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  • 99
    Thermo Fisher glutathione s transferase gst fusion proteins
    RhoA signaling contributed to the JNK-regulated cell differentiation and polarity formation. First, mDPCs were infected with RhoA mutant adenoviruses (RhoA WT or RhoA Q63L) for 48h, then were cultured with DMSO or <t>SP600125(10μM).</t> (A) After 24 h with SP600125 treatment, <t>GST-Pull</t> down assays were performed, RhoA Q63Lcould partly up-regulated the RhoA activity with SP600125 treatment. (B) After 5 days with SP600125 treatment, ALPase staining showed that RhoA Q63L could partly rescue SP600125-regulated inhibition of mDPCs differentiation. (C) After 10 h with SP600125 treatment, cell immunofluorescence of GM130 for the cells along scratch line were analyzed, and RhoA Q63L could partly rescue SP600125-regulated decreased of mDPCs polarity formation. * P
    Glutathione S Transferase Gst Fusion Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 4028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    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 1184 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.
    Gst Fusion Proteins, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 98/100, based on 13367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glutathione transferase gst fusion proteins
    Force-induced Rac activation. (A) FLNa-repleted A7 and FLNa-deficient M2 cells were subjected to mechanical force for 30 min and 2 h as described previously. Cell extracts were then incubated with <t>GST-PBD</t> or GST-RBD immobilized on glutathione-Sepharose
    Glutathione Transferase Gst Fusion Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher b per gst fusion protein purification kit
    Force-induced Rac activation. (A) FLNa-repleted A7 and FLNa-deficient M2 cells were subjected to mechanical force for 30 min and 2 h as described previously. Cell extracts were then incubated with <t>GST-PBD</t> or GST-RBD immobilized on glutathione-Sepharose
    B Per Gst Fusion Protein Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stratagene glutathione s transferase gst fusion proteins
    Force-induced Rac activation. (A) FLNa-repleted A7 and FLNa-deficient M2 cells were subjected to mechanical force for 30 min and 2 h as described previously. Cell extracts were then incubated with <t>GST-PBD</t> or GST-RBD immobilized on glutathione-Sepharose
    Glutathione S Transferase Gst Fusion Proteins, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Syntaxin gst fusion proteins
    Effect of phosphorylation of the <t>synprint</t> N- and C-terminal halves on interactions with syntaxin and SNAP-25. Control <t>GST,</t> GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with either α 1B (718–859) or α 1B (832–963) phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–859) with PKC (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , CaM KII phosphorylation of α 1B (718–859). C , PKC phosphorylation of α 1B (832–963). D , CaM KII phosphorylation of α 1B (832–963).
    Gst Fusion Proteins, supplied by Syntaxin, used in various techniques. Bioz Stars score: 92/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc gst fusion protein
    Complex formation of Grb10 and <t>Akt.</t> (A) Products of an anti-Grb10 immunoprecipitation (IP) using lysates from Mo7e cells (left panel) and from K562 cells (right panel) were separated by SDS-PAGE and immunoblotted (IB) with the antibodies indicated (top two panels). A control immunoprecipitation (bottom panels) using nonspecific rαm IgG was simultaneously performed, and the product was immunoblotted with α-Akt. (B) Lysates from nonstimulated COS1 cells transiently expressing HA epitope-tagged WT Akt (4 μg) and various FLAG epitope-tagged Grb10 constructs (4 μg) were subjected to an anti-FLAG immunoprecipitation (top two panels) and an anti-HA immunoprecipitation (bottom two panels). A control immunoprecipitation with nonspecific rabbit rαm IgG was performed (third panel from top). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies indicated. Lysates of the same cells were separated by SDS-PAGE and immunoblotted with an anti-Akt antibody to confirm equal expression levels of Akt (fourth panel from top). (C) SCF-induced association between c-kit and Grb10 and constitutive association between Grb10 and Akt are independent of Akt activity. An anti-FLAG immunoprecipitation (lanes 5 to 8) and a control immunoprecipitation (lanes 1 to 4) using nonspecific rαm IgG were performed using lysates from COS1 cells transfected with SS-EYFP-kitWT (4 μg), WT Akt (2 μg), and FLAG-tagged Grb10WT (4 μg). The cells were treated with SCF and PI3-K inhibitors prior to lysis as indicated. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies as indicated (top three panels). Lysates of the same cells were separated by SDS-PAGE and immunoblotted with a phosphospecific anti-Akt antibody to confirm the activation status of Akt and with an anti-Akt antibody to confirm equal expression levels of Akt. (D) <t>GST-Akt</t> binds to Grb10 and Grb10ΔPH. After preaclearing the lysates from COS1 cells expressing FLAG-tagged Grb10WT (lane 1) and Grb10ΔPH (lane 2) with glutathione beads, 2 μg of GST-Akt or only GST bound to glutathione beads was incubated at 4°C in equal volumes of lysates for 1 h. The beads were washed, and bound fractions were subjected to SDS-PAGE and transferred to PVDF membranes. Bound Grb10 protein was visualized by Western blotting using an anti-FLAG antibody. Lane 3 contains equal amounts of the GST-Akt protein to control cross-reactivity of the anti-FLAG antibody with GST-Akt fragments. This figure shows the results of two separate experiments.
    Gst Fusion Protein, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene glutathione transferase gst fusion protein
    Complex formation of Grb10 and <t>Akt.</t> (A) Products of an anti-Grb10 immunoprecipitation (IP) using lysates from Mo7e cells (left panel) and from K562 cells (right panel) were separated by SDS-PAGE and immunoblotted (IB) with the antibodies indicated (top two panels). A control immunoprecipitation (bottom panels) using nonspecific rαm IgG was simultaneously performed, and the product was immunoblotted with α-Akt. (B) Lysates from nonstimulated COS1 cells transiently expressing HA epitope-tagged WT Akt (4 μg) and various FLAG epitope-tagged Grb10 constructs (4 μg) were subjected to an anti-FLAG immunoprecipitation (top two panels) and an anti-HA immunoprecipitation (bottom two panels). A control immunoprecipitation with nonspecific rabbit rαm IgG was performed (third panel from top). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies indicated. Lysates of the same cells were separated by SDS-PAGE and immunoblotted with an anti-Akt antibody to confirm equal expression levels of Akt (fourth panel from top). (C) SCF-induced association between c-kit and Grb10 and constitutive association between Grb10 and Akt are independent of Akt activity. An anti-FLAG immunoprecipitation (lanes 5 to 8) and a control immunoprecipitation (lanes 1 to 4) using nonspecific rαm IgG were performed using lysates from COS1 cells transfected with SS-EYFP-kitWT (4 μg), WT Akt (2 μg), and FLAG-tagged Grb10WT (4 μg). The cells were treated with SCF and PI3-K inhibitors prior to lysis as indicated. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies as indicated (top three panels). Lysates of the same cells were separated by SDS-PAGE and immunoblotted with a phosphospecific anti-Akt antibody to confirm the activation status of Akt and with an anti-Akt antibody to confirm equal expression levels of Akt. (D) <t>GST-Akt</t> binds to Grb10 and Grb10ΔPH. After preaclearing the lysates from COS1 cells expressing FLAG-tagged Grb10WT (lane 1) and Grb10ΔPH (lane 2) with glutathione beads, 2 μg of GST-Akt or only GST bound to glutathione beads was incubated at 4°C in equal volumes of lysates for 1 h. The beads were washed, and bound fractions were subjected to SDS-PAGE and transferred to PVDF membranes. Bound Grb10 protein was visualized by Western blotting using an anti-FLAG antibody. Lane 3 contains equal amounts of the GST-Akt protein to control cross-reactivity of the anti-FLAG antibody with GST-Akt fragments. This figure shows the results of two separate experiments.
    Glutathione Transferase Gst Fusion Protein, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher b per gst fusion protein spin purification kit
    Complex formation of Grb10 and <t>Akt.</t> (A) Products of an anti-Grb10 immunoprecipitation (IP) using lysates from Mo7e cells (left panel) and from K562 cells (right panel) were separated by SDS-PAGE and immunoblotted (IB) with the antibodies indicated (top two panels). A control immunoprecipitation (bottom panels) using nonspecific rαm IgG was simultaneously performed, and the product was immunoblotted with α-Akt. (B) Lysates from nonstimulated COS1 cells transiently expressing HA epitope-tagged WT Akt (4 μg) and various FLAG epitope-tagged Grb10 constructs (4 μg) were subjected to an anti-FLAG immunoprecipitation (top two panels) and an anti-HA immunoprecipitation (bottom two panels). A control immunoprecipitation with nonspecific rabbit rαm IgG was performed (third panel from top). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies indicated. Lysates of the same cells were separated by SDS-PAGE and immunoblotted with an anti-Akt antibody to confirm equal expression levels of Akt (fourth panel from top). (C) SCF-induced association between c-kit and Grb10 and constitutive association between Grb10 and Akt are independent of Akt activity. An anti-FLAG immunoprecipitation (lanes 5 to 8) and a control immunoprecipitation (lanes 1 to 4) using nonspecific rαm IgG were performed using lysates from COS1 cells transfected with SS-EYFP-kitWT (4 μg), WT Akt (2 μg), and FLAG-tagged Grb10WT (4 μg). The cells were treated with SCF and PI3-K inhibitors prior to lysis as indicated. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies as indicated (top three panels). Lysates of the same cells were separated by SDS-PAGE and immunoblotted with a phosphospecific anti-Akt antibody to confirm the activation status of Akt and with an anti-Akt antibody to confirm equal expression levels of Akt. (D) <t>GST-Akt</t> binds to Grb10 and Grb10ΔPH. After preaclearing the lysates from COS1 cells expressing FLAG-tagged Grb10WT (lane 1) and Grb10ΔPH (lane 2) with glutathione beads, 2 μg of GST-Akt or only GST bound to glutathione beads was incubated at 4°C in equal volumes of lysates for 1 h. The beads were washed, and bound fractions were subjected to SDS-PAGE and transferred to PVDF membranes. Bound Grb10 protein was visualized by Western blotting using an anti-FLAG antibody. Lane 3 contains equal amounts of the GST-Akt protein to control cross-reactivity of the anti-FLAG antibody with GST-Akt fragments. This figure shows the results of two separate experiments.
    B Per Gst Fusion Protein Spin Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher gst fusion proteins
    Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained <t>SDS–polyacrylamide</t> gel showing the expression and expected size of the <t>GST–polι</t> fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.
    Gst Fusion Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1751 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Stratagene recombinant gst fusion proteins
    Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained <t>SDS–polyacrylamide</t> gel showing the expression and expected size of the <t>GST–polι</t> fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.
    Recombinant Gst Fusion Proteins, supplied by Stratagene, used in various techniques. Bioz Stars score: 91/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Syntaxin glutathione s transferase gst fusion proteins
    Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained <t>SDS–polyacrylamide</t> gel showing the expression and expected size of the <t>GST–polι</t> fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.
    Glutathione S Transferase Gst Fusion Proteins, supplied by Syntaxin, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Upstate Biotechnology Inc glutathione s transferase gst fusion protein
    Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained <t>SDS–polyacrylamide</t> gel showing the expression and expected size of the <t>GST–polι</t> fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.
    Glutathione S Transferase Gst Fusion Protein, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 91/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glutathione s transferase gst fusion protein/product/Upstate Biotechnology Inc
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    Cytoskeleton Inc gst fusion protein
    Thrombin-stimulated RhoA activity in BCEC lysates as a function of time. Transient RhoA activation in BCEC after treatment with thrombin (2 U/ml). Freshly prepapred lysates from BCEC before thrombin exposure (0 min) and after 0.5 min, 1 min, 3 min, 5 min and 15 min after thrombin exposure were assessed. Active RhoA was isolated using <t>GST-Rhotekin-RBD</t> beads following the manufacturer’s protocol. In vitro GTP S-loaded RhoA was used as a positive control (square inset). Both the active RhoA levels as well as the total RhoA levels were analyzed by immunoblotting using anti-RhoA. The double lines indicate samples were taken from a different part from the same blot and exposure. (B) Quantitative analysis of 3 independent experiments, in which the activated RhoA signals were normalized to the signal of the maximal activatable RhoA obtained using GTP S treatment. Data represent mean S.E.M.
    Gst Fusion Protein, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 93/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RhoA signaling contributed to the JNK-regulated cell differentiation and polarity formation. First, mDPCs were infected with RhoA mutant adenoviruses (RhoA WT or RhoA Q63L) for 48h, then were cultured with DMSO or SP600125(10μM). (A) After 24 h with SP600125 treatment, GST-Pull down assays were performed, RhoA Q63Lcould partly up-regulated the RhoA activity with SP600125 treatment. (B) After 5 days with SP600125 treatment, ALPase staining showed that RhoA Q63L could partly rescue SP600125-regulated inhibition of mDPCs differentiation. (C) After 10 h with SP600125 treatment, cell immunofluorescence of GM130 for the cells along scratch line were analyzed, and RhoA Q63L could partly rescue SP600125-regulated decreased of mDPCs polarity formation. * P

    Journal: bioRxiv

    Article Title: C-Jun N-terminal kinase (JNK) pathway activation is essential for dental papilla cells polarization

    doi: 10.1101/2020.05.18.101782

    Figure Lengend Snippet: RhoA signaling contributed to the JNK-regulated cell differentiation and polarity formation. First, mDPCs were infected with RhoA mutant adenoviruses (RhoA WT or RhoA Q63L) for 48h, then were cultured with DMSO or SP600125(10μM). (A) After 24 h with SP600125 treatment, GST-Pull down assays were performed, RhoA Q63Lcould partly up-regulated the RhoA activity with SP600125 treatment. (B) After 5 days with SP600125 treatment, ALPase staining showed that RhoA Q63L could partly rescue SP600125-regulated inhibition of mDPCs differentiation. (C) After 10 h with SP600125 treatment, cell immunofluorescence of GM130 for the cells along scratch line were analyzed, and RhoA Q63L could partly rescue SP600125-regulated decreased of mDPCs polarity formation. * P

    Article Snippet: RhoA pull-down assay Ad-RhoA WT or Ad-RhoA Q63L were transfected into mDPCs for 48 h, following SP600125 treatment for 24 h, then GST Pull-down assay with a glutathione transferase (GST) fusion protein containing the RhoA binding domain of rhotekin (rhotekin-GST) was performed by the manufacturer’s protocol using GTPase Pull-Down kit (Thermo Scientific, Waltham, MA, USA).

    Techniques: Cell Differentiation, Infection, Mutagenesis, Cell Culture, Activity Assay, Staining, Inhibition, Immunofluorescence

    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

    Binding of homologs of ComE1 from P. multocida (PM1665), H. influenzae (Hi1008), A. actinomycetemcomitans (Aa1426), A. pleuropneumoniae (APL_1406), M. haemolytica (MhORF35) or M. succiniproducens (Ms0826) expressed as GST-fusions to pUC19 DNA (A) or Fn (B) measured by direct binding ELISA. Optical density values at 492 nm were converted to estimates of the concentration of bound protein by reference to a standard curve for each protein.

    Journal: PLoS ONE

    Article Title: Pasteurellaceae ComE1 Proteins Combine the Properties of Fibronectin Adhesins and DNA Binding Competence Proteins

    doi: 10.1371/journal.pone.0003991

    Figure Lengend Snippet: Binding of homologs of ComE1 from P. multocida (PM1665), H. influenzae (Hi1008), A. actinomycetemcomitans (Aa1426), A. pleuropneumoniae (APL_1406), M. haemolytica (MhORF35) or M. succiniproducens (Ms0826) expressed as GST-fusions to pUC19 DNA (A) or Fn (B) measured by direct binding ELISA. Optical density values at 492 nm were converted to estimates of the concentration of bound protein by reference to a standard curve for each protein.

    Article Snippet: Competition ELISA For inhibition ELISAs, recombinant GST-fusion proteins were pre-incubated for 1 h at 37°C with soluble Fn (Sigma), bacterial chromosomal DNA or pUC19 DNA.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Binding of pUC19 to ComE1. Binding of 1 nM of recombinant GST-ComE1 from P. multocida to immobilised Fn or pUC19 DNA measured by direct binding ELISA. The data are presented as the mean±SEM of triplicate wells. The data shown are representative of three separate experiments.

    Journal: PLoS ONE

    Article Title: Pasteurellaceae ComE1 Proteins Combine the Properties of Fibronectin Adhesins and DNA Binding Competence Proteins

    doi: 10.1371/journal.pone.0003991

    Figure Lengend Snippet: Binding of pUC19 to ComE1. Binding of 1 nM of recombinant GST-ComE1 from P. multocida to immobilised Fn or pUC19 DNA measured by direct binding ELISA. The data are presented as the mean±SEM of triplicate wells. The data shown are representative of three separate experiments.

    Article Snippet: Competition ELISA For inhibition ELISAs, recombinant GST-fusion proteins were pre-incubated for 1 h at 37°C with soluble Fn (Sigma), bacterial chromosomal DNA or pUC19 DNA.

    Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

    Schematic representation of the structural features of ComE1 (A). Binding of fragments of ComE1 from P. multocida (expressed as GST fusion proteins) to pUC19 DNA measured by direct binding ELISA (B). Increasing concentrations of rGST-ComE1 (open circles), the C-terminal 64 residues of ComE1 (closed circles), the two HHH domains of ComE1 (closed triangles) or a combination of the conserved VNINTA motif plus the first HHH domain (open triangles), were added to wells coated with Fn. Optical density values at 492 nm were converted to estimates of the concentration of bound protein by reference to a standard curve for each protein.

    Journal: PLoS ONE

    Article Title: Pasteurellaceae ComE1 Proteins Combine the Properties of Fibronectin Adhesins and DNA Binding Competence Proteins

    doi: 10.1371/journal.pone.0003991

    Figure Lengend Snippet: Schematic representation of the structural features of ComE1 (A). Binding of fragments of ComE1 from P. multocida (expressed as GST fusion proteins) to pUC19 DNA measured by direct binding ELISA (B). Increasing concentrations of rGST-ComE1 (open circles), the C-terminal 64 residues of ComE1 (closed circles), the two HHH domains of ComE1 (closed triangles) or a combination of the conserved VNINTA motif plus the first HHH domain (open triangles), were added to wells coated with Fn. Optical density values at 492 nm were converted to estimates of the concentration of bound protein by reference to a standard curve for each protein.

    Article Snippet: Competition ELISA For inhibition ELISAs, recombinant GST-fusion proteins were pre-incubated for 1 h at 37°C with soluble Fn (Sigma), bacterial chromosomal DNA or pUC19 DNA.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Binding of ComE1 to single-stranded DNA. Binding of 1 nM of recombinant GST-ComE1 from P. multocida to immobilised DNA fragments or single-stranded DNA (ssDNA) measured by direct binding ELISA. The data are presented as the mean±SEM of triplicate wells. The data shown are representative of three separate experiments.

    Journal: PLoS ONE

    Article Title: Pasteurellaceae ComE1 Proteins Combine the Properties of Fibronectin Adhesins and DNA Binding Competence Proteins

    doi: 10.1371/journal.pone.0003991

    Figure Lengend Snippet: Binding of ComE1 to single-stranded DNA. Binding of 1 nM of recombinant GST-ComE1 from P. multocida to immobilised DNA fragments or single-stranded DNA (ssDNA) measured by direct binding ELISA. The data are presented as the mean±SEM of triplicate wells. The data shown are representative of three separate experiments.

    Article Snippet: Competition ELISA For inhibition ELISAs, recombinant GST-fusion proteins were pre-incubated for 1 h at 37°C with soluble Fn (Sigma), bacterial chromosomal DNA or pUC19 DNA.

    Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

    Mutational analysis of RacC. ( a ) Alignment of various Rac proteins. The position of the mutations generated is indicated as well as of the Switch I and II regions. ( b ) Surface potential of D. discoideum Rac1a and RacC. The three dimensional structure of Rac1a and RacC is modeled using SWISSMODEL and the surface potential calculated using APBS plug-in in Pymol. Red, negative, blue, positive charge. ( c ) Interactions of RacC Mut1-5 with GFP-CRN7 WT. The RacC mutant proteins were expressed as GST fusions, bound to Glutathione-Sepharose beads, loaded with GDP or GTPγS and used to pull down GFP-CRN7 WT from cell lysates. C, represents the protein amounts in 2 × 10 5 cells; I, aliquot of the cell lysate used in the assay. The western blots were probed with mAb K3-184-2. ( d ) Binding affinities of different Rac mutants to GFP-CRN7. Bar chart shows the binding of RacC wild type and mutant proteins loaded with GDP or GTPγS to GFP-CRN7. Between three and five experiments were carried out. Relative intensities are shown as arbitrary units ± SD. The differences for wild type RacC (WT), Mut2 and Mut3 were statistically significant (P

    Journal: Scientific Reports

    Article Title: Coronin7 regulates WASP and SCAR through CRIB mediated interaction with Rac proteins

    doi: 10.1038/srep14437

    Figure Lengend Snippet: Mutational analysis of RacC. ( a ) Alignment of various Rac proteins. The position of the mutations generated is indicated as well as of the Switch I and II regions. ( b ) Surface potential of D. discoideum Rac1a and RacC. The three dimensional structure of Rac1a and RacC is modeled using SWISSMODEL and the surface potential calculated using APBS plug-in in Pymol. Red, negative, blue, positive charge. ( c ) Interactions of RacC Mut1-5 with GFP-CRN7 WT. The RacC mutant proteins were expressed as GST fusions, bound to Glutathione-Sepharose beads, loaded with GDP or GTPγS and used to pull down GFP-CRN7 WT from cell lysates. C, represents the protein amounts in 2 × 10 5 cells; I, aliquot of the cell lysate used in the assay. The western blots were probed with mAb K3-184-2. ( d ) Binding affinities of different Rac mutants to GFP-CRN7. Bar chart shows the binding of RacC wild type and mutant proteins loaded with GDP or GTPγS to GFP-CRN7. Between three and five experiments were carried out. Relative intensities are shown as arbitrary units ± SD. The differences for wild type RacC (WT), Mut2 and Mut3 were statistically significant (P

    Article Snippet: Pull downs were performed with Glutathione Sepharose 4B beads (GE Healthcare) carrying GST tagged fusion proteins, immunoprecipitations were done with Protein A-Sepharose 4B beads (Sigma) carrying appropriate antibodies.

    Techniques: Generated, Mutagenesis, Western Blot, Binding Assay

    The CRIB domains of CRN7 and Rac GTPase interactions of CRN7. (aa) Domain structure of coronin and CRN7. (ab) Sequence alignment of the CRIB domain in coronin and CRN7 and selected proteins. The consensus sequence is shown above the alignment. The position within the proteins is indicated. (ac) Model of CRN7. The 3D structure was predicted as described 11 . The CRIB domains are in shown red. A magnification of the region containing the CRIB domain and the putative F-actin binding site in WD2 is shown below. The conserved K residue is indicated in a stick model in magenta. ( b ) CRN7 interacts primarily with GDP-bound Rac. GST and GST-Rac proteins loaded with GDP or GTPγS were bound to Glutathione Sepharose beads and incubated with lysates of corB − cells expressing GFP-CRN7 WT. The bound proteins were analyzed by western blotting. Probing was with GFP specific mAb K3-184-2. ( c ) The N- and the C-terminal half of CRN7 interact with Rac GTPases. The precipitated proteins were detected with mAb K3-184-2, the GST-fusions with polyclonal GST-specific antibodies. GST control (GST CTL) and beads control (Beads CTL) are shown. ( d ) CRN7 interacts directly with Rac proteins. Bacterially expressed GST-CRN7-NT encompassing the PST-domain was cleaved from the GST part and used in pull down assays with GST-Rac GTPases. The precipitated proteins were analyzed in western blots. CRN7-NT was detected with mAb K67-31-5.

    Journal: Scientific Reports

    Article Title: Coronin7 regulates WASP and SCAR through CRIB mediated interaction with Rac proteins

    doi: 10.1038/srep14437

    Figure Lengend Snippet: The CRIB domains of CRN7 and Rac GTPase interactions of CRN7. (aa) Domain structure of coronin and CRN7. (ab) Sequence alignment of the CRIB domain in coronin and CRN7 and selected proteins. The consensus sequence is shown above the alignment. The position within the proteins is indicated. (ac) Model of CRN7. The 3D structure was predicted as described 11 . The CRIB domains are in shown red. A magnification of the region containing the CRIB domain and the putative F-actin binding site in WD2 is shown below. The conserved K residue is indicated in a stick model in magenta. ( b ) CRN7 interacts primarily with GDP-bound Rac. GST and GST-Rac proteins loaded with GDP or GTPγS were bound to Glutathione Sepharose beads and incubated with lysates of corB − cells expressing GFP-CRN7 WT. The bound proteins were analyzed by western blotting. Probing was with GFP specific mAb K3-184-2. ( c ) The N- and the C-terminal half of CRN7 interact with Rac GTPases. The precipitated proteins were detected with mAb K3-184-2, the GST-fusions with polyclonal GST-specific antibodies. GST control (GST CTL) and beads control (Beads CTL) are shown. ( d ) CRN7 interacts directly with Rac proteins. Bacterially expressed GST-CRN7-NT encompassing the PST-domain was cleaved from the GST part and used in pull down assays with GST-Rac GTPases. The precipitated proteins were analyzed in western blots. CRN7-NT was detected with mAb K67-31-5.

    Article Snippet: Pull downs were performed with Glutathione Sepharose 4B beads (GE Healthcare) carrying GST tagged fusion proteins, immunoprecipitations were done with Protein A-Sepharose 4B beads (Sigma) carrying appropriate antibodies.

    Techniques: Sequencing, Binding Assay, Incubation, Expressing, Western Blot, CTL Assay

    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

    Autoinhibition of Jak2 in E. coli . Gst-fusion proteins for JH1 (JH1-Gst) and JH1–2 (JH1–2-Gst) domains of Jak2 were expressed in bacterial cells as described in MATERIALS AND METHODS. The cells were lysed by boiling in reducing Laemmli sample buffer. Lysates were separated in 7.5% SDS-PAGE, and analyzed in anti-HA (right) and anti-phosphotyrosine (left) immunoblots. Arrows indicate the migration of JH1-Gst and JH1–2-Gst. The mobilities of the molecular mass markers (in kilodaltons) are shown on the right.

    Journal: Molecular Biology of the Cell

    Article Title: Autoinhibition of Jak2 Tyrosine Kinase Is Dependent on Specific Regions in Its Pseudokinase Domain

    doi: 10.1091/mbc.E02-06-0342

    Figure Lengend Snippet: Autoinhibition of Jak2 in E. coli . Gst-fusion proteins for JH1 (JH1-Gst) and JH1–2 (JH1–2-Gst) domains of Jak2 were expressed in bacterial cells as described in MATERIALS AND METHODS. The cells were lysed by boiling in reducing Laemmli sample buffer. Lysates were separated in 7.5% SDS-PAGE, and analyzed in anti-HA (right) and anti-phosphotyrosine (left) immunoblots. Arrows indicate the migration of JH1-Gst and JH1–2-Gst. The mobilities of the molecular mass markers (in kilodaltons) are shown on the right.

    Article Snippet: Overnight cultures were diluted 1:200 in Luria media containing ampicillin and incubated at 37°C for 3 h. A sample from JH1–2-Gst culture was taken after 15-min induction of Gst fusion protein expression with 1 mM isopropyl β- d -thiogalactoside (final concentration) (Sigma-Aldrich, St. Louis, MO).

    Techniques: SDS Page, Western Blot, Migration

    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

    Expression and purification of mature CpdB. The recombinant protein was expressed in BL21 cells transformed with pGEX-6P-3-cpdB and induced by IPTG. The GST-CpdB fusion protein was purified by affinity adsorption to GSH-Sepharose. Mature CpdB separated from the GST tag was recovered from the gel after in-column digestion with PreScission. (A) Denaturing gel electrophoresis analysis. L–and L+, bacterial lysates before and after IPTG induction. S and P, supernatant and precipitate of the L+ lysate. M, molecular weight markers (phosphorylase B, 97400; bovine serum albumin, 66200; ovoalbumin, 45000). E, fraction excluded from the GSH-Sepharose column during application of the PreScission protease. 1–4, fractions collected after intracolumn proteolysis. X and Y, bands corresponding to the GST-CpdB fusion and to GPLGS-CpdB obtained by proteolysis. (B) Phosphohydrolytic activities on the indicated substrates, measured in the fractions collected from the GSH-Sepharose column.

    Journal: PLoS ONE

    Article Title: The Characterization of Escherichia coli CpdB as a Recombinant Protein Reveals that, besides Having the Expected 3´-Nucleotidase and 2´,3´-Cyclic Mononucleotide Phosphodiesterase Activities, It Is Also Active as Cyclic Dinucleotide Phosphodiesterase

    doi: 10.1371/journal.pone.0157308

    Figure Lengend Snippet: Expression and purification of mature CpdB. The recombinant protein was expressed in BL21 cells transformed with pGEX-6P-3-cpdB and induced by IPTG. The GST-CpdB fusion protein was purified by affinity adsorption to GSH-Sepharose. Mature CpdB separated from the GST tag was recovered from the gel after in-column digestion with PreScission. (A) Denaturing gel electrophoresis analysis. L–and L+, bacterial lysates before and after IPTG induction. S and P, supernatant and precipitate of the L+ lysate. M, molecular weight markers (phosphorylase B, 97400; bovine serum albumin, 66200; ovoalbumin, 45000). E, fraction excluded from the GSH-Sepharose column during application of the PreScission protease. 1–4, fractions collected after intracolumn proteolysis. X and Y, bands corresponding to the GST-CpdB fusion and to GPLGS-CpdB obtained by proteolysis. (B) Phosphohydrolytic activities on the indicated substrates, measured in the fractions collected from the GSH-Sepharose column.

    Article Snippet: Therefore, mature CpdB can be expressed from the IPTG-inducible tac promoter of pGEX-6P-3-cpdB as a GST fusion protein which can be cut with PreScission (GE Healthcare Life Sciences), yielding mature CpdB with a N-terminal extension of 5 amino acids (GPLGS).

    Techniques: Expressing, Purification, Recombinant, Transformation Assay, Adsorption, Nucleic Acid Electrophoresis, Molecular Weight

    Force-induced Rac activation. (A) FLNa-repleted A7 and FLNa-deficient M2 cells were subjected to mechanical force for 30 min and 2 h as described previously. Cell extracts were then incubated with GST-PBD or GST-RBD immobilized on glutathione-Sepharose

    Journal: Molecular Biology of the Cell

    Article Title: The Role of FilGAP-Filamin A Interactions in Mechanoprotection

    doi: 10.1091/mbc.E08-08-0872

    Figure Lengend Snippet: Force-induced Rac activation. (A) FLNa-repleted A7 and FLNa-deficient M2 cells were subjected to mechanical force for 30 min and 2 h as described previously. Cell extracts were then incubated with GST-PBD or GST-RBD immobilized on glutathione-Sepharose

    Article Snippet: The cell lysates were cleared, and supernatants were sampled for the determination of total GTPases or incubated with glutathione transferase (GST) fusion proteins (Millipore) corresponding to the p21-binding domain (PBD, expressed in Escherichia coli and bound to glutathione agarose) or the rhotekin-binding domain (RBD, expressed in E. coli and bound to glutathione agarose).

    Techniques: Activation Assay, Incubation

    Effect of phosphorylation of the synprint N- and C-terminal halves on interactions with syntaxin and SNAP-25. Control GST, GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with either α 1B (718–859) or α 1B (832–963) phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–859) with PKC (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , CaM KII phosphorylation of α 1B (718–859). C , PKC phosphorylation of α 1B (832–963). D , CaM KII phosphorylation of α 1B (832–963).

    Journal: The Journal of Neuroscience

    Article Title: Phosphorylation of the Synaptic Protein Interaction Site on N-type Calcium Channels Inhibits Interactions with SNARE Proteins

    doi: 10.1523/JNEUROSCI.17-18-06929.1997

    Figure Lengend Snippet: Effect of phosphorylation of the synprint N- and C-terminal halves on interactions with syntaxin and SNAP-25. Control GST, GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with either α 1B (718–859) or α 1B (832–963) phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–859) with PKC (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , CaM KII phosphorylation of α 1B (718–859). C , PKC phosphorylation of α 1B (832–963). D , CaM KII phosphorylation of α 1B (832–963).

    Article Snippet: No binding to a control GST protein (25 kDa) was observed, and anti-GST-tag immunoblotting confirmed that the phosphorylation state of the synprint polypeptide did not interfere with the immobilization of the GST fusion proteins.

    Techniques: Incubation, Chick Chorioallantoic Membrane Assay, SDS Page, Binding Assay

    Effect of phosphorylation of synprint polypeptides on interactions with syntaxin and SNAP-25. Control GST, GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with α 1B (718–963) phosphorylated with PKA, PKG, PKC, or CaM KII or were treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–963) with PKA (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , PKG phosphorylation. C , PKC phosphorylation. D , CaM KII phosphorylation. E , Both PKC and CaM KII phosphorylation. Chemiluminescent signals for this and all subsequent experiments were within the linear range of the detection system.

    Journal: The Journal of Neuroscience

    Article Title: Phosphorylation of the Synaptic Protein Interaction Site on N-type Calcium Channels Inhibits Interactions with SNARE Proteins

    doi: 10.1523/JNEUROSCI.17-18-06929.1997

    Figure Lengend Snippet: Effect of phosphorylation of synprint polypeptides on interactions with syntaxin and SNAP-25. Control GST, GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with α 1B (718–963) phosphorylated with PKA, PKG, PKC, or CaM KII or were treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–963) with PKA (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , PKG phosphorylation. C , PKC phosphorylation. D , CaM KII phosphorylation. E , Both PKC and CaM KII phosphorylation. Chemiluminescent signals for this and all subsequent experiments were within the linear range of the detection system.

    Article Snippet: No binding to a control GST protein (25 kDa) was observed, and anti-GST-tag immunoblotting confirmed that the phosphorylation state of the synprint polypeptide did not interfere with the immobilization of the GST fusion proteins.

    Techniques: Incubation, Chick Chorioallantoic Membrane Assay, SDS Page, Binding Assay

    Effect of phosphorylation of syntaxin and SNAP-25 on interactions with synprint polypeptides. Control GST, GST-syntaxin 1A, or GST-SNAP-25 was immobilized on glutathione-Sepharose, phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase, and then incubated with α 1B (718–963). Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of control GST (25 kDa), GST-syntaxin 1A (60 kDa), and GST-SNAP-25 (50 kDa) with PKC (+) or a control buffer (−), followed by incubation with α 1B (718–963) and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , Phosphorylation with CaM KII.

    Journal: The Journal of Neuroscience

    Article Title: Phosphorylation of the Synaptic Protein Interaction Site on N-type Calcium Channels Inhibits Interactions with SNARE Proteins

    doi: 10.1523/JNEUROSCI.17-18-06929.1997

    Figure Lengend Snippet: Effect of phosphorylation of syntaxin and SNAP-25 on interactions with synprint polypeptides. Control GST, GST-syntaxin 1A, or GST-SNAP-25 was immobilized on glutathione-Sepharose, phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase, and then incubated with α 1B (718–963). Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of control GST (25 kDa), GST-syntaxin 1A (60 kDa), and GST-SNAP-25 (50 kDa) with PKC (+) or a control buffer (−), followed by incubation with α 1B (718–963) and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , Phosphorylation with CaM KII.

    Article Snippet: No binding to a control GST protein (25 kDa) was observed, and anti-GST-tag immunoblotting confirmed that the phosphorylation state of the synprint polypeptide did not interfere with the immobilization of the GST fusion proteins.

    Techniques: Chick Chorioallantoic Membrane Assay, Incubation, SDS Page

    Complex formation of Grb10 and Akt. (A) Products of an anti-Grb10 immunoprecipitation (IP) using lysates from Mo7e cells (left panel) and from K562 cells (right panel) were separated by SDS-PAGE and immunoblotted (IB) with the antibodies indicated (top two panels). A control immunoprecipitation (bottom panels) using nonspecific rαm IgG was simultaneously performed, and the product was immunoblotted with α-Akt. (B) Lysates from nonstimulated COS1 cells transiently expressing HA epitope-tagged WT Akt (4 μg) and various FLAG epitope-tagged Grb10 constructs (4 μg) were subjected to an anti-FLAG immunoprecipitation (top two panels) and an anti-HA immunoprecipitation (bottom two panels). A control immunoprecipitation with nonspecific rabbit rαm IgG was performed (third panel from top). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies indicated. Lysates of the same cells were separated by SDS-PAGE and immunoblotted with an anti-Akt antibody to confirm equal expression levels of Akt (fourth panel from top). (C) SCF-induced association between c-kit and Grb10 and constitutive association between Grb10 and Akt are independent of Akt activity. An anti-FLAG immunoprecipitation (lanes 5 to 8) and a control immunoprecipitation (lanes 1 to 4) using nonspecific rαm IgG were performed using lysates from COS1 cells transfected with SS-EYFP-kitWT (4 μg), WT Akt (2 μg), and FLAG-tagged Grb10WT (4 μg). The cells were treated with SCF and PI3-K inhibitors prior to lysis as indicated. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies as indicated (top three panels). Lysates of the same cells were separated by SDS-PAGE and immunoblotted with a phosphospecific anti-Akt antibody to confirm the activation status of Akt and with an anti-Akt antibody to confirm equal expression levels of Akt. (D) GST-Akt binds to Grb10 and Grb10ΔPH. After preaclearing the lysates from COS1 cells expressing FLAG-tagged Grb10WT (lane 1) and Grb10ΔPH (lane 2) with glutathione beads, 2 μg of GST-Akt or only GST bound to glutathione beads was incubated at 4°C in equal volumes of lysates for 1 h. The beads were washed, and bound fractions were subjected to SDS-PAGE and transferred to PVDF membranes. Bound Grb10 protein was visualized by Western blotting using an anti-FLAG antibody. Lane 3 contains equal amounts of the GST-Akt protein to control cross-reactivity of the anti-FLAG antibody with GST-Akt fragments. This figure shows the results of two separate experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Role for the Adaptor Protein Grb10 in the Activation of Akt

    doi: 10.1128/MCB.22.4.979-991.2002

    Figure Lengend Snippet: Complex formation of Grb10 and Akt. (A) Products of an anti-Grb10 immunoprecipitation (IP) using lysates from Mo7e cells (left panel) and from K562 cells (right panel) were separated by SDS-PAGE and immunoblotted (IB) with the antibodies indicated (top two panels). A control immunoprecipitation (bottom panels) using nonspecific rαm IgG was simultaneously performed, and the product was immunoblotted with α-Akt. (B) Lysates from nonstimulated COS1 cells transiently expressing HA epitope-tagged WT Akt (4 μg) and various FLAG epitope-tagged Grb10 constructs (4 μg) were subjected to an anti-FLAG immunoprecipitation (top two panels) and an anti-HA immunoprecipitation (bottom two panels). A control immunoprecipitation with nonspecific rabbit rαm IgG was performed (third panel from top). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies indicated. Lysates of the same cells were separated by SDS-PAGE and immunoblotted with an anti-Akt antibody to confirm equal expression levels of Akt (fourth panel from top). (C) SCF-induced association between c-kit and Grb10 and constitutive association between Grb10 and Akt are independent of Akt activity. An anti-FLAG immunoprecipitation (lanes 5 to 8) and a control immunoprecipitation (lanes 1 to 4) using nonspecific rαm IgG were performed using lysates from COS1 cells transfected with SS-EYFP-kitWT (4 μg), WT Akt (2 μg), and FLAG-tagged Grb10WT (4 μg). The cells were treated with SCF and PI3-K inhibitors prior to lysis as indicated. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies as indicated (top three panels). Lysates of the same cells were separated by SDS-PAGE and immunoblotted with a phosphospecific anti-Akt antibody to confirm the activation status of Akt and with an anti-Akt antibody to confirm equal expression levels of Akt. (D) GST-Akt binds to Grb10 and Grb10ΔPH. After preaclearing the lysates from COS1 cells expressing FLAG-tagged Grb10WT (lane 1) and Grb10ΔPH (lane 2) with glutathione beads, 2 μg of GST-Akt or only GST bound to glutathione beads was incubated at 4°C in equal volumes of lysates for 1 h. The beads were washed, and bound fractions were subjected to SDS-PAGE and transferred to PVDF membranes. Bound Grb10 protein was visualized by Western blotting using an anti-FLAG antibody. Lane 3 contains equal amounts of the GST-Akt protein to control cross-reactivity of the anti-FLAG antibody with GST-Akt fragments. This figure shows the results of two separate experiments.

    Article Snippet: A purified GST fusion protein of full-length Akt was obtained from a commercial source (Upstate Biotechnology).

    Techniques: Immunoprecipitation, SDS Page, Expressing, FLAG-tag, Construct, Activity Assay, Transfection, Lysis, Activation Assay, Incubation, Western Blot

    Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained SDS–polyacrylamide gel showing the expression and expected size of the GST–polι fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.

    Journal: The EMBO Journal

    Article Title: Localization of DNA polymerases ? and ? to the replication machinery is tightly co-ordinated in human cells

    doi: 10.1093/emboj/cdf618

    Figure Lengend Snippet: Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained SDS–polyacrylamide gel showing the expression and expected size of the GST–polι fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.

    Article Snippet: Purified GST fusion proteins were separated by 4–20% SDS–PAGE (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes (Invitrogen).

    Techniques: In Vivo, Transformation Assay, Incubation, Western Blot, Staining, Expressing, Negative Control, Pull Down Assay, In Vitro, SDS Page, Binding Assay, Infection

    Western analysis of GST-Syntaxin 7 binding to endosomes and lysosomes in vitro. Purified lysosomes or endosomes (0.5 mg/ml) were incubated in the in vitro fusion system in the presence of 20 μg/ml GST-Syntaxin 7. After incubation at 37°C, fusion was assessed as described in MATERIALS AND METHODS. Samples of endosomes or lysosomes from the fusion assay were pelleted and washed with fusion buffer. Vesicle pellets were solubilized by the addition of 0.05% Triton X-100 containing 10 mM Tris-HCl, pH 7.5, and 50 mM NaCl at 0°C for 30 min followed by centrifugation at 40,000 × g for 30 min. Supernatants were incubated with glutathione–agarose beads at 0°C for 2 h or overnight, beads were pelleted and washed, and GST protein was eluted with 20 mM glutathione according to the Pierce protocol. Eluted samples were run on 4–20% SDS-PAGE, and Western analysis was performed with the use of affinity-purified rabbit anti-Syntaxin 7 (1:1000) as the primary antibody followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000). A represents samples from lysosomes and B represents endosomal samples. Lanes A represent purified GST-Syntaxin 7 eluted from glutathione–agarose. Lanes B represent the supernatant from pelleted fusion reactions. Lanes C represent lysates after incubation with glutathione–agarose. Lanes D represent a glutathione–agarose bead wash after lysate incubation. Lanes E represent glutathione eluates from the lysosome or endosome glutathione–agarose beads. The arrow represents GST-Syntaxin 7 eluted from only purified lysosomes.

    Journal: Molecular Biology of the Cell

    Article Title: Syntaxin 7 and VAMP-7 are Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptors Required for Late Endosome-Lysosome and Homotypic Lysosome Fusion in Alveolar Macrophages

    doi:

    Figure Lengend Snippet: Western analysis of GST-Syntaxin 7 binding to endosomes and lysosomes in vitro. Purified lysosomes or endosomes (0.5 mg/ml) were incubated in the in vitro fusion system in the presence of 20 μg/ml GST-Syntaxin 7. After incubation at 37°C, fusion was assessed as described in MATERIALS AND METHODS. Samples of endosomes or lysosomes from the fusion assay were pelleted and washed with fusion buffer. Vesicle pellets were solubilized by the addition of 0.05% Triton X-100 containing 10 mM Tris-HCl, pH 7.5, and 50 mM NaCl at 0°C for 30 min followed by centrifugation at 40,000 × g for 30 min. Supernatants were incubated with glutathione–agarose beads at 0°C for 2 h or overnight, beads were pelleted and washed, and GST protein was eluted with 20 mM glutathione according to the Pierce protocol. Eluted samples were run on 4–20% SDS-PAGE, and Western analysis was performed with the use of affinity-purified rabbit anti-Syntaxin 7 (1:1000) as the primary antibody followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000). A represents samples from lysosomes and B represents endosomal samples. Lanes A represent purified GST-Syntaxin 7 eluted from glutathione–agarose. Lanes B represent the supernatant from pelleted fusion reactions. Lanes C represent lysates after incubation with glutathione–agarose. Lanes D represent a glutathione–agarose bead wash after lysate incubation. Lanes E represent glutathione eluates from the lysosome or endosome glutathione–agarose beads. The arrow represents GST-Syntaxin 7 eluted from only purified lysosomes.

    Article Snippet: Transformed bacteria were induced with isopropyl β- d -galactopyranoside, and recombinant GST/Syntaxin 7 was purified by glutathione agarose chromatography according to GST fusion protein protocols from Pierce Chemical (Rockford, IL).

    Techniques: Western Blot, Binding Assay, In Vitro, Purification, Incubation, Single Vesicle Fusion Assay, Centrifugation, SDS Page, Affinity Purification

    Thrombin-stimulated RhoA activity in BCEC lysates as a function of time. Transient RhoA activation in BCEC after treatment with thrombin (2 U/ml). Freshly prepapred lysates from BCEC before thrombin exposure (0 min) and after 0.5 min, 1 min, 3 min, 5 min and 15 min after thrombin exposure were assessed. Active RhoA was isolated using GST-Rhotekin-RBD beads following the manufacturer’s protocol. In vitro GTP S-loaded RhoA was used as a positive control (square inset). Both the active RhoA levels as well as the total RhoA levels were analyzed by immunoblotting using anti-RhoA. The double lines indicate samples were taken from a different part from the same blot and exposure. (B) Quantitative analysis of 3 independent experiments, in which the activated RhoA signals were normalized to the signal of the maximal activatable RhoA obtained using GTP S treatment. Data represent mean S.E.M.

    Journal: PLoS ONE

    Article Title: RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

    doi: 10.1371/journal.pone.0042074

    Figure Lengend Snippet: Thrombin-stimulated RhoA activity in BCEC lysates as a function of time. Transient RhoA activation in BCEC after treatment with thrombin (2 U/ml). Freshly prepapred lysates from BCEC before thrombin exposure (0 min) and after 0.5 min, 1 min, 3 min, 5 min and 15 min after thrombin exposure were assessed. Active RhoA was isolated using GST-Rhotekin-RBD beads following the manufacturer’s protocol. In vitro GTP S-loaded RhoA was used as a positive control (square inset). Both the active RhoA levels as well as the total RhoA levels were analyzed by immunoblotting using anti-RhoA. The double lines indicate samples were taken from a different part from the same blot and exposure. (B) Quantitative analysis of 3 independent experiments, in which the activated RhoA signals were normalized to the signal of the maximal activatable RhoA obtained using GTP S treatment. Data represent mean S.E.M.

    Article Snippet: RhoA-activity Assay Activation of Rho GTPase was examined by GST pull-down of GTP-bound Rho proteins by using the Rho-binding domain (RBD) of Rhothekin protein (an effector of RhoA) expressed as a GST-fusion protein and coupled to agarose beads (RT02, Cytoskeleton, Inc.) to capture the active (GTP-bound) Rho proteins in BCEC-lysates.

    Techniques: Activity Assay, Activation Assay, Isolation, In Vitro, Positive Control

    Thrombin-stimulated RhoA activity in BCEC after C3-pretreatment. Western-blot analysis of RhoA activity after BCEC exposure to thrombin (0.5, 1.5 and 15 min) obtained from lysates of untreated BCEC and BCEC pretreated for 3 hours with C3 at 37°C. Active RhoA fraction was isolated from freshly prepared BCEC lysates using GST-Rhotekin-RBD beads following the manufacturer’s protocol (upper blot). In vitro GTP S-loaded RhoA was used as a positive control. The total RhoA levels were determined in all lysates (lower blot).

    Journal: PLoS ONE

    Article Title: RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

    doi: 10.1371/journal.pone.0042074

    Figure Lengend Snippet: Thrombin-stimulated RhoA activity in BCEC after C3-pretreatment. Western-blot analysis of RhoA activity after BCEC exposure to thrombin (0.5, 1.5 and 15 min) obtained from lysates of untreated BCEC and BCEC pretreated for 3 hours with C3 at 37°C. Active RhoA fraction was isolated from freshly prepared BCEC lysates using GST-Rhotekin-RBD beads following the manufacturer’s protocol (upper blot). In vitro GTP S-loaded RhoA was used as a positive control. The total RhoA levels were determined in all lysates (lower blot).

    Article Snippet: RhoA-activity Assay Activation of Rho GTPase was examined by GST pull-down of GTP-bound Rho proteins by using the Rho-binding domain (RBD) of Rhothekin protein (an effector of RhoA) expressed as a GST-fusion protein and coupled to agarose beads (RT02, Cytoskeleton, Inc.) to capture the active (GTP-bound) Rho proteins in BCEC-lysates.

    Techniques: Activity Assay, Western Blot, Isolation, In Vitro, Positive Control