1. ( A ) COS cells were transfected with no DNA ( mock ), or 20 μg/dish pCMV5-c-kit ( c-kit ), or 20 μg/dish pCMV5-tr-kit ( tr-kit ). C-kit–transfected cells were incubated for the final 10 min with or without 100 ng/ml SCF. Cell extracts were either analyzed immediately in Western blot (50 μg in each lane) with an anti-kit antibody ( right side of the panel ), or incubated for 2 h with a GST-PLCγ1-SH2SH2SH3 fusion protein linked to glutathione–agarose beads. Proteins bound to the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody ( left side of the panel ). ( B ) Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were immunoprecipitated using an anti-kit antibody preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to the one expected for tr-kit is present in all the samples, regardless of tr-kit presence, indicating that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. ( C ) Cell extracts (50 μg) from the same samples shown in B were analyzed in Western blot using an anti-kit antibody. All panels are representative of at least three separate experiments.

    Journal: The Journal of Cell Biology

    Article Title: Involvement of Phospholipase C?1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

    doi:

    Figure Lengend Snippet: Tr-kit does not stably associate with PLCγ1. ( A ) COS cells were transfected with no DNA ( mock ), or 20 μg/dish pCMV5-c-kit ( c-kit ), or 20 μg/dish pCMV5-tr-kit ( tr-kit ). C-kit–transfected cells were incubated for the final 10 min with or without 100 ng/ml SCF. Cell extracts were either analyzed immediately in Western blot (50 μg in each lane) with an anti-kit antibody ( right side of the panel ), or incubated for 2 h with a GST-PLCγ1-SH2SH2SH3 fusion protein linked to glutathione–agarose beads. Proteins bound to the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody ( left side of the panel ). ( B ) Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were immunoprecipitated using an anti-kit antibody preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to the one expected for tr-kit is present in all the samples, regardless of tr-kit presence, indicating that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. ( C ) Cell extracts (50 μg) from the same samples shown in B were analyzed in Western blot using an anti-kit antibody. All panels are representative of at least three separate experiments.

    Article Snippet: Control GST protein, GST-PLCγ1 SH2-SH2, and GST-PLCγ1 SH3 fusion proteins were produced as described by , affinity purified on glutathione Sepharose, extensively dialyzed against PBS, and concentrated to 10 mg/ml using a 10-kD cutoff Centricon ( Millipore Corp. , Bedford, MA).

    Techniques: Stable Transfection, Transfection, Incubation, Western Blot, Expressing, Immunoprecipitation

    Expression of PLCγ1 in MII-arrested mouse eggs, and schematic representation of the bacterial fusion proteins containing different PLCγ1 domains. In the schematic representation of PLCγ1 on the top of this figure, brackets identify the regions of PLCγ1 recognized by the two antibodies used for microinjection experiments shown in Table II . PH , pleckstrin homology domain; Ca 2 + , EF-hand domain (calcium binding motif); X and Y , split catalytic domain; P and H , split pleckstrin homology domain; SH2 and SH3 , src-homology domains; C2 , Ca 2+ -dependent lipid binding domain. ( A ) Western blot from extracts of COS cells transfected with a PLCγ1 expression vector (50 μg), and from extracts of 50 mouse eggs, probed with the anti-PLCγ1bd antibody, described in the Materials and Methods section, and used for the microinjection experiments shown in Table II . ( B ) Bacterial fusion proteins containing different PLCγ1 domains used for the experiments shown in Table I . The Coomassie blue staining of a 10% SDS-PAGE gel loaded with affinity-purified GST fusion proteins is shown on the right: lane 1 , GST; lane 2 , GST-PLCγ1-SH2SH2SH3; lane 3 , GST-PLCγ1-SH2SH2; lane 4 , GST-PLCγ1-SH3.

    Journal: The Journal of Cell Biology

    Article Title: Involvement of Phospholipase C?1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

    doi:

    Figure Lengend Snippet: Expression of PLCγ1 in MII-arrested mouse eggs, and schematic representation of the bacterial fusion proteins containing different PLCγ1 domains. In the schematic representation of PLCγ1 on the top of this figure, brackets identify the regions of PLCγ1 recognized by the two antibodies used for microinjection experiments shown in Table II . PH , pleckstrin homology domain; Ca 2 + , EF-hand domain (calcium binding motif); X and Y , split catalytic domain; P and H , split pleckstrin homology domain; SH2 and SH3 , src-homology domains; C2 , Ca 2+ -dependent lipid binding domain. ( A ) Western blot from extracts of COS cells transfected with a PLCγ1 expression vector (50 μg), and from extracts of 50 mouse eggs, probed with the anti-PLCγ1bd antibody, described in the Materials and Methods section, and used for the microinjection experiments shown in Table II . ( B ) Bacterial fusion proteins containing different PLCγ1 domains used for the experiments shown in Table I . The Coomassie blue staining of a 10% SDS-PAGE gel loaded with affinity-purified GST fusion proteins is shown on the right: lane 1 , GST; lane 2 , GST-PLCγ1-SH2SH2SH3; lane 3 , GST-PLCγ1-SH2SH2; lane 4 , GST-PLCγ1-SH3.

    Article Snippet: Control GST protein, GST-PLCγ1 SH2-SH2, and GST-PLCγ1 SH3 fusion proteins were produced as described by , affinity purified on glutathione Sepharose, extensively dialyzed against PBS, and concentrated to 10 mg/ml using a 10-kD cutoff Centricon ( Millipore Corp. , Bedford, MA).

    Techniques: Expressing, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Staining, SDS Page, Affinity Purification

    The SH3 domain, and not the SH2 domains, of PLCγ1 inhibits tr-kit–induced cortical granules exocytosis and pronuclear formation in mouse eggs. Eggs were co-injected with cell extracts (200 μg/ml) containing recombinant tr-kit and 500 μg/ml (final concentration in the egg: ∼10 μg/ml) of either GST-PLCγ1-SH3 ( A ) or GST-PLCγ1-SH2SH2 ( B ) and fixed 4 h after injection. Tr-kit–induced cortical granule reaction was inhibited by co-injection of GST-PLCγ1-SH3, but not by GST-PLCγ1-SH2SH2, with a similar rate as for pronuclear formation (see Table I ) in three separate experiments. Double staining of chromatin (Hoechst dye) and cortical granules (TRITC-labeled lectin) was performed as described under Materials and Methods. Bar, 30 μm.

    Journal: The Journal of Cell Biology

    Article Title: Involvement of Phospholipase C?1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

    doi:

    Figure Lengend Snippet: The SH3 domain, and not the SH2 domains, of PLCγ1 inhibits tr-kit–induced cortical granules exocytosis and pronuclear formation in mouse eggs. Eggs were co-injected with cell extracts (200 μg/ml) containing recombinant tr-kit and 500 μg/ml (final concentration in the egg: ∼10 μg/ml) of either GST-PLCγ1-SH3 ( A ) or GST-PLCγ1-SH2SH2 ( B ) and fixed 4 h after injection. Tr-kit–induced cortical granule reaction was inhibited by co-injection of GST-PLCγ1-SH3, but not by GST-PLCγ1-SH2SH2, with a similar rate as for pronuclear formation (see Table I ) in three separate experiments. Double staining of chromatin (Hoechst dye) and cortical granules (TRITC-labeled lectin) was performed as described under Materials and Methods. Bar, 30 μm.

    Article Snippet: Control GST protein, GST-PLCγ1 SH2-SH2, and GST-PLCγ1 SH3 fusion proteins were produced as described by , affinity purified on glutathione Sepharose, extensively dialyzed against PBS, and concentrated to 10 mg/ml using a 10-kD cutoff Centricon ( Millipore Corp. , Bedford, MA).

    Techniques: Injection, Recombinant, Concentration Assay, Double Staining, Labeling

    Glutathione-S-transferase (GST) pull-down assays to identify potential FurA targets. Purified FurA-GST fusion protein was incubated with a crude extract from Anabaena. Then, Glutathione (GSH)-Sepharose beads were added to recover FurA-GST with the potential

    Journal: Antioxidants & Redox Signaling

    Article Title: Unraveling the Redox Properties of the Global Regulator FurA from Anabaena sp. PCC 7120: Disulfide Reductase Activity Based on Its CXXC Motifs

    doi: 10.1089/ars.2013.5376

    Figure Lengend Snippet: Glutathione-S-transferase (GST) pull-down assays to identify potential FurA targets. Purified FurA-GST fusion protein was incubated with a crude extract from Anabaena. Then, Glutathione (GSH)-Sepharose beads were added to recover FurA-GST with the potential

    Article Snippet: After washing with buffer A, FurA-GST protein was eluted with 50 m M Tris/HCl pH 8 containing 30 m M reduced GSH (Sigma) and concentrated by centrifugation in 10 kDa Centricon devices (Millipore).

    Techniques: Purification, Incubation

    Analysis of glutathionylation of FurA in vitro . Prereduced FurA was treated or not with H 2 O 2 before being incubated with biotinylated GSH ethyl ester (BioGEE). Western blotting analysis was performed to detect biotinylated GSH, using GST as positive control

    Journal: Antioxidants & Redox Signaling

    Article Title: Unraveling the Redox Properties of the Global Regulator FurA from Anabaena sp. PCC 7120: Disulfide Reductase Activity Based on Its CXXC Motifs

    doi: 10.1089/ars.2013.5376

    Figure Lengend Snippet: Analysis of glutathionylation of FurA in vitro . Prereduced FurA was treated or not with H 2 O 2 before being incubated with biotinylated GSH ethyl ester (BioGEE). Western blotting analysis was performed to detect biotinylated GSH, using GST as positive control

    Article Snippet: After washing with buffer A, FurA-GST protein was eluted with 50 m M Tris/HCl pH 8 containing 30 m M reduced GSH (Sigma) and concentrated by centrifugation in 10 kDa Centricon devices (Millipore).

    Techniques: In Vitro, Incubation, Western Blot, Positive Control

    CEACAM1-4L is a direct substrate of EGFR. ( A ) After treatment of MDA-MB468 cells with EGF (100 nM) (+) or buffer alone (_) for 5 minutes, cells were lysed and EGFR was extracted by immunoprecipitation with α-EGFR. GST fusion to intracellular peptides of wild type (WT CC1; lanes 5 and 6) or Y488F mutant (Y488F CC1; lanes 3 and 4) CEACAM1-4L (GST-CC1 int ) were incubated with equal amounts of EGFR immunoprecipitates in the presence of [γ- 32 P]ATP for 10 minutes prior to the addition of SDS buffer and analysis by 6_12% SDS-PAGE and autoradiography for the detection of phosphorylated proteins. Peptide-free GST was included as control to account for nonspecific association (Alone; lanes 1 and 2). ( B ) The amount of GST peptides from the experiment in A , measured by Ponceau S staining. The results obtained were consistent in at least three experiments. The two gels in A are from the same SDS-PAGE gel and differ in the exposure time of the autoradiogram for optimal visualization of both EGFR and GST bands.

    Journal: Journal of Clinical Investigation

    Article Title: CEACAM1 modulates epidermal growth factor receptor-mediated cell proliferation

    doi: 10.1172/JCI200421786

    Figure Lengend Snippet: CEACAM1-4L is a direct substrate of EGFR. ( A ) After treatment of MDA-MB468 cells with EGF (100 nM) (+) or buffer alone (_) for 5 minutes, cells were lysed and EGFR was extracted by immunoprecipitation with α-EGFR. GST fusion to intracellular peptides of wild type (WT CC1; lanes 5 and 6) or Y488F mutant (Y488F CC1; lanes 3 and 4) CEACAM1-4L (GST-CC1 int ) were incubated with equal amounts of EGFR immunoprecipitates in the presence of [γ- 32 P]ATP for 10 minutes prior to the addition of SDS buffer and analysis by 6_12% SDS-PAGE and autoradiography for the detection of phosphorylated proteins. Peptide-free GST was included as control to account for nonspecific association (Alone; lanes 1 and 2). ( B ) The amount of GST peptides from the experiment in A , measured by Ponceau S staining. The results obtained were consistent in at least three experiments. The two gels in A are from the same SDS-PAGE gel and differ in the exposure time of the autoradiogram for optimal visualization of both EGFR and GST bands.

    Article Snippet: GST fusion peptides (10 μg) were phosphorylated by preactivated EGFR protein tyrosine kinase (0.25 U; Calbiochem) (with 50 μM ATP, 75 mM MgCl2 , 50 mM MnCl2 , and 0.5 mg/ml BSA) in the presence of 70 μCi [γ-32 P]ATP and were allowed to bind (60 minutes at 4°C) prior to being washed in HNTG buffer (150 mM HEPES, pH 7.6, 50 mM NaCl, and 10% glycerol) contai ning 1% Triton X-100 and analyzed by electrophoresis ( ).

    Techniques: Multiple Displacement Amplification, Immunoprecipitation, Mutagenesis, Incubation, SDS Page, Autoradiography, Staining

    CEACAM1-4L does not directly interact with EGFR. ( A ) Sepharose-coupled GST fusion to intracellular peptides of WT (lanes 3 and 4) or mutant (lanes 5_12 and 15_18) CEACAM1-4L were incubated with (+) or without (_) purified EGFR in the presence of [γ- 32 P]ATP. Peptide-free GST was included as control to account for nonspecific association (lanes 1, 2, 13, and 14). After protein binding, the Sepharose pellets were analyzed by 6_12% SDS-PAGE and autoradiography. ( B ) Proteins in A were immunoblotted with α-EGFR for examination of the association of EGFR with CEACAM1-4L. ( C ) The amount of GST peptides from the experiment in A was measured by Ponceau S staining. The results obtained were consistent in at least three experiments.

    Journal: Journal of Clinical Investigation

    Article Title: CEACAM1 modulates epidermal growth factor receptor-mediated cell proliferation

    doi: 10.1172/JCI200421786

    Figure Lengend Snippet: CEACAM1-4L does not directly interact with EGFR. ( A ) Sepharose-coupled GST fusion to intracellular peptides of WT (lanes 3 and 4) or mutant (lanes 5_12 and 15_18) CEACAM1-4L were incubated with (+) or without (_) purified EGFR in the presence of [γ- 32 P]ATP. Peptide-free GST was included as control to account for nonspecific association (lanes 1, 2, 13, and 14). After protein binding, the Sepharose pellets were analyzed by 6_12% SDS-PAGE and autoradiography. ( B ) Proteins in A were immunoblotted with α-EGFR for examination of the association of EGFR with CEACAM1-4L. ( C ) The amount of GST peptides from the experiment in A was measured by Ponceau S staining. The results obtained were consistent in at least three experiments.

    Article Snippet: GST fusion peptides (10 μg) were phosphorylated by preactivated EGFR protein tyrosine kinase (0.25 U; Calbiochem) (with 50 μM ATP, 75 mM MgCl2 , 50 mM MnCl2 , and 0.5 mg/ml BSA) in the presence of 70 μCi [γ-32 P]ATP and were allowed to bind (60 minutes at 4°C) prior to being washed in HNTG buffer (150 mM HEPES, pH 7.6, 50 mM NaCl, and 10% glycerol) contai ning 1% Triton X-100 and analyzed by electrophoresis ( ).

    Techniques: Mutagenesis, Incubation, Purification, Protein Binding, SDS Page, Autoradiography, Staining

    Binding of α 1B , α 1A(rbA), and α 1A(BI) to SNARE proteins. GST-fusion proteins containing synaptotagmin, syntaxin, SNAP-25, or GST alone (50 pmol) were incubated with glutathione-Sepharose beads. After 60 min, the beads were washed three times with Tris-saline buffer (100 mM Tris⋅HCl/140 mM NaCl/0.1% Triton X-100, pH 8). Purified His-tagged Ca 2+ channel fusion proteins from α 1B (50 pmol), α 1A(rbA) (1.3 nmol), or α 1A(BI) (300 pmol) were added to the beads as indicated in the figure. The mixture was then incubated at 4°C for 3 h on a rotating mixer. The Tris-saline buffer containing 15 μM Ca 2+ was used for the binding and for the washes. The fusion proteins were then eluted with 20 μl elution buffer (15 mM reduced-glutathione in Tris-saline buffer, pH 8). The eluate was boiled in Tricine sample buffer for 2 min, and the proteins were separated on 10–20% Tricine–SDS/PAGE gradient gels and electrophoretically transferred overnight to nitrocellulose. Separated bands were immunoblotted with T7 monoclonal antibody and visualized with the ECL system (Amersham). Concentrations of GST fusion protein in each condition were visualized by staining with Ponceau S. A positive control indicates the level of staining with the T7 mAb for 2.5 pmol of α 1B , α 1A(rbA) , or α 1A(BI) added directly to the gel (control). The appearance of multiple bands in this and subsequent figures (e.g., in α 1A(BI) samples) is because of minor proteolytic cleavage products of the fusion proteins that retain binding activity.

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

    Article Title: Ca2+-dependent and -independent interactions of the isoforms of the ?1A subunit of brain Ca2+ channels with presynaptic SNARE proteins

    doi:

    Figure Lengend Snippet: Binding of α 1B , α 1A(rbA), and α 1A(BI) to SNARE proteins. GST-fusion proteins containing synaptotagmin, syntaxin, SNAP-25, or GST alone (50 pmol) were incubated with glutathione-Sepharose beads. After 60 min, the beads were washed three times with Tris-saline buffer (100 mM Tris⋅HCl/140 mM NaCl/0.1% Triton X-100, pH 8). Purified His-tagged Ca 2+ channel fusion proteins from α 1B (50 pmol), α 1A(rbA) (1.3 nmol), or α 1A(BI) (300 pmol) were added to the beads as indicated in the figure. The mixture was then incubated at 4°C for 3 h on a rotating mixer. The Tris-saline buffer containing 15 μM Ca 2+ was used for the binding and for the washes. The fusion proteins were then eluted with 20 μl elution buffer (15 mM reduced-glutathione in Tris-saline buffer, pH 8). The eluate was boiled in Tricine sample buffer for 2 min, and the proteins were separated on 10–20% Tricine–SDS/PAGE gradient gels and electrophoretically transferred overnight to nitrocellulose. Separated bands were immunoblotted with T7 monoclonal antibody and visualized with the ECL system (Amersham). Concentrations of GST fusion protein in each condition were visualized by staining with Ponceau S. A positive control indicates the level of staining with the T7 mAb for 2.5 pmol of α 1B , α 1A(rbA) , or α 1A(BI) added directly to the gel (control). The appearance of multiple bands in this and subsequent figures (e.g., in α 1A(BI) samples) is because of minor proteolytic cleavage products of the fusion proteins that retain binding activity.

    Article Snippet: Concentrations of GST-tagged fusion protein immobilized to Sepharose beads were quantitated either by staining with Ponceau S (Sigma) or by immunoblotting with anti-GST antisera (Pharmacia LKB) after stripping of the nitrocellulose membrane ( ).

    Techniques: Binding Assay, Incubation, Purification, SDS Page, Staining, Positive Control, Activity Assay

    Protein A does not cause translocation arrest in the absence of GST as a spacer. Differing amounts (2 and 4 μl) of PRS of a reticulocyte lysate translation mixture containing newly synthesized [ 35 S]methionine-labeled pPPrA were incubated with isolated yeast mitochondria. Following import, mitochondrial pellets were resuspended in buffer and either left untreated (lanes 2 and 3) or treated for 30 min on ice with trypsin (lanes 4 and 5). Samples were analyzed by SDS/PAGE followed by autoradiography. Lane 1 shows 0.4 μl of PRS in the absence of mitochondria. Lane 6 shows the migration position of mPut.

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

    Article Title: In vivo zippering of inner and outer mitochondrial membranes by a stable translocation intermediate

    doi:

    Figure Lengend Snippet: Protein A does not cause translocation arrest in the absence of GST as a spacer. Differing amounts (2 and 4 μl) of PRS of a reticulocyte lysate translation mixture containing newly synthesized [ 35 S]methionine-labeled pPPrA were incubated with isolated yeast mitochondria. Following import, mitochondrial pellets were resuspended in buffer and either left untreated (lanes 2 and 3) or treated for 30 min on ice with trypsin (lanes 4 and 5). Samples were analyzed by SDS/PAGE followed by autoradiography. Lane 1 shows 0.4 μl of PRS in the absence of mitochondria. Lane 6 shows the migration position of mPut.

    Article Snippet: The Kpn I– Bam HI fragment of the resulting plasmid, pBluescript/GST-protein A, was then subcloned into the same sites of pNYHM170 [pPut in pET3a (Novagen; ref. )] to obtain the plasmid pET3a/pPut-GST-protein A.

    Techniques: Translocation Assay, Synthesized, Labeling, Incubation, Isolation, SDS Page, Autoradiography, Migration

    RelB and RelE proteins interact directly in vitro and in vivo. For Western (A and B) and far-Western (C) analyses, the following samples were subjected to SDS-15% PAGE gel and transferred to PVDF membranes. Lane 1, purified RelB::GST; lane 2,

    Journal:

    Article Title: Three Mycobacterium tuberculosis Rel Toxin-Antitoxin Modules Inhibit Mycobacterial Growth and Are Expressed in Infected Human Macrophages ▿

    doi: 10.1128/JB.01318-08

    Figure Lengend Snippet: RelB and RelE proteins interact directly in vitro and in vivo. For Western (A and B) and far-Western (C) analyses, the following samples were subjected to SDS-15% PAGE gel and transferred to PVDF membranes. Lane 1, purified RelB::GST; lane 2,

    Article Snippet: Purified RelB-GST or RelE-GST protein (140 μg) was emulsified in TiterMax Gold (Sigma-Aldrich, St. Louis, MO) and used to immunize two New Zealand White rabbits by subcutaneous injections.

    Techniques: In Vitro, In Vivo, Western Blot, Polyacrylamide Gel Electrophoresis, Purification

    Effects of kinase inhibitors on Claspin and Chk1 phosphorylation The extract was incubated with radiolabelled Claspin 679–1332 , radiolabelled GST-CKBD, OA+poly(dA/dT) 70 and the following additions: staurosporine (staur), UCN01, roscovitine (rosco) and caffeine (upper panels); Gö6976, Ro318220, Bim1, SB203580, rapamycin (Rapa), PD98059 and H89 (lower panels). Proteins were detected by immunoblotting with antibodies raised against Chk1 (anti-Chk1) or specific phosphorylation sites on Chk1 (α-p345, α-p317 and α-p296). Claspin 679–1332 and GST–CKBD proteins were detected by autoradiography.

    Journal: Biochemical Journal

    Article Title: DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system

    doi: 10.1042/BJ20041966

    Figure Lengend Snippet: Effects of kinase inhibitors on Claspin and Chk1 phosphorylation The extract was incubated with radiolabelled Claspin 679–1332 , radiolabelled GST-CKBD, OA+poly(dA/dT) 70 and the following additions: staurosporine (staur), UCN01, roscovitine (rosco) and caffeine (upper panels); Gö6976, Ro318220, Bim1, SB203580, rapamycin (Rapa), PD98059 and H89 (lower panels). Proteins were detected by immunoblotting with antibodies raised against Chk1 (anti-Chk1) or specific phosphorylation sites on Chk1 (α-p345, α-p317 and α-p296). Claspin 679–1332 and GST–CKBD proteins were detected by autoradiography.

    Article Snippet: Antibodies against human Chk1 used for immunoprecipitation were raised in sheep inoculated with GST (glutathione S-transferase)–Chk1 [ ] and purified against the protein coupled with Reactigel beads (Bio-Rad Laboratories, Hemel Hempstead, Herts., U.K.).

    Techniques: Incubation, Autoradiography

    Plasma α-GST levels 4 h after reperfusion and 90 min of ischemia, nt: non-treated group; IP: ischemic preconditioning; IPsel: selective ischemic preconditioning, LA: lipoic acid. a P

    Journal:

    Article Title: Protective effects of ischemic preconditioning and application of lipoic acid prior to 90 min of hepatic ischemia in a rat model

    doi: 10.3748/wjg.v13.i27.3692

    Figure Lengend Snippet: Plasma α-GST levels 4 h after reperfusion and 90 min of ischemia, nt: non-treated group; IP: ischemic preconditioning; IPsel: selective ischemic preconditioning, LA: lipoic acid. a P

    Article Snippet: α-GST in plasma 4 h after the onset of reperfusion was measured by absorption using a microplate reader (Bio Rad).

    Techniques:

    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

    Fine Mapping of H2AX/CARP-1 interacting epitopes. The Gst-tagged H2AX protein, and various Gst-tagged H2AX peptides and His-TAT-HA-tagged CARP-1 peptides were purified following expression in E. coli BL-21 cells essentially as described in Section 2 . ( A ) Gst-H2AX protein was immobilized on glutathione sepharose followed by incubation indicated CARP-1 peptides. Following stringent washing, the bound proteins were analyzed with WB using anti-HA (upper) or anti-Gst (middle) antibodies. The lower blot shows respective HA-tagged CARP-1 peptides used as input. ( B ) Indicated Gst-tagged H2AX peptides were firstly immobilized on glutathione sepharose followed by incubation with His-TAT-HA-CARP-1 (631–660) peptide. Stringent washing and WB analyses were performed as in panel A. ( C,D ) Approximately 1 mg of cell lysate from each of the indicated cells stably expressing EGFP, EGFP-CARP-1 (636–650), or EGFP-H2AX (1–35) was subjected to immunoprecipitation using anti-EGFP antibodies. The immunoprecipitates were then analyzed with WB by probing the membrane with anti-EGFP, anti-H2AX, or anti-CARP-1 (α2) antibodies as noted in the upper boxes in ( C , D ). The whole-cell lysates were separately analyzed with WB to determine endogenous H2AX and CARP-1 levels in the respective cell line (lower boxes in ( C , D )). The presence of respective proteins is indicated by an arrowhead on the left side of each blot. The approximate location of various molecular-weight markers is indicated on the right side of each blot; kDa, kilodalton.

    Journal: Cancers

    Article Title: A H2AX–CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage

    doi: 10.3390/cancers11020221

    Figure Lengend Snippet: Fine Mapping of H2AX/CARP-1 interacting epitopes. The Gst-tagged H2AX protein, and various Gst-tagged H2AX peptides and His-TAT-HA-tagged CARP-1 peptides were purified following expression in E. coli BL-21 cells essentially as described in Section 2 . ( A ) Gst-H2AX protein was immobilized on glutathione sepharose followed by incubation indicated CARP-1 peptides. Following stringent washing, the bound proteins were analyzed with WB using anti-HA (upper) or anti-Gst (middle) antibodies. The lower blot shows respective HA-tagged CARP-1 peptides used as input. ( B ) Indicated Gst-tagged H2AX peptides were firstly immobilized on glutathione sepharose followed by incubation with His-TAT-HA-CARP-1 (631–660) peptide. Stringent washing and WB analyses were performed as in panel A. ( C,D ) Approximately 1 mg of cell lysate from each of the indicated cells stably expressing EGFP, EGFP-CARP-1 (636–650), or EGFP-H2AX (1–35) was subjected to immunoprecipitation using anti-EGFP antibodies. The immunoprecipitates were then analyzed with WB by probing the membrane with anti-EGFP, anti-H2AX, or anti-CARP-1 (α2) antibodies as noted in the upper boxes in ( C , D ). The whole-cell lysates were separately analyzed with WB to determine endogenous H2AX and CARP-1 levels in the respective cell line (lower boxes in ( C , D )). The presence of respective proteins is indicated by an arrowhead on the left side of each blot. The approximate location of various molecular-weight markers is indicated on the right side of each blot; kDa, kilodalton.

    Article Snippet: Antibodies for Gst-tag, enhanced GFP (EGFP), Myc-tag, phospho, and total H2AX were purchased from Cell Signaling (Beverley, MA, USA).

    Techniques: Purification, Expressing, Incubation, Western Blot, Stable Transfection, Immunoprecipitation, Molecular Weight

    MLIP specifically binds lamin A/C. A , purified recombinant MLIP binds directly within the first 230 amino acids of recombinant lamins A and C in an in vitro immunoprecipitation assay. Purified recombinant His 6 -MLIP and GST-lamin A were mixed together (as indicated) with complexes precipitated through the His tag. Western blot analysis was performed using anti-GST (Cell Signaling) and anti-MLIP polyclonal antibodies. A 1:10 dilution of the total starting material was run on the same gel ( left panels ). The assay was repeated two additional times with similar results. B , MLIP peptide neutralization of the anti-MLIP serum demonstrates the specificity of the MLIP antibodies in Western blot analysis. C , MLIP co-immunoprecipitates with lamin A/C from adult mouse hearts. Total mouse heart lysates were divided 45%:45%:10% with 45% incubated with either anti-MLIP serum or MLIP peptide neutralized anti-MLIP serum ( Control ). The input control for the immunoprecipitation represents ∼2% of total heart lysate.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of a Novel Muscle A-type Lamin-interacting Protein (MLIP) *

    doi: 10.1074/jbc.M110.165548

    Figure Lengend Snippet: MLIP specifically binds lamin A/C. A , purified recombinant MLIP binds directly within the first 230 amino acids of recombinant lamins A and C in an in vitro immunoprecipitation assay. Purified recombinant His 6 -MLIP and GST-lamin A were mixed together (as indicated) with complexes precipitated through the His tag. Western blot analysis was performed using anti-GST (Cell Signaling) and anti-MLIP polyclonal antibodies. A 1:10 dilution of the total starting material was run on the same gel ( left panels ). The assay was repeated two additional times with similar results. B , MLIP peptide neutralization of the anti-MLIP serum demonstrates the specificity of the MLIP antibodies in Western blot analysis. C , MLIP co-immunoprecipitates with lamin A/C from adult mouse hearts. Total mouse heart lysates were divided 45%:45%:10% with 45% incubated with either anti-MLIP serum or MLIP peptide neutralized anti-MLIP serum ( Control ). The input control for the immunoprecipitation represents ∼2% of total heart lysate.

    Article Snippet: Western blot analysis was performed using anti-GST (Cell Signaling) and anti-MLIP polyclonal antibodies.

    Techniques: Purification, Recombinant, In Vitro, Immunoprecipitation, Western Blot, Neutralization, Incubation

    IFT25 interacts physically with IFT27. Purified GST-tagged IFT27 and MBP-tagged IFT25 were used for in vitro binding assay. The left panel shows that immobilized GST-IFT27 protein on the beads could retain MBP-IFT25 protein but not the control protein, MBP. The right panel shows that immobilized MBP-IFT25 protein on beads could retain GST-IFT27 protein but not the control protein, GST. The molecular marker is labeled on the left of each figure. From top to bottom for both panels, the first figure is the Coomassie blue-stained gel. The second and third figures represent the immunoblots probed with antibodies against IFT25 and IFT27, respectively. The fourth one is the immunoblots probed with antibodies against either MBP (left panel) or GST (right panel). The loading materials for each lane of the gels are shown in the tables at the top of each panel. S stands for supernatant and P for bead pellet.

    Journal: PLoS ONE

    Article Title: Intraflagellar Transport (IFT) Protein IFT25 Is a Phosphoprotein Component of IFT Complex B and Physically Interacts with IFT27 in Chlamydomonas

    doi: 10.1371/journal.pone.0005384

    Figure Lengend Snippet: IFT25 interacts physically with IFT27. Purified GST-tagged IFT27 and MBP-tagged IFT25 were used for in vitro binding assay. The left panel shows that immobilized GST-IFT27 protein on the beads could retain MBP-IFT25 protein but not the control protein, MBP. The right panel shows that immobilized MBP-IFT25 protein on beads could retain GST-IFT27 protein but not the control protein, GST. The molecular marker is labeled on the left of each figure. From top to bottom for both panels, the first figure is the Coomassie blue-stained gel. The second and third figures represent the immunoblots probed with antibodies against IFT25 and IFT27, respectively. The fourth one is the immunoblots probed with antibodies against either MBP (left panel) or GST (right panel). The loading materials for each lane of the gels are shown in the tables at the top of each panel. S stands for supernatant and P for bead pellet.

    Article Snippet: Other antibodies used in this study include antibodies against α-tubulin (clone B-5-1-2, ascites fluid; Sigma); IC69 (clone 1869A; Sigma); acetylated tubulin (clone 6-11B-1, ascites fluid; sigma); MBP (New England biolabs), and GST (clone 9AT106, ABGENT).

    Techniques: Purification, In Vitro, Binding Assay, Marker, Labeling, Staining, Western Blot

    Scd6 does not destabilize Pab1-4G or 4A-4G interactions A) 7-methyl-GTP sepharose pull downs were performed to look at interaction of GST-eIF4G/eIF4E complex with recombinant Pab1 in presence of Scd6 or Scd6ΔRGG protein as described in materials and methods. 8ug of GST-eIF4G/eIF4E was incubated with wt or mutant Scd6 protein for 1h at 4°C with end-to-end rotation. Following this Pab1 was added to reaction mixture and incubated for 1h with end-to-end rotation. Finally, 7-methyl-GTP sepharose was added to reaction mix and incubated for 2h. Washing was performed as described in materials and methods. eIF4G and Pab1 were detected with polyclonal antibodies. B) Glutathione pull down assay to analyze effect of purified Scd6 on eIF4A-eIF4E/G interaction. Incubation and washing conditions were same as mentioned in A. eIF4G and eIF4A were detected with polyclonal antibodies. The anti-eIF4A antibody cross-reacts with purified Scd6 (contains both His and FLAG-tag). The middle panel depicts ponceau-stained blot with clearly visible wt and mutant Scd6 proteins. This was done since eIF4A and Scd6ΔRGG run at similar position (in top panel). Typically 10% of total reaction was loaded in the ‘input’ lanes and 50 or 100% of total pulled-down material was loaded in ‘pellet’ lanes.

    Journal: Molecular cell

    Article Title: Scd6 targets eIF4G to repress translation: RGG-motif proteins as a class of eIF4G-binding proteins

    doi: 10.1016/j.molcel.2011.11.026

    Figure Lengend Snippet: Scd6 does not destabilize Pab1-4G or 4A-4G interactions A) 7-methyl-GTP sepharose pull downs were performed to look at interaction of GST-eIF4G/eIF4E complex with recombinant Pab1 in presence of Scd6 or Scd6ΔRGG protein as described in materials and methods. 8ug of GST-eIF4G/eIF4E was incubated with wt or mutant Scd6 protein for 1h at 4°C with end-to-end rotation. Following this Pab1 was added to reaction mixture and incubated for 1h with end-to-end rotation. Finally, 7-methyl-GTP sepharose was added to reaction mix and incubated for 2h. Washing was performed as described in materials and methods. eIF4G and Pab1 were detected with polyclonal antibodies. B) Glutathione pull down assay to analyze effect of purified Scd6 on eIF4A-eIF4E/G interaction. Incubation and washing conditions were same as mentioned in A. eIF4G and eIF4A were detected with polyclonal antibodies. The anti-eIF4A antibody cross-reacts with purified Scd6 (contains both His and FLAG-tag). The middle panel depicts ponceau-stained blot with clearly visible wt and mutant Scd6 proteins. This was done since eIF4A and Scd6ΔRGG run at similar position (in top panel). Typically 10% of total reaction was loaded in the ‘input’ lanes and 50 or 100% of total pulled-down material was loaded in ‘pellet’ lanes.

    Article Snippet: Western analysis was performed using anti-GST (Abgent), anti-Flag M2 (Sigma), anti-His (Abcam), anti-4G and anti-4E (John McCarthy), anti-Pab1 (Alan Jacobson) antibodies or a polyclonal anti-Scd6 raised in rabbits to the purified protein (Cocalico Biologicals).

    Techniques: Recombinant, Incubation, Mutagenesis, Pull Down Assay, Purification, FLAG-tag, Staining

    Scd6 binds eIF4G ). D) Glutathione sepharose pull downs performed to look at interaction of purified GST-4G/E with recombinant Scd6 and Scd6ΔRGG mutant protein. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. E) Glutathione sepharose pull downs were performed to look at interaction of GST-4G in bacterial extracts with recombinant Scd6 and Scd6ΔRGG mutant protein. 6ug of each protein was used in 200ul reaction mixture. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. Typically 10% of total reaction was loaded in the ‘input’ lanes and 100% of total pulled-down material was loaded in ‘pellet’ lanes.

    Journal: Molecular cell

    Article Title: Scd6 targets eIF4G to repress translation: RGG-motif proteins as a class of eIF4G-binding proteins

    doi: 10.1016/j.molcel.2011.11.026

    Figure Lengend Snippet: Scd6 binds eIF4G ). D) Glutathione sepharose pull downs performed to look at interaction of purified GST-4G/E with recombinant Scd6 and Scd6ΔRGG mutant protein. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. E) Glutathione sepharose pull downs were performed to look at interaction of GST-4G in bacterial extracts with recombinant Scd6 and Scd6ΔRGG mutant protein. 6ug of each protein was used in 200ul reaction mixture. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. Typically 10% of total reaction was loaded in the ‘input’ lanes and 100% of total pulled-down material was loaded in ‘pellet’ lanes.

    Article Snippet: Western analysis was performed using anti-GST (Abgent), anti-Flag M2 (Sigma), anti-His (Abcam), anti-4G and anti-4E (John McCarthy), anti-Pab1 (Alan Jacobson) antibodies or a polyclonal anti-Scd6 raised in rabbits to the purified protein (Cocalico Biologicals).

    Techniques: Purification, Recombinant, Mutagenesis

    IRGMd binds to cardiolipin and causes loss of mitochondrial membrane potential a . Splice variants of IRGM. G1-G5, GTPase motifs. The S47N mutation is indicated in blue. b. HeLa cells were transfected with IRGMb, IRGMd-WT (wild type IRGMd, unaltered) or IRGM-S47N mutant (SN) for 48 h, labeled with MTR and imaged by live microscopy. c. Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative. d . Quantification of GFP + MTR + cells. e,f . Analysis of GST-IRGMd binding to lipids by lipid protein binding dot blots (e) and cardiolipin bound agarose bead pull down assay (f; details in Methods). g. IRGMd, IRGMdS47 mutant, and IRGMb isoform concentration-dependent analysis of binding to lipids on strip blots (numbers identifying lipids correspond to the legend in panel e). GST control is in Suppl. Fig. S1d . Membranes in e-g were probed with anti-GST antibody.

    Journal: Nature cell biology

    Article Title: Human IRGM Regulates Autophagy and Its Cell-Autonomous Immunity Functions Through Mitochondria

    doi: 10.1038/ncb2119

    Figure Lengend Snippet: IRGMd binds to cardiolipin and causes loss of mitochondrial membrane potential a . Splice variants of IRGM. G1-G5, GTPase motifs. The S47N mutation is indicated in blue. b. HeLa cells were transfected with IRGMb, IRGMd-WT (wild type IRGMd, unaltered) or IRGM-S47N mutant (SN) for 48 h, labeled with MTR and imaged by live microscopy. c. Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative. d . Quantification of GFP + MTR + cells. e,f . Analysis of GST-IRGMd binding to lipids by lipid protein binding dot blots (e) and cardiolipin bound agarose bead pull down assay (f; details in Methods). g. IRGMd, IRGMdS47 mutant, and IRGMb isoform concentration-dependent analysis of binding to lipids on strip blots (numbers identifying lipids correspond to the legend in panel e). GST control is in Suppl. Fig. S1d . Membranes in e-g were probed with anti-GST antibody.

    Article Snippet: Filters were incubated with 90 nM of GST (Abcam) or GST-IRGM for 1 h in blocking buffer, followed by three washes with PBS, 0.1% Tween-20.

    Techniques: Mutagenesis, Transfection, Labeling, Microscopy, Fluorescence, Binding Assay, Protein Binding, Pull Down Assay, Concentration Assay, Stripping Membranes

    TMEM74 associates with ATG16L1 via its C-terminal and influences the interaction between ATG5 and ATG16L1. ( a ) Schematic representations of WT ATG16L1 and its mutants: ATG16L1(1–320), and ATG16L1 △(1–320) . ( b ) HeLa cells were co-transfected with GFP-ATG16L1 and mCherry-TMEM74 for 24 h, Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( c ) HeLa cells were co-transfected with GFP-TMEM74 and mCherry-ATG16L1 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, ATG16L1 was detected in the washed beads using an anti-ATG16L1 IgG by western blotting. ( d ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG16L1, GFP-ATG16L1 was detected in the washed beads by western blotting. ( e , f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG16L1(1–320), or GFP-ATG16L1 △(1–320) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 were detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG16L1 for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, ATG5-ATG12 complex pulled down was detected in the immunoprecipitates using anti-ATG5 by western blotting. Quantification of ATG5-ATG12 pulled down relative to GFP-ATG16L1 was shown as column. Data are means±S.D. of three experiments. * P

    Journal: Cell Death & Disease

    Article Title: TMEM74 promotes tumor cell survival by inducing autophagy via interactions with ATG16L1 and ATG9A

    doi: 10.1038/cddis.2017.370

    Figure Lengend Snippet: TMEM74 associates with ATG16L1 via its C-terminal and influences the interaction between ATG5 and ATG16L1. ( a ) Schematic representations of WT ATG16L1 and its mutants: ATG16L1(1–320), and ATG16L1 △(1–320) . ( b ) HeLa cells were co-transfected with GFP-ATG16L1 and mCherry-TMEM74 for 24 h, Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( c ) HeLa cells were co-transfected with GFP-TMEM74 and mCherry-ATG16L1 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, ATG16L1 was detected in the washed beads using an anti-ATG16L1 IgG by western blotting. ( d ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG16L1, GFP-ATG16L1 was detected in the washed beads by western blotting. ( e , f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG16L1(1–320), or GFP-ATG16L1 △(1–320) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 were detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG16L1 for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, ATG5-ATG12 complex pulled down was detected in the immunoprecipitates using anti-ATG5 by western blotting. Quantification of ATG5-ATG12 pulled down relative to GFP-ATG16L1 was shown as column. Data are means±S.D. of three experiments. * P

    Article Snippet: Antibodies and reagents Polyclonal antibodies against TMEM74 produced and purified by our lab; anti-caspase-3 (CST, Boston, USA, 9665S); anti-Actin (Tianjin Sungene Biotech, Tianjin, China, KM9001); anti-Flag (Tianjin Sungene Biotech, KM8002); anti-Myc (Tianjin Sungene Biotech, KM8003); Anti-GST (MultiSciences, Hangzhou, China, ab004-040); anti-GFP (Abcam, Cambridge, UK, ab1218), anti-GFP (Sigma Aldrich, Darmstadt, German, SAB4301138); Anti-GAPDH (Tianjin Sungene LK9002); anti-ATG5 (CST, 4445); anti-ATG7 (CST,4445); anti-ATG16L1 (CST,4445); anti-ATG3 (CST,4445); anti-Beclin1 (CST,4445); anti-ULK1 (Abcam, ab120243); anti-Apg10 (Abcam, ab124711); anti-PI3KC3 (Abcam, ab124905); anti-p62 (CST, 5114); anti-p-AKT (CST,4051S); anti-AKT (CST, 4691S); anti-p-PI3K(CST, 4228P); anti-PI3K p55 (CST, 11889S); anti-p-p70S6K (CST,9234S); anti-p70S6K (CST, 2708S); anti-p-mTOR (CST, 2971S); anti-mTOR (CST,2983S); anti-p-p38MAPK (CST, 4511S); anti-p38MAPK (CST, 8690S); anti-p-AMPKα (CST, 2531S); anti-AMPKα (CST, 2532S); anti-p-GSK3β (CST, 9323S); anti-GSK3β (CST, 12456S); anti-p-eIF2α (CST, 3398P); anti-eIF2α (CST, 5324S); anti-WIPI1 (CST, 12124S); anti-LC3B (Sigma Aldrich, L7543); HRP-conjugated goat anti-rabbit secondary antibody (Biodragon, Beijing, China, BF03008); HRP-conjugated goat anti-mouse secondary antibody (Biodragon, BF03001); Bafilomycin.A1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-201550A); Rabbit control IgG (Biodragon, BF01001); Mouse control IgG (Biodragon, BF01116); BL21 competent cells (Solarbio, Beijing, China, C1400-10); CCK-8 (DOJINDO, Shanghai, China, CK04); AnnexinV-PI apoptosis detection kit (DOJINDO, AD10); LY294002 (Abcam, ab120243); ATP detection kit (Beyotime Biotechnology, Nanjing, China, S0026); MG-132 (Abcam, ab146600); CQ (Sigma Aldrich, C6628); Rapamycin (Beyotime Biotechnology, S1842); No-glucose DMEM medium (BasalMedia (Yuanpei Biotech), Shanghai, China, L160); EBSS (Sigma Aldrich, E7510); Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA, L3000015).

    Techniques: Transfection, Western Blot, Incubation

    TMEM74 associates with ATG9A via its N-terminal and influences the interaction between ATG9 and WIPI1. ( a ) HeLa cells were co-transfected with GFP-ATG9A and mCherry-TMEM74 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( b ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG9A, GFP-ATG9A was detected in the washed beads using an anti-GFP IgG by western blotting. ( c ) Schematic representations of WT-ATG9A and its mutants: ATG9A(1–495), and ATG9A △(1–495) , and ATG9A △(153–289) . ( d – f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG9A(1–495), GFP-ATG9A △(1–495) , or GFP-ATG9A △(153–289) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 was detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG9A for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, WIPI1 pulled down was detected in the immunoprecipitates using the anti-WIPI1 antibody by western blotting. Quantification of WIPI1 pulled down relative to GFP-ATG9A was shown as column. Data are means±S.D. of three experiments. * P

    Journal: Cell Death & Disease

    Article Title: TMEM74 promotes tumor cell survival by inducing autophagy via interactions with ATG16L1 and ATG9A

    doi: 10.1038/cddis.2017.370

    Figure Lengend Snippet: TMEM74 associates with ATG9A via its N-terminal and influences the interaction between ATG9 and WIPI1. ( a ) HeLa cells were co-transfected with GFP-ATG9A and mCherry-TMEM74 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( b ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG9A, GFP-ATG9A was detected in the washed beads using an anti-GFP IgG by western blotting. ( c ) Schematic representations of WT-ATG9A and its mutants: ATG9A(1–495), and ATG9A △(1–495) , and ATG9A △(153–289) . ( d – f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG9A(1–495), GFP-ATG9A △(1–495) , or GFP-ATG9A △(153–289) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 was detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG9A for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, WIPI1 pulled down was detected in the immunoprecipitates using the anti-WIPI1 antibody by western blotting. Quantification of WIPI1 pulled down relative to GFP-ATG9A was shown as column. Data are means±S.D. of three experiments. * P

    Article Snippet: Antibodies and reagents Polyclonal antibodies against TMEM74 produced and purified by our lab; anti-caspase-3 (CST, Boston, USA, 9665S); anti-Actin (Tianjin Sungene Biotech, Tianjin, China, KM9001); anti-Flag (Tianjin Sungene Biotech, KM8002); anti-Myc (Tianjin Sungene Biotech, KM8003); Anti-GST (MultiSciences, Hangzhou, China, ab004-040); anti-GFP (Abcam, Cambridge, UK, ab1218), anti-GFP (Sigma Aldrich, Darmstadt, German, SAB4301138); Anti-GAPDH (Tianjin Sungene LK9002); anti-ATG5 (CST, 4445); anti-ATG7 (CST,4445); anti-ATG16L1 (CST,4445); anti-ATG3 (CST,4445); anti-Beclin1 (CST,4445); anti-ULK1 (Abcam, ab120243); anti-Apg10 (Abcam, ab124711); anti-PI3KC3 (Abcam, ab124905); anti-p62 (CST, 5114); anti-p-AKT (CST,4051S); anti-AKT (CST, 4691S); anti-p-PI3K(CST, 4228P); anti-PI3K p55 (CST, 11889S); anti-p-p70S6K (CST,9234S); anti-p70S6K (CST, 2708S); anti-p-mTOR (CST, 2971S); anti-mTOR (CST,2983S); anti-p-p38MAPK (CST, 4511S); anti-p38MAPK (CST, 8690S); anti-p-AMPKα (CST, 2531S); anti-AMPKα (CST, 2532S); anti-p-GSK3β (CST, 9323S); anti-GSK3β (CST, 12456S); anti-p-eIF2α (CST, 3398P); anti-eIF2α (CST, 5324S); anti-WIPI1 (CST, 12124S); anti-LC3B (Sigma Aldrich, L7543); HRP-conjugated goat anti-rabbit secondary antibody (Biodragon, Beijing, China, BF03008); HRP-conjugated goat anti-mouse secondary antibody (Biodragon, BF03001); Bafilomycin.A1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-201550A); Rabbit control IgG (Biodragon, BF01001); Mouse control IgG (Biodragon, BF01116); BL21 competent cells (Solarbio, Beijing, China, C1400-10); CCK-8 (DOJINDO, Shanghai, China, CK04); AnnexinV-PI apoptosis detection kit (DOJINDO, AD10); LY294002 (Abcam, ab120243); ATP detection kit (Beyotime Biotechnology, Nanjing, China, S0026); MG-132 (Abcam, ab146600); CQ (Sigma Aldrich, C6628); Rapamycin (Beyotime Biotechnology, S1842); No-glucose DMEM medium (BasalMedia (Yuanpei Biotech), Shanghai, China, L160); EBSS (Sigma Aldrich, E7510); Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA, L3000015).

    Techniques: Transfection, Western Blot, Incubation