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  • 99
    Thermo Fisher glutathione s transferase gst
    CD97 binds β-catenin. (A) The N-terminal (NTF) and C-terminal fragment (CTF) of CD97 were associated in DLD1 cells (left panel) and in the colon of Tg(villin-CD97) mice (right panel). Input control and immunoprecipitates (IP), obtained either with IgG or with CD97 NTF- and CTF-specific antibodies (Abs), were analyzed with a CD97 Ab detecting the other fragment, as indicated. (B,C) CD97 and β-catenin co-immunoprecipitated in CD97-positive DLD1 cells [ (B) , left panel] and in colonic lysates of Tg(villin-CD97) mice [ (C) , left panel]. HEK293 cells, only slightly positive for CD97 [ (B) , right panel], and CD97 knock-out (Ko) mice [ (C) , right panel] served as negative controls. (D) Next to β-catenin, CD97 co-immunoprecipitated with E-cadherin (Ecad) in DLD1 cells (upper picture) and with Ecad and p120-catenin in Tg(villin-CD97) mice (lower picture). (E) upper panel: beads loaded with glutathione S-transferase <t>(GST)</t> or β-catenin-GST were incubated with lysates of HT1080 CD97(EGF125), CD97(EGF125/TM2) cells with a C-terminal truncated CD97 ( 15 ), and mock cells. Bound proteins were eluted and CD97 was detected with NTF or CTF CD97-specific Abs by western blotting. Pulldown could be demonstrated only with β-catenin-GST and lysates of HT1080 cells expressing full-length (TM7), but not expressing C-terminal truncated (TM2) CD97. In the lower panel the respective elution controls for β-catenin and GST are shown.
    Glutathione S Transferase Gst, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glutathione s transferase gst
    Effect of mitochondrial DNA (mtDNA) depletion on the transcripts of the antioxidant defense enzymes. The total RNA from the control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts was prepared. The transcript level of the antioxidant defense enzymes was quantified by RT-PCR (A) and q RT-PCR (B). β-Actin was used as the control. All results represent the mean ± SEM from five independent experiments. GR, glutathione reductase; GPx, glutathione peroxidase; <t>GST,</t> glutathione S-transferase; SOD, superoxide dismutase. *** p
    Glutathione S Transferase Gst, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher glutathione s transferase gst protein
    <t>REP15</t> colocalizes with Rab15-GTP on early endosomal membranes. (A) Equal amounts of REP15 expressed as a fusion protein with <t>GST</t> (GST-REP15) or GST only were examined by Western analysis using a REP15 polyclonal antibody. (B) Lysates prepared from control
    Glutathione S Transferase Gst Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher glutathione s transferase
    <t>REP15</t> colocalizes with Rab15-GTP on early endosomal membranes. (A) Equal amounts of REP15 expressed as a fusion protein with <t>GST</t> (GST-REP15) or GST only were examined by Western analysis using a REP15 polyclonal antibody. (B) Lysates prepared from control
    Glutathione S Transferase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenScript glutathione s transferase gst
    <t>REP15</t> colocalizes with Rab15-GTP on early endosomal membranes. (A) Equal amounts of REP15 expressed as a fusion protein with <t>GST</t> (GST-REP15) or GST only were examined by Western analysis using a REP15 polyclonal antibody. (B) Lysates prepared from control
    Glutathione S Transferase Gst, supplied by GenScript, used in various techniques. Bioz Stars score: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glutathione s transferase gst assay kit
    Phylogram of <t>glutathione-S-transferases</t> of P. americana and other insects. Ad, Anopheles dirus ; Ag, Anopheles gambiae ; Bg, Blattella germanica ; Bm, Bombyx mori ; Dm, Drosophila melanogaster ; Dp, Dermatophagoides pteronyssinus ; Lc, Lucilia cuprina ; Lm, Locusta migratoria ; Ms, Manduca sexta ; Nl, Nilaparvata lugens ; Pa, Periplaneta americana .
    Glutathione S Transferase Gst Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Aven Tools glutathione s transferase gst
    Tudor domains of TDRD3 and SMN recognize methylated <t>Aven.</t> ( A ) Recombinant Tudor domains of TDRD3, SPF30, and SMN were fused <t>GST</t> and used in ‘pull-down’ assays with HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-AvenΔRGG. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG antibodies. ( B , C ) Lysates from HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-AvenΔRGG were IP with anti-FLAG antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting. ( D ) Aven interaction with TDRD3 and SMN was reduced in cells deficient for PRMT1 using siRNAs. FLAG-Aven was co-expressed with either siControl or siPRMT1 in U2OS cells. Anti-FLAG antibody immunoprecipitations were performed and the presence of endogenous TDRD3 and SMN monitored by immunoblotting following separation by SDS-PAGE. ( E ) PRMT1 FL/−;CreERT MEFs treated with OHT for 6 days or left untreated were lysed and IP with anti-Aven antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting (upper panels). DOI: http://dx.doi.org/10.7554/eLife.06234.007
    Glutathione S Transferase Gst, supplied by Aven Tools, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abnova glutathione s transferase gst
    Tudor domains of TDRD3 and SMN recognize methylated <t>Aven.</t> ( A ) Recombinant Tudor domains of TDRD3, SPF30, and SMN were fused <t>GST</t> and used in ‘pull-down’ assays with HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-AvenΔRGG. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG antibodies. ( B , C ) Lysates from HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-AvenΔRGG were IP with anti-FLAG antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting. ( D ) Aven interaction with TDRD3 and SMN was reduced in cells deficient for PRMT1 using siRNAs. FLAG-Aven was co-expressed with either siControl or siPRMT1 in U2OS cells. Anti-FLAG antibody immunoprecipitations were performed and the presence of endogenous TDRD3 and SMN monitored by immunoblotting following separation by SDS-PAGE. ( E ) PRMT1 FL/−;CreERT MEFs treated with OHT for 6 days or left untreated were lysed and IP with anti-Aven antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting (upper panels). DOI: http://dx.doi.org/10.7554/eLife.06234.007
    Glutathione S Transferase Gst, supplied by Abnova, used in various techniques. Bioz Stars score: 89/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti glutathione s transferase gst
    Effect of inositol-(1,4,5)triphosphate (IP 3 ) sponge inhibitor on Ca 2+ signaling in MSNs that overexpress HAP1A. (A) Immunoblots of YAC128 MSN cultures that overexpressed the glutathione S -transferase <t>(GST)-tagged</t> IP 3 R1 ligand binding region (226–604 amino acid region) with a point mutation (R441Q: m49) cloned into the pUltra-Chili vector, indicated as p49-GST, and the negative control with a point mutation (K508A: m30), indicated as p30-GST cloned into the pUltra-Chili vector or control (non-transduced) MSNs. MSN cultures on DIV14 from YAC128 mice that overexpressed p49-dTomato and p30-dTomato as an IP 3 sponge and control respectively were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. (B) DHPG-induced Ca 2+ release from the ER in YAC128 MSNs that overexpressed p49-dTomato or p30-dTomato as a control for the IP 3 sponge inhibitor. (C) Protocol to measure DHPG-induced ER Ca 2+ release and DHPG-induced SOCE using the eight-well system. MSN cultures on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP or pLenti-GFP and p49-dTomato or p30-dTomato were loaded with Fura-2AM and incubated in Ca 2 -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (D) Effect of p49-dTomato or p30-dTomato on DHPG-induced ER Ca 2+ release in YAC128 MSNs that overexpressed HAP1A-pLenti-GFP or pLenti-GFP. (E) Effect of p49-dTomato or p30-dTomato on DHPG-induced SOCE in YAC128 MSNs that overexpressed HAP1A-pLenti-GFP or pLenti-GFP. (F) To avoid the firing-effect in MSNs, 1 μM tetrodotoxin (TTX) was added during the Ca 2+ measurement protocol. In the presence of TTX, DHPG-induced ER Ca 2+ release and DHPG-induced SOCE were measured using the eight-well system. MSN cultures on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP or pLenti-GFP and p49-dTomato or p30-dTomato were loaded with Fura-2AM and incubated in Ca 2+ -free medium. Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. Normalized delta ratio was analyzed as average from three experiments (B,D) . * p
    Anti Glutathione S Transferase Gst, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione s transferase gst
    CRMP2 and CRMP4 form complexes in growth cones. (a) Growth cones lysates from rat brain were subjected to glutathione S-transferase- <t>(GST-)</t> pulldown assays using GST-CRMP2 or GST-CRMP4. The retrieved sediments were subjected to western blot analysis using the CRMP4 or CRMP2 antibodies. The GAPDH antibody was used to show equal loading. (b) Growth cones lysates from rat brain were subjected to coimmunoprecipitation (Co-IP) assays with rabbit CRMP2 and CRMP4 antibodies, and the resulting sediments were subjected to western blot analysis with mouse CRMP4 or CRMP2 antibodies. (c) Rabbit anti-CRMP2 and mouse anti-CRMP4 antibodies were used to detect endogenous proteins in the growth cones of hippocampal neurons. Scale bar: 10 μ m.
    Glutathione S Transferase Gst, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1936 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kaneka Corp glutathione s transferase gst
    CRMP2 and CRMP4 form complexes in growth cones. (a) Growth cones lysates from rat brain were subjected to glutathione S-transferase- <t>(GST-)</t> pulldown assays using GST-CRMP2 or GST-CRMP4. The retrieved sediments were subjected to western blot analysis using the CRMP4 or CRMP2 antibodies. The GAPDH antibody was used to show equal loading. (b) Growth cones lysates from rat brain were subjected to coimmunoprecipitation (Co-IP) assays with rabbit CRMP2 and CRMP4 antibodies, and the resulting sediments were subjected to western blot analysis with mouse CRMP4 or CRMP2 antibodies. (c) Rabbit anti-CRMP2 and mouse anti-CRMP4 antibodies were used to detect endogenous proteins in the growth cones of hippocampal neurons. Scale bar: 10 μ m.
    Glutathione S Transferase Gst, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology glutathione s transferase gst
    CRMP2 and CRMP4 form complexes in growth cones. (a) Growth cones lysates from rat brain were subjected to glutathione S-transferase- <t>(GST-)</t> pulldown assays using GST-CRMP2 or GST-CRMP4. The retrieved sediments were subjected to western blot analysis using the CRMP4 or CRMP2 antibodies. The GAPDH antibody was used to show equal loading. (b) Growth cones lysates from rat brain were subjected to coimmunoprecipitation (Co-IP) assays with rabbit CRMP2 and CRMP4 antibodies, and the resulting sediments were subjected to western blot analysis with mouse CRMP4 or CRMP2 antibodies. (c) Rabbit anti-CRMP2 and mouse anti-CRMP4 antibodies were used to detect endogenous proteins in the growth cones of hippocampal neurons. Scale bar: 10 μ m.
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    Millipore gst
    Schematic diagram of the GHS-based detoxification pathway in plants. MCB is used as a model substrate for conjugation to <t>GSH</t> by a <t>GST</t> in the cytoplasm and subsequent sequestration in the vacuole by a glutathione S- ).
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    Millipore glutathione s transferase gst antibodies
    Schematic diagram of the GHS-based detoxification pathway in plants. MCB is used as a model substrate for conjugation to <t>GSH</t> by a <t>GST</t> in the cytoplasm and subsequent sequestration in the vacuole by a glutathione S- ).
    Glutathione S Transferase Gst Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc glutathione s transferase gst
    Schematic diagram of the GHS-based detoxification pathway in plants. MCB is used as a model substrate for conjugation to <t>GSH</t> by a <t>GST</t> in the cytoplasm and subsequent sequestration in the vacuole by a glutathione S- ).
    Glutathione S Transferase Gst, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cayman Chemical glutathione s transferase gst
    Schematic diagram of the GHS-based detoxification pathway in plants. MCB is used as a model substrate for conjugation to <t>GSH</t> by a <t>GST</t> in the cytoplasm and subsequent sequestration in the vacuole by a glutathione S- ).
    Glutathione S Transferase Gst, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam glutathione s transferase gst
    Schematic diagram of the GHS-based detoxification pathway in plants. MCB is used as a model substrate for conjugation to <t>GSH</t> by a <t>GST</t> in the cytoplasm and subsequent sequestration in the vacuole by a glutathione S- ).
    Glutathione S Transferase Gst, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc glutathione s transferase gst
    Schematic diagram of the GHS-based detoxification pathway in plants. MCB is used as a model substrate for conjugation to <t>GSH</t> by a <t>GST</t> in the cytoplasm and subsequent sequestration in the vacuole by a glutathione S- ).
    Glutathione S Transferase Gst, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 89/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti glutathione s transferase
    DCC1 interacts with CA2. A, Interaction between DCC1 and CA2 in the yeast two-hybrid assay. Activation was observed at 3 d on selection plates (synthetic dextrose [SD]-Leu-Trp-His-Ade) with X-α-gal. B, Interaction between DCC1 and CA2 in the pull-down assay. The 10% input and <t>GST</t> pull-down proteins were detected by <t>immunoblotting</t> using anti-His antibody (top row). CA2-GST and GST proteins were detected by immunoblotting using anti-GST antibody (bottom row). C, Interaction between DCC1 and CA2 in firefly luciferase complementation assays in transiently transfected leaf of N. benthamiana . D, Interaction between DCC1 and CA2 in coimmunoprecipitation assays. The 10% input and immunoprecipitated proteins with anti-GFP (GFP-IP) were detected by immunoblotting using an anti-MYC antibody (top row). The 10% input and DCC1-GFP proteins were detected by immunoblotting using an anti-GFP antibody (bottom row).
    Anti Glutathione S Transferase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology glutathione s transferase gst prb
    (A) Autoradiographic image of electrophoretically separated [γ- 32 P]ATP-labeled <t>GST-pRB</t> fusion protein. Synchronized HeLa cells were mock infected (lanes 1, 2, 4, 6, and 8) or infected with HSV-1(F) (lanes 3, 5, 7, and 9). The cells were harvested at the times indicated, solubilized, electrophoretically separated in denaturing gel, transferred to a nitrocellulose sheet, and reacted with polyclonal anti-cdk4. Immunoprecipitates were incubated with the substrate GST-pRB in a kinase buffer supplemented with [γ- 32 P]ATP. The reaction mixtures were subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and subjected to autoradiography. The radiolabeled product was collected on a phosphorimager. (B) Lanes 10 to 12, autoradiographic image of electrophoretically separated GST-pRB labeled with [γ- 32 P]ATP by cdk4 immune precipitated from synchronized HeLa cells harvested 12 h after mock infection (lane 10) or infection with HSV-1(F) (lane 11) or R7914 (lane 12). Experimental details were the same as for panel A. Lanes 13 to 15, autoradiographic image of electrophoretically separated histone H1 labeled with [γ- 32 P]ATP by cdk2 immune precipitated from synchronized HeLa cells 12 h after mock infection (lane 13) or infection with HSV-1(F) (lane 14) or with R7914 (lane 15). Experimental details were the same as for panel A.
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    Bio-Rad glutathione s transferase gst
    (A) Autoradiographic image of electrophoretically separated [γ- 32 P]ATP-labeled <t>GST-pRB</t> fusion protein. Synchronized HeLa cells were mock infected (lanes 1, 2, 4, 6, and 8) or infected with HSV-1(F) (lanes 3, 5, 7, and 9). The cells were harvested at the times indicated, solubilized, electrophoretically separated in denaturing gel, transferred to a nitrocellulose sheet, and reacted with polyclonal anti-cdk4. Immunoprecipitates were incubated with the substrate GST-pRB in a kinase buffer supplemented with [γ- 32 P]ATP. The reaction mixtures were subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and subjected to autoradiography. The radiolabeled product was collected on a phosphorimager. (B) Lanes 10 to 12, autoradiographic image of electrophoretically separated GST-pRB labeled with [γ- 32 P]ATP by cdk4 immune precipitated from synchronized HeLa cells harvested 12 h after mock infection (lane 10) or infection with HSV-1(F) (lane 11) or R7914 (lane 12). Experimental details were the same as for panel A. Lanes 13 to 15, autoradiographic image of electrophoretically separated histone H1 labeled with [γ- 32 P]ATP by cdk2 immune precipitated from synchronized HeLa cells 12 h after mock infection (lane 13) or infection with HSV-1(F) (lane 14) or with R7914 (lane 15). Experimental details were the same as for panel A.
    Glutathione S Transferase Gst, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA glutathione s transferase gst tag
    (A) Autoradiographic image of electrophoretically separated [γ- 32 P]ATP-labeled <t>GST-pRB</t> fusion protein. Synchronized HeLa cells were mock infected (lanes 1, 2, 4, 6, and 8) or infected with HSV-1(F) (lanes 3, 5, 7, and 9). The cells were harvested at the times indicated, solubilized, electrophoretically separated in denaturing gel, transferred to a nitrocellulose sheet, and reacted with polyclonal anti-cdk4. Immunoprecipitates were incubated with the substrate GST-pRB in a kinase buffer supplemented with [γ- 32 P]ATP. The reaction mixtures were subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and subjected to autoradiography. The radiolabeled product was collected on a phosphorimager. (B) Lanes 10 to 12, autoradiographic image of electrophoretically separated GST-pRB labeled with [γ- 32 P]ATP by cdk4 immune precipitated from synchronized HeLa cells harvested 12 h after mock infection (lane 10) or infection with HSV-1(F) (lane 11) or R7914 (lane 12). Experimental details were the same as for panel A. Lanes 13 to 15, autoradiographic image of electrophoretically separated histone H1 labeled with [γ- 32 P]ATP by cdk2 immune precipitated from synchronized HeLa cells 12 h after mock infection (lane 13) or infection with HSV-1(F) (lane 14) or with R7914 (lane 15). Experimental details were the same as for panel A.
    Glutathione S Transferase Gst Tag, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione s transferase gst column
    (A) Autoradiographic image of electrophoretically separated [γ- 32 P]ATP-labeled <t>GST-pRB</t> fusion protein. Synchronized HeLa cells were mock infected (lanes 1, 2, 4, 6, and 8) or infected with HSV-1(F) (lanes 3, 5, 7, and 9). The cells were harvested at the times indicated, solubilized, electrophoretically separated in denaturing gel, transferred to a nitrocellulose sheet, and reacted with polyclonal anti-cdk4. Immunoprecipitates were incubated with the substrate GST-pRB in a kinase buffer supplemented with [γ- 32 P]ATP. The reaction mixtures were subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and subjected to autoradiography. The radiolabeled product was collected on a phosphorimager. (B) Lanes 10 to 12, autoradiographic image of electrophoretically separated GST-pRB labeled with [γ- 32 P]ATP by cdk4 immune precipitated from synchronized HeLa cells harvested 12 h after mock infection (lane 10) or infection with HSV-1(F) (lane 11) or R7914 (lane 12). Experimental details were the same as for panel A. Lanes 13 to 15, autoradiographic image of electrophoretically separated histone H1 labeled with [γ- 32 P]ATP by cdk2 immune precipitated from synchronized HeLa cells 12 h after mock infection (lane 13) or infection with HSV-1(F) (lane 14) or with R7914 (lane 15). Experimental details were the same as for panel A.
    Glutathione S Transferase Gst Column, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher glutathione s transferase gst pak1
    Mena associates with activated Rac1, and loss of Mena in cardiomyocytes significantly increases Rac1 activity. A : cardiomyocytes were incubated with <t>GST-Pak1-p21</t> binding domain (PBD) or glutathione- S -transferase (GST) alone. After pulldown, active Rac1
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    Cytoskeleton Inc glutathione s transferase rhotekin gst
    Mena associates with activated Rac1, and loss of Mena in cardiomyocytes significantly increases Rac1 activity. A : cardiomyocytes were incubated with <t>GST-Pak1-p21</t> binding domain (PBD) or glutathione- S -transferase (GST) alone. After pulldown, active Rac1
    Glutathione S Transferase Rhotekin Gst, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glutathione s transferase gst rhotekin
    Silencing of integrin β1 inhibits IGFBP3-induced migration A. The activities of Cdc42, Rac1 and RhoA were detected in IGBBP3 knockdown cells (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). Equal amounts of input protein were subjected to Western blot using anti-Cdc42 (total Cdc42), anti-Rac1 (total Rac1), and anti-RhoA (total RhoA) antibodies. Equal amounts of protein were incubated with <t>GST-PAK1</t> (detection of active Cdc42 or Rac1) and <t>GST-Rhotekin</t> (detection of active RhoA). Complexes were collected with gluthathione-Sepharose and resolved by Western blot. GTPγS was served as a positive control. Anti-GST antibodies were served as a loading control. B. The density of each band was measured by image J and normalized with the controls (LN1–1 pLKO-GFP). The activities of active small GTPase were conducted by dividing the density of active small GTPase to that of GST loading controls. The relative activities were obtained when the activity of active small GTPase in LN1–1 pLKO-GFP were set to 1. C. Representative data shows the relative migration activity of OEC-M1 cells with dominant-negative Cdc42 (Cdc42dn) and the corresponding controls (PB). The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB treated with treated with IGFBP3 and PB-Cdc42dn cells with/without IGFBP3 treatment with that in OEC-M1 PB cells. D. Representative data shows the relative migration activity of IGFBP3 expressing cells (OEC-M1 IGFBP3) and the corresponding controls (OEC-M1 PB) with anti-integrin β1 (200 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB and IGFBP3 cells treated with anti-integrin β1 or OEC-M1 IGFBP3 cells treated with IgG antibodies (200 ng/ml) to that in OEC-M1 PB cells treated with IgG antibodies. E. Immunoblot analysis of integrin β1 protein in OEC-M1 cells with ITGB1 shRNA expression (OEC-M1 ITGB1 sh3 and sh4) and vector controls (OEC-M1 pLKO-GFP). α-tubulin serves as an internal control. F. Representative data shows the relative migration activity of OEC-M1 pLKO-GFP, ITGB1 sh3 and sh4 cells upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 with ITGB1 knockdown and IGFBP3 treatment with that in the control cells. G. Representative data showed the relative migration activity of OEC-M1 treated with 10, 20 uM of PD98059 (PD) and dimethyl sulfoxide (DMSO) upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell/per field in OEC-M1 treated with PD98059 and IGFBP3 with that in the control cells. H. Immunoblot analysis revealed knockdown of ITGB1 inhibited IGFBP3-induced ERK phosphorylation at different time points in OEC-M1 cells (upper panel, 0, untreated; 10, 10 min; 30, 30 min for 100 ng/ml IGFBP3 treatment). The ratio of phosphorylated ERK/total ERK was obtained by dividing the intensity of phosphorylated ERK to that of total ERK. The relative expression was obtained when the ration of untreated cells were set to 1 (lower panel). Bar, SE; ** p
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    Image Search Results


    CD97 binds β-catenin. (A) The N-terminal (NTF) and C-terminal fragment (CTF) of CD97 were associated in DLD1 cells (left panel) and in the colon of Tg(villin-CD97) mice (right panel). Input control and immunoprecipitates (IP), obtained either with IgG or with CD97 NTF- and CTF-specific antibodies (Abs), were analyzed with a CD97 Ab detecting the other fragment, as indicated. (B,C) CD97 and β-catenin co-immunoprecipitated in CD97-positive DLD1 cells [ (B) , left panel] and in colonic lysates of Tg(villin-CD97) mice [ (C) , left panel]. HEK293 cells, only slightly positive for CD97 [ (B) , right panel], and CD97 knock-out (Ko) mice [ (C) , right panel] served as negative controls. (D) Next to β-catenin, CD97 co-immunoprecipitated with E-cadherin (Ecad) in DLD1 cells (upper picture) and with Ecad and p120-catenin in Tg(villin-CD97) mice (lower picture). (E) upper panel: beads loaded with glutathione S-transferase (GST) or β-catenin-GST were incubated with lysates of HT1080 CD97(EGF125), CD97(EGF125/TM2) cells with a C-terminal truncated CD97 ( 15 ), and mock cells. Bound proteins were eluted and CD97 was detected with NTF or CTF CD97-specific Abs by western blotting. Pulldown could be demonstrated only with β-catenin-GST and lysates of HT1080 cells expressing full-length (TM7), but not expressing C-terminal truncated (TM2) CD97. In the lower panel the respective elution controls for β-catenin and GST are shown.

    Journal: Frontiers in Oncology

    Article Title: The Interaction of CD97/ADGRE5 With β-Catenin in Adherens Junctions Is Lost During Colorectal Carcinogenesis

    doi: 10.3389/fonc.2018.00182

    Figure Lengend Snippet: CD97 binds β-catenin. (A) The N-terminal (NTF) and C-terminal fragment (CTF) of CD97 were associated in DLD1 cells (left panel) and in the colon of Tg(villin-CD97) mice (right panel). Input control and immunoprecipitates (IP), obtained either with IgG or with CD97 NTF- and CTF-specific antibodies (Abs), were analyzed with a CD97 Ab detecting the other fragment, as indicated. (B,C) CD97 and β-catenin co-immunoprecipitated in CD97-positive DLD1 cells [ (B) , left panel] and in colonic lysates of Tg(villin-CD97) mice [ (C) , left panel]. HEK293 cells, only slightly positive for CD97 [ (B) , right panel], and CD97 knock-out (Ko) mice [ (C) , right panel] served as negative controls. (D) Next to β-catenin, CD97 co-immunoprecipitated with E-cadherin (Ecad) in DLD1 cells (upper picture) and with Ecad and p120-catenin in Tg(villin-CD97) mice (lower picture). (E) upper panel: beads loaded with glutathione S-transferase (GST) or β-catenin-GST were incubated with lysates of HT1080 CD97(EGF125), CD97(EGF125/TM2) cells with a C-terminal truncated CD97 ( 15 ), and mock cells. Bound proteins were eluted and CD97 was detected with NTF or CTF CD97-specific Abs by western blotting. Pulldown could be demonstrated only with β-catenin-GST and lysates of HT1080 cells expressing full-length (TM7), but not expressing C-terminal truncated (TM2) CD97. In the lower panel the respective elution controls for β-catenin and GST are shown.

    Article Snippet: Antibodies (Abs) The following Abs were used: Ecad (sc-7870, Santa Cruz, Heidelberg, Germany), glutathione S-transferase (GST) (MA4-004; Thermo Fisher Scientific, Darmstadt, Germany), α-catenin (GTX22981, GeneTex, Irvine, CA, USA), β-catenin (sc-7199; Santa Cruz; 610153; BD Transduction Laboratories, Heidelberg, Germany), p120-catenin (sc-13957, Santa Cruz), α-tubulin (T9026, Sigma-Aldrich, Munich, Germany), and ZO-1 (61-7300, Thermo Fisher).

    Techniques: Mouse Assay, Immunoprecipitation, Knock-Out, Incubation, Western Blot, Expressing

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

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

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

    doi: 10.4196/kjpp.2019.23.6.519

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

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

    Techniques: Reverse Transcription Polymerase Chain Reaction

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

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

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

    doi: 10.4196/kjpp.2019.23.6.519

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

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

    Techniques: Activity Assay

    Comparison of the differences in the protein expression of (A) glutathione-S-transferase (GST) and (B) mRNA expression of interleukin-1β (IL-1β), (C) tumor necrosis factor-α (TNF-α), and (D) cyclooxygenase-2 (COX-2). Figure 3A shows the representative band indicative of changes in the expression of GST by western blot analysis. Figure 3B, C, and D show results obtained by extracting mRNA via reverse transcription-PCR assay to measure the expression of each gene. All results were obtained with 3 independent experiments and values are displayed as mean±SD. * and † indicate P

    Journal: Intestinal Research

    Article Title: Expression of Heat Shock Proteins and Cytokines in Response to Ethanol Induced Damage in the Small Intestine of ICR Mice

    doi: 10.5217/ir.2014.12.3.205

    Figure Lengend Snippet: Comparison of the differences in the protein expression of (A) glutathione-S-transferase (GST) and (B) mRNA expression of interleukin-1β (IL-1β), (C) tumor necrosis factor-α (TNF-α), and (D) cyclooxygenase-2 (COX-2). Figure 3A shows the representative band indicative of changes in the expression of GST by western blot analysis. Figure 3B, C, and D show results obtained by extracting mRNA via reverse transcription-PCR assay to measure the expression of each gene. All results were obtained with 3 independent experiments and values are displayed as mean±SD. * and † indicate P

    Article Snippet: Once transferred, polyvinylidene fluoride membranes were blocked with 5% blocking buffer (i.e., non-fat dry milk) for 30 minutes and then incubated with primary antibody diluted into blocking buffer for 16 hours at 4℃ (HSP40, HSP70, and HSP90 antibody, 1:1,000, Stressgen, Ann Arbor, MI, USA; HSP32 [heme oxygenase-1, HO-1] antibody, 1:1,000, Abcam, Cambridge, MA, USA; glutathione-S-transferase [GST] antibody, 1:1,000, Sigma, St. Louis, MO, USA; β-actin antibody, 1:2,500, Sigma, St. Louis, MO, USA).

    Techniques: Expressing, Western Blot, Polymerase Chain Reaction

    Foetal muscles. Alkaline phosphatase (A) , glutathione-S-transferase (B) , catalase (C) , glutathione (D) , malondialdehyde (E) levels and the MDA/GST ratio (F) of the foetal muscle from Walker 256 carcinoma-bearing pregnant rats that were subjected to a leucine-rich or control diet. Legend: C – control rats; L – rats fed a leucine-rich diet; W – tumour-bearing rats; LW – tumour-bearing rats fed a leucine-rich diet; Cp – rats subjected to a similar, paired-nutrition intake of rats in group W; and Lp – rats subjected to leucine-rich diet and nutrition that was paired to the intake of rats in Group LW. * P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Leucine-rich diet supplementation modulates foetal muscle protein metabolism impaired by Walker-256 tumour

    doi: 10.1186/1477-7827-12-2

    Figure Lengend Snippet: Foetal muscles. Alkaline phosphatase (A) , glutathione-S-transferase (B) , catalase (C) , glutathione (D) , malondialdehyde (E) levels and the MDA/GST ratio (F) of the foetal muscle from Walker 256 carcinoma-bearing pregnant rats that were subjected to a leucine-rich or control diet. Legend: C – control rats; L – rats fed a leucine-rich diet; W – tumour-bearing rats; LW – tumour-bearing rats fed a leucine-rich diet; Cp – rats subjected to a similar, paired-nutrition intake of rats in group W; and Lp – rats subjected to leucine-rich diet and nutrition that was paired to the intake of rats in Group LW. * P

    Article Snippet: Aliquots of the muscle-homogenate supernatant were analysed for glutathione-S-transferase (GST) activity based on the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB; Sigma) with glutathione, and the activity was expressed as nmol·μg protein-1 ·min-1 using an extinction coefficient of 9.6 as previously described [ ].

    Techniques: Multiple Displacement Amplification

    REP15 colocalizes with Rab15-GTP on early endosomal membranes. (A) Equal amounts of REP15 expressed as a fusion protein with GST (GST-REP15) or GST only were examined by Western analysis using a REP15 polyclonal antibody. (B) Lysates prepared from control

    Journal:

    Article Title: Rab15 Effector Protein: A Novel Protein for Receptor Recycling from the Endocytic Recycling Compartment D⃞

    doi: 10.1091/mbc.E05-03-0204

    Figure Lengend Snippet: REP15 colocalizes with Rab15-GTP on early endosomal membranes. (A) Equal amounts of REP15 expressed as a fusion protein with GST (GST-REP15) or GST only were examined by Western analysis using a REP15 polyclonal antibody. (B) Lysates prepared from control

    Article Snippet: The resulting REP15 anti-sera was purified by initial passage over GST immobilized on AminoLink coupling gel, and the flow through was subsequently purified against GST-REP15 immobilized to AminoLink coupling gel (Pierce Chemical, Rockville, IL).

    Techniques: Western Blot

    Generation and characterization of a polyclonal antibody against exon 33L. A , The 22-amino acid polypeptides encoded by exon 33L were fused with GST ( GST-33L ) and purified for rabbit immunization. BSA standards of 1, 2, and 5 μg are shown. M ,

    Journal: The Journal of Biological Chemistry

    Article Title: Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart *

    doi: 10.1074/jbc.M114.594911

    Figure Lengend Snippet: Generation and characterization of a polyclonal antibody against exon 33L. A , The 22-amino acid polypeptides encoded by exon 33L were fused with GST ( GST-33L ) and purified for rabbit immunization. BSA standards of 1, 2, and 5 μg are shown. M ,

    Article Snippet: Serum collected after immunization was preabsorbed with GST protein first to remove GST antibodies, and 33L polyclonal antibody was affinity-purified from immobilized 33L protein with an IgG elution buffer (Pierce).

    Techniques: Purification

    Zfp296 is a component of heterochromatin in mammalian cells. ( A ) Immunofluorescence staining of an E9.75 wild-type embryo for Zfp296. Nuclei were counterstained with DAPI. Scale bar = 10 μm. Right panel shows a magnified view. Scale bar = 2 μm. ( B ) Domain structure and deletion mutants of Zfp296 used in this study. Mouse Zfp296 consists of 445 amino acid residues and has one C2H2 zinc finger domain and five CCHC zinc finger domains. ΔZF 2–3 has an internal deletion of amino acid residues 213–267. ( C ) Immunofluorescence staining of primary MEFs transfected with a plasmid vector expressing Flag-tagged wild-type Zfp296 or its mutant constructs shown in ( B ). Nuclei were stained with an anti-Flag antibody followed by a fluorescein-labeled second antibody, counterstained with DAPI, and observed by fluorescence microscopy. ( D ) GST pull-down assays. Control GST protein or GST-Zfp296 fusion protein was incubated with ES cell nuclear extracts, and the pulled-down samples were subjected to LC-MS/MS shotgun proteomics. Venn diagram shows the number of proteins identified in two experiments (minimum value of total spectrum counts was normalized to 2, 2-fold change cut-off). *p

    Journal: Scientific Reports

    Article Title: Zfp296 negatively regulates H3K9 methylation in embryonic development as a component of heterochromatin

    doi: 10.1038/s41598-017-12772-y

    Figure Lengend Snippet: Zfp296 is a component of heterochromatin in mammalian cells. ( A ) Immunofluorescence staining of an E9.75 wild-type embryo for Zfp296. Nuclei were counterstained with DAPI. Scale bar = 10 μm. Right panel shows a magnified view. Scale bar = 2 μm. ( B ) Domain structure and deletion mutants of Zfp296 used in this study. Mouse Zfp296 consists of 445 amino acid residues and has one C2H2 zinc finger domain and five CCHC zinc finger domains. ΔZF 2–3 has an internal deletion of amino acid residues 213–267. ( C ) Immunofluorescence staining of primary MEFs transfected with a plasmid vector expressing Flag-tagged wild-type Zfp296 or its mutant constructs shown in ( B ). Nuclei were stained with an anti-Flag antibody followed by a fluorescein-labeled second antibody, counterstained with DAPI, and observed by fluorescence microscopy. ( D ) GST pull-down assays. Control GST protein or GST-Zfp296 fusion protein was incubated with ES cell nuclear extracts, and the pulled-down samples were subjected to LC-MS/MS shotgun proteomics. Venn diagram shows the number of proteins identified in two experiments (minimum value of total spectrum counts was normalized to 2, 2-fold change cut-off). *p

    Article Snippet: GST pull-down experiments and mass spectrometry Recombinant GST-Zfp296 or GST proteins were purified in hypertonic buffer using glutathione magnetic beads (Pierce).

    Techniques: Immunofluorescence, Staining, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Construct, Labeling, Fluorescence, Microscopy, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Phylogram of glutathione-S-transferases of P. americana and other insects. Ad, Anopheles dirus ; Ag, Anopheles gambiae ; Bg, Blattella germanica ; Bm, Bombyx mori ; Dm, Drosophila melanogaster ; Dp, Dermatophagoides pteronyssinus ; Lc, Lucilia cuprina ; Lm, Locusta migratoria ; Ms, Manduca sexta ; Nl, Nilaparvata lugens ; Pa, Periplaneta americana .

    Journal: Scientific Reports

    Article Title: Glutathione S-transferase (GST) of American Cockroach, Periplaneta americana: Classes, Isoforms, and Allergenicity

    doi: 10.1038/s41598-017-18759-z

    Figure Lengend Snippet: Phylogram of glutathione-S-transferases of P. americana and other insects. Ad, Anopheles dirus ; Ag, Anopheles gambiae ; Bg, Blattella germanica ; Bm, Bombyx mori ; Dm, Drosophila melanogaster ; Dp, Dermatophagoides pteronyssinus ; Lc, Lucilia cuprina ; Lm, Locusta migratoria ; Ms, Manduca sexta ; Nl, Nilaparvata lugens ; Pa, Periplaneta americana .

    Article Snippet: Determination of enzymatic activity of GST preparations Enzymatic activities of the nGST and the rGST were determined by using glutathione S -transferase assay kit (Sigma-Aldrich., MO, USA) which the 1-chloro-2,4-dinitrobenzene (CDNB) was used as the enzymatic substrate.

    Techniques: Mass Spectrometry

    Growth kinetics of yeast clones and GrB mRNA expression. (A) Cultures of P. pastoris carrying pPIC9-GrB, pPIC9-GST-furS-GrB, pPIC9-MBP-furS-GrB, or empty pPIC9 vector were induced with methanol. Samples were taken every 24 h, and OD 600 was determined as a measure for cell density. (B) Quantification of GrB mRNA levels. RNA was isolated from yeast cells carrying pPIC9-GrB, pPIC9-GST-furS-GrB, pPIC9-MBP-furS-GrB, or empty pPIC9 vector after induction with methanol for one day. cDNA was synthesized and analyzed by quantitative real-time PCR including GAPDH for normalization. Means of duplicate samples are shown. Error bars indicate SD.

    Journal: PLoS ONE

    Article Title: Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

    doi: 10.1371/journal.pone.0014404

    Figure Lengend Snippet: Growth kinetics of yeast clones and GrB mRNA expression. (A) Cultures of P. pastoris carrying pPIC9-GrB, pPIC9-GST-furS-GrB, pPIC9-MBP-furS-GrB, or empty pPIC9 vector were induced with methanol. Samples were taken every 24 h, and OD 600 was determined as a measure for cell density. (B) Quantification of GrB mRNA levels. RNA was isolated from yeast cells carrying pPIC9-GrB, pPIC9-GST-furS-GrB, pPIC9-MBP-furS-GrB, or empty pPIC9 vector after induction with methanol for one day. cDNA was synthesized and analyzed by quantitative real-time PCR including GAPDH for normalization. Means of duplicate samples are shown. Error bars indicate SD.

    Article Snippet: Glutathione-S-transferase activity assay Enzymatic activity of MBP-fur-GST and MBP-furS-GST proteins was analyzed using a GST assay kit (Sigma-Aldrich) following the manufacturer's recommendations.

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Isolation, Synthesized, Real-time Polymerase Chain Reaction

    Analysis of GrB secreted into the culture medium. (A) Supernatants of yeast cultures harboring pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected at the indicated time points after induction with methanol, and analyzed by immunoblotting with GrB-specific antibody. Band intensities were quantified by determining mean gray values (MGV) relative to the highest value obtained. (B) Expression of an ErbB2 protein fragment. Yeast cells carrying pPIC9-ErbB2 222 or pPIC9-MBP-furS-ErbB2 222 were induced with methanol, and ErbB2 protein in culture supernatants was analyzed by immunoblotting with ErbB2-specific antibody.

    Journal: PLoS ONE

    Article Title: Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

    doi: 10.1371/journal.pone.0014404

    Figure Lengend Snippet: Analysis of GrB secreted into the culture medium. (A) Supernatants of yeast cultures harboring pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected at the indicated time points after induction with methanol, and analyzed by immunoblotting with GrB-specific antibody. Band intensities were quantified by determining mean gray values (MGV) relative to the highest value obtained. (B) Expression of an ErbB2 protein fragment. Yeast cells carrying pPIC9-ErbB2 222 or pPIC9-MBP-furS-ErbB2 222 were induced with methanol, and ErbB2 protein in culture supernatants was analyzed by immunoblotting with ErbB2-specific antibody.

    Article Snippet: Glutathione-S-transferase activity assay Enzymatic activity of MBP-fur-GST and MBP-furS-GST proteins was analyzed using a GST assay kit (Sigma-Aldrich) following the manufacturer's recommendations.

    Techniques: Expressing

    Secretion of GST and MBP into the culture medium. The presence of GST and MBP in yeast culture supernatants after induction for the indicated time periods was analyzed by immunoblotting with GST-specific (A) or MBP-specific antibodies (B). (C) Intracellular accumulation of GrB and GrB fusion proteins was investigated by analysis of cell lysates with GrB-specific antibody. The positions of the different GrB proteins are indicated by arrows. Samples analyzed were from the same cultures examined for secretion of GrB as shown in Fig. 2A .

    Journal: PLoS ONE

    Article Title: Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

    doi: 10.1371/journal.pone.0014404

    Figure Lengend Snippet: Secretion of GST and MBP into the culture medium. The presence of GST and MBP in yeast culture supernatants after induction for the indicated time periods was analyzed by immunoblotting with GST-specific (A) or MBP-specific antibodies (B). (C) Intracellular accumulation of GrB and GrB fusion proteins was investigated by analysis of cell lysates with GrB-specific antibody. The positions of the different GrB proteins are indicated by arrows. Samples analyzed were from the same cultures examined for secretion of GrB as shown in Fig. 2A .

    Article Snippet: Glutathione-S-transferase activity assay Enzymatic activity of MBP-fur-GST and MBP-furS-GST proteins was analyzed using a GST assay kit (Sigma-Aldrich) following the manufacturer's recommendations.

    Techniques:

    Expression of GrB in the yeast Pichia pastoris . (A) Constructs for expression of human granzyme B, and MBP-GrB and GST-GrB fusion proteins. AOX1 , methanol-inducible alcohol oxidase I promoter; SP, α-factor signal peptide; furS, furin recognition motif; M, Myc tag; H, polyhistidine tag. (B) Culture supernatants of yeast clones carrying pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected after induction with methanol for 3 days, and expression of GrB was analyzed by immunoblotting with GrB-specific antibody. Each lane represents an individual clone. Clones marked with an asterisk were used in subsequent experiments. (C) For comparison, culture supernatants of clones carrying the different expression constructs were analyzed together in a single immunoblot experiment as indicated. Supernatant of a yeast clone carrying empty pPIC9 served as a control.

    Journal: PLoS ONE

    Article Title: Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

    doi: 10.1371/journal.pone.0014404

    Figure Lengend Snippet: Expression of GrB in the yeast Pichia pastoris . (A) Constructs for expression of human granzyme B, and MBP-GrB and GST-GrB fusion proteins. AOX1 , methanol-inducible alcohol oxidase I promoter; SP, α-factor signal peptide; furS, furin recognition motif; M, Myc tag; H, polyhistidine tag. (B) Culture supernatants of yeast clones carrying pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected after induction with methanol for 3 days, and expression of GrB was analyzed by immunoblotting with GrB-specific antibody. Each lane represents an individual clone. Clones marked with an asterisk were used in subsequent experiments. (C) For comparison, culture supernatants of clones carrying the different expression constructs were analyzed together in a single immunoblot experiment as indicated. Supernatant of a yeast clone carrying empty pPIC9 served as a control.

    Article Snippet: Glutathione-S-transferase activity assay Enzymatic activity of MBP-fur-GST and MBP-furS-GST proteins was analyzed using a GST assay kit (Sigma-Aldrich) following the manufacturer's recommendations.

    Techniques: Expressing, Construct, Clone Assay

    Processing and enzymatic activity of MBP-GST fusion proteins. (A) Culture supernatants of representative yeast clones carrying pPIC9-MBP-fur-GST or pPIC9-MBP-furS-GST were collected after induction with methanol for 3 days, and expression and processing of the MBP-GST fusion proteins was analyzed by immunoblotting with GST-specific (left panel) or MBP-specific antibody (right panel). Supernatant of yeast cells carrying empty pPIC9 served as a control. (B) Enzymatic activity of MBP-GST fusion proteins and kinetics of conjugation of glutathione to synthetic CDNB substrate was analyzed in a colorimetric assay. The MBP-fur-GST or MBP-furS-GST containing supernatants analyzed in (A) were tested as indicated. Commercially available recombinant GST was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 served as negative control. Means of triplicate samples are shown. Error bars indicate SEM.

    Journal: PLoS ONE

    Article Title: Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

    doi: 10.1371/journal.pone.0014404

    Figure Lengend Snippet: Processing and enzymatic activity of MBP-GST fusion proteins. (A) Culture supernatants of representative yeast clones carrying pPIC9-MBP-fur-GST or pPIC9-MBP-furS-GST were collected after induction with methanol for 3 days, and expression and processing of the MBP-GST fusion proteins was analyzed by immunoblotting with GST-specific (left panel) or MBP-specific antibody (right panel). Supernatant of yeast cells carrying empty pPIC9 served as a control. (B) Enzymatic activity of MBP-GST fusion proteins and kinetics of conjugation of glutathione to synthetic CDNB substrate was analyzed in a colorimetric assay. The MBP-fur-GST or MBP-furS-GST containing supernatants analyzed in (A) were tested as indicated. Commercially available recombinant GST was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 served as negative control. Means of triplicate samples are shown. Error bars indicate SEM.

    Article Snippet: Glutathione-S-transferase activity assay Enzymatic activity of MBP-fur-GST and MBP-furS-GST proteins was analyzed using a GST assay kit (Sigma-Aldrich) following the manufacturer's recommendations.

    Techniques: Activity Assay, Clone Assay, Expressing, Conjugation Assay, Colorimetric Assay, Recombinant, Positive Control, Negative Control

    Measurement of GST activity and metabolite profiling of wheat roots subjected to Fe starvation. (A) Glutathione S -transferase activity of wheat roots under Fe starvation (–Fe) and control (+Fe) conditions. (B) Metabolite profiling of amino acids, sugars, polyols, organic acids, and related compounds. Change in abundance of significant ( P

    Journal: Journal of Experimental Botany

    Article Title: Integrative analysis of hexaploid wheat roots identifies signature components during iron starvation

    doi: 10.1093/jxb/erz358

    Figure Lengend Snippet: Measurement of GST activity and metabolite profiling of wheat roots subjected to Fe starvation. (A) Glutathione S -transferase activity of wheat roots under Fe starvation (–Fe) and control (+Fe) conditions. (B) Metabolite profiling of amino acids, sugars, polyols, organic acids, and related compounds. Change in abundance of significant ( P

    Article Snippet: The estimation was done using the glutathione S -transferases assay kit (Sigma, USA).

    Techniques: Activity Assay

    Tudor domains of TDRD3 and SMN recognize methylated Aven. ( A ) Recombinant Tudor domains of TDRD3, SPF30, and SMN were fused GST and used in ‘pull-down’ assays with HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-AvenΔRGG. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG antibodies. ( B , C ) Lysates from HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-AvenΔRGG were IP with anti-FLAG antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting. ( D ) Aven interaction with TDRD3 and SMN was reduced in cells deficient for PRMT1 using siRNAs. FLAG-Aven was co-expressed with either siControl or siPRMT1 in U2OS cells. Anti-FLAG antibody immunoprecipitations were performed and the presence of endogenous TDRD3 and SMN monitored by immunoblotting following separation by SDS-PAGE. ( E ) PRMT1 FL/−;CreERT MEFs treated with OHT for 6 days or left untreated were lysed and IP with anti-Aven antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting (upper panels). DOI: http://dx.doi.org/10.7554/eLife.06234.007

    Journal: eLife

    Article Title: Aven recognition of RNA G-quadruplexes regulates translation of the mixed lineage leukemia protooncogenes

    doi: 10.7554/eLife.06234

    Figure Lengend Snippet: Tudor domains of TDRD3 and SMN recognize methylated Aven. ( A ) Recombinant Tudor domains of TDRD3, SPF30, and SMN were fused GST and used in ‘pull-down’ assays with HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-AvenΔRGG. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG antibodies. ( B , C ) Lysates from HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-AvenΔRGG were IP with anti-FLAG antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting. ( D ) Aven interaction with TDRD3 and SMN was reduced in cells deficient for PRMT1 using siRNAs. FLAG-Aven was co-expressed with either siControl or siPRMT1 in U2OS cells. Anti-FLAG antibody immunoprecipitations were performed and the presence of endogenous TDRD3 and SMN monitored by immunoblotting following separation by SDS-PAGE. ( E ) PRMT1 FL/−;CreERT MEFs treated with OHT for 6 days or left untreated were lysed and IP with anti-Aven antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting (upper panels). DOI: http://dx.doi.org/10.7554/eLife.06234.007

    Article Snippet: An in vitro methylation assay was performed using a glutathione-S-transferase (GST)-Aven RGG/RG fusion protein incubated with recombinant GST-PRMT1 in the presence of (methyl -3 H)-S-adenosyl-L-methionine.

    Techniques: Methylation, Recombinant, Expressing, SDS Page, Immunoprecipitation

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

    Journal: Frontiers in Cellular Neuroscience

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

    doi: 10.3389/fncel.2018.00381

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

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

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

    CRMP2 and CRMP4 form complexes in growth cones. (a) Growth cones lysates from rat brain were subjected to glutathione S-transferase- (GST-) pulldown assays using GST-CRMP2 or GST-CRMP4. The retrieved sediments were subjected to western blot analysis using the CRMP4 or CRMP2 antibodies. The GAPDH antibody was used to show equal loading. (b) Growth cones lysates from rat brain were subjected to coimmunoprecipitation (Co-IP) assays with rabbit CRMP2 and CRMP4 antibodies, and the resulting sediments were subjected to western blot analysis with mouse CRMP4 or CRMP2 antibodies. (c) Rabbit anti-CRMP2 and mouse anti-CRMP4 antibodies were used to detect endogenous proteins in the growth cones of hippocampal neurons. Scale bar: 10 μ m.

    Journal: Neural Plasticity

    Article Title: CRMP4 and CRMP2 Interact to Coordinate Cytoskeleton Dynamics, Regulating Growth Cone Development and Axon Elongation

    doi: 10.1155/2015/947423

    Figure Lengend Snippet: CRMP2 and CRMP4 form complexes in growth cones. (a) Growth cones lysates from rat brain were subjected to glutathione S-transferase- (GST-) pulldown assays using GST-CRMP2 or GST-CRMP4. The retrieved sediments were subjected to western blot analysis using the CRMP4 or CRMP2 antibodies. The GAPDH antibody was used to show equal loading. (b) Growth cones lysates from rat brain were subjected to coimmunoprecipitation (Co-IP) assays with rabbit CRMP2 and CRMP4 antibodies, and the resulting sediments were subjected to western blot analysis with mouse CRMP4 or CRMP2 antibodies. (c) Rabbit anti-CRMP2 and mouse anti-CRMP4 antibodies were used to detect endogenous proteins in the growth cones of hippocampal neurons. Scale bar: 10 μ m.

    Article Snippet: The CRMPs or truncated CRMPs tagged with glutathione S-transferase (GST) at the N-terminus were generated by subcloning into the pGEX-4T-1 vector (Amersham Pharmacia Biotech, Buckinghamshire, UK).

    Techniques: Western Blot, Co-Immunoprecipitation Assay

    CRMP2 and CRMP4 interact with tubulin and actin. (a) Bacterial recombinant glutathione S-transferase- (GST-) CRMPs were purified and subjected to GST-pulldown assays with growth cone extracts from rat brain. Coomassie blue staining of GST and GST-CRMPs (bottom) showed equal loading of bait proteins. The pulldown sediments were subjected to western blot assays with tubulin and actin antibodies using GAPDH as the lysate input control. Each result is representative of three to five separate experiments with similar results. (b) Growth cone lysates from rat brain were subjected to coimmunoprecipitation (Co-IP) with tubulin or actin antibody and then processed for western blot (WB) analysis with the indicated antibodies. (c) Growth cones lysates were immunoprecipitated with CRMP2 or CRMP4 antibodies and processed for WB assays to detect the indicated proteins using the appropriate antibodies.

    Journal: Neural Plasticity

    Article Title: CRMP4 and CRMP2 Interact to Coordinate Cytoskeleton Dynamics, Regulating Growth Cone Development and Axon Elongation

    doi: 10.1155/2015/947423

    Figure Lengend Snippet: CRMP2 and CRMP4 interact with tubulin and actin. (a) Bacterial recombinant glutathione S-transferase- (GST-) CRMPs were purified and subjected to GST-pulldown assays with growth cone extracts from rat brain. Coomassie blue staining of GST and GST-CRMPs (bottom) showed equal loading of bait proteins. The pulldown sediments were subjected to western blot assays with tubulin and actin antibodies using GAPDH as the lysate input control. Each result is representative of three to five separate experiments with similar results. (b) Growth cone lysates from rat brain were subjected to coimmunoprecipitation (Co-IP) with tubulin or actin antibody and then processed for western blot (WB) analysis with the indicated antibodies. (c) Growth cones lysates were immunoprecipitated with CRMP2 or CRMP4 antibodies and processed for WB assays to detect the indicated proteins using the appropriate antibodies.

    Article Snippet: The CRMPs or truncated CRMPs tagged with glutathione S-transferase (GST) at the N-terminus were generated by subcloning into the pGEX-4T-1 vector (Amersham Pharmacia Biotech, Buckinghamshire, UK).

    Techniques: Recombinant, Purification, Staining, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

    ( A ) Western blot of preS-pulldowned proteins; ( B ) Western blot of SCCA1-pulldowned proteins; ( C ) Western blot of FTL-pulldowned proteins. For A, MBP-preS was pre-incubated with either GST-FTL or GST before mixing with amylose beads; for B, MBP-SCCA1 was pre-incubated with either GST-FTL or GST before mixing with amylose beads; for C, HA-tagged preS and SCCA1 were coexpressed with His-tagged FTL protein in HepG2 cells before immunoprecipitation by anti-His-tag antibody. Abbreviations: MBP, maltose binding protein; GST, glutathione-S-transferase; FTL, ferritin light chain; SCCA1, squamous cell carcinoma antigen 1.

    Journal: International Journal of Nanomedicine

    Article Title: Ferritin light chain and squamous cell carcinoma antigen 1 are coreceptors for cellular attachment and entry of hepatitis B virus

    doi: 10.2147/IJN.S27803

    Figure Lengend Snippet: ( A ) Western blot of preS-pulldowned proteins; ( B ) Western blot of SCCA1-pulldowned proteins; ( C ) Western blot of FTL-pulldowned proteins. For A, MBP-preS was pre-incubated with either GST-FTL or GST before mixing with amylose beads; for B, MBP-SCCA1 was pre-incubated with either GST-FTL or GST before mixing with amylose beads; for C, HA-tagged preS and SCCA1 were coexpressed with His-tagged FTL protein in HepG2 cells before immunoprecipitation by anti-His-tag antibody. Abbreviations: MBP, maltose binding protein; GST, glutathione-S-transferase; FTL, ferritin light chain; SCCA1, squamous cell carcinoma antigen 1.

    Article Snippet: Protein–protein interaction assay For the in vitro protein–protein interaction study, fusion proteins of preS, FTL, and SCCA1 with glutathione-S-transferase (GST)-tag, maltose binding protein (MBP)-tag, and histidine (His)-tag were expressed in E. coli through pGEX-4T-1 (carrying GST-tag, Amersham Biosciences, Amersham, UK), pMal-c2x (carrying MBP-tag, New England Biolabs, Ipswich, MA), and pET-28a (carrying His-tag, Merck, Darmstadt, Germany), respectively.

    Techniques: Western Blot, Incubation, Immunoprecipitation, Binding Assay

    ( A ) Determination of regions on FTL for interaction with preS and SCCA1. GST-tagged FTL deletion mutant proteins, each with deletion of one of the five α-helices (a to e), were allowed to bind to MBP-preS and His-SCCA1, and absorbed with Gutathione–Sepharose TM beads. Proteins absorbed on Glutathione–Sepharose beads were subjected to Western blotting with anti-MBP antibody (detecting MBP-preS, upper), or with His-tag antisera (detecting His-SCCA1, down). ( B ) Verification of FTL-binding activity of N-terminal 1–11 amino acids of preS2. For the upper, pulldown assay was done by mixing GST-FTL with MBP-preS2 (lane 1) or MBP-preS2Δ (1–11) (deletion of N-terminal 1–11 amino acids of preS2, lane 2), and the proteins recovered by Glutathione–Sepharose beads were subjected to Western blot with anti-MBP antibody. For the down, pulldown assay was done by mixing GST-FTL with MBP (lane 1) or MBP-peptide (1–11) (MBP with the peptide of 11 amino acids from N-terminus of preS2, lane2), and the proteins recovered by Glutathione-Sepharose beads were subjected to Western blot with anti-MBP antibody. Abbreviations: FTL, ferritin light chain; MBP, maltose binding protein; SCCA1, squamous cell carcinoma antigen 1; GST, glutathione-S-transferase.

    Journal: International Journal of Nanomedicine

    Article Title: Ferritin light chain and squamous cell carcinoma antigen 1 are coreceptors for cellular attachment and entry of hepatitis B virus

    doi: 10.2147/IJN.S27803

    Figure Lengend Snippet: ( A ) Determination of regions on FTL for interaction with preS and SCCA1. GST-tagged FTL deletion mutant proteins, each with deletion of one of the five α-helices (a to e), were allowed to bind to MBP-preS and His-SCCA1, and absorbed with Gutathione–Sepharose TM beads. Proteins absorbed on Glutathione–Sepharose beads were subjected to Western blotting with anti-MBP antibody (detecting MBP-preS, upper), or with His-tag antisera (detecting His-SCCA1, down). ( B ) Verification of FTL-binding activity of N-terminal 1–11 amino acids of preS2. For the upper, pulldown assay was done by mixing GST-FTL with MBP-preS2 (lane 1) or MBP-preS2Δ (1–11) (deletion of N-terminal 1–11 amino acids of preS2, lane 2), and the proteins recovered by Glutathione–Sepharose beads were subjected to Western blot with anti-MBP antibody. For the down, pulldown assay was done by mixing GST-FTL with MBP (lane 1) or MBP-peptide (1–11) (MBP with the peptide of 11 amino acids from N-terminus of preS2, lane2), and the proteins recovered by Glutathione-Sepharose beads were subjected to Western blot with anti-MBP antibody. Abbreviations: FTL, ferritin light chain; MBP, maltose binding protein; SCCA1, squamous cell carcinoma antigen 1; GST, glutathione-S-transferase.

    Article Snippet: Protein–protein interaction assay For the in vitro protein–protein interaction study, fusion proteins of preS, FTL, and SCCA1 with glutathione-S-transferase (GST)-tag, maltose binding protein (MBP)-tag, and histidine (His)-tag were expressed in E. coli through pGEX-4T-1 (carrying GST-tag, Amersham Biosciences, Amersham, UK), pMal-c2x (carrying MBP-tag, New England Biolabs, Ipswich, MA), and pET-28a (carrying His-tag, Merck, Darmstadt, Germany), respectively.

    Techniques: Mutagenesis, Western Blot, Binding Assay, Activity Assay

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

    Journal: Plant Physiology

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

    doi: 10.1104/pp.104.056168

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

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

    Techniques: Conjugation Assay

    DCC1 interacts with CA2. A, Interaction between DCC1 and CA2 in the yeast two-hybrid assay. Activation was observed at 3 d on selection plates (synthetic dextrose [SD]-Leu-Trp-His-Ade) with X-α-gal. B, Interaction between DCC1 and CA2 in the pull-down assay. The 10% input and GST pull-down proteins were detected by immunoblotting using anti-His antibody (top row). CA2-GST and GST proteins were detected by immunoblotting using anti-GST antibody (bottom row). C, Interaction between DCC1 and CA2 in firefly luciferase complementation assays in transiently transfected leaf of N. benthamiana . D, Interaction between DCC1 and CA2 in coimmunoprecipitation assays. The 10% input and immunoprecipitated proteins with anti-GFP (GFP-IP) were detected by immunoblotting using an anti-MYC antibody (top row). The 10% input and DCC1-GFP proteins were detected by immunoblotting using an anti-GFP antibody (bottom row).

    Journal: Plant Physiology

    Article Title: Thioredoxin-Mediated ROS Homeostasis Explains Natural Variation in Plant Regeneration 1Thioredoxin-Mediated ROS Homeostasis Explains Natural Variation in Plant Regeneration 1 [OPEN]

    doi: 10.1104/pp.17.00633

    Figure Lengend Snippet: DCC1 interacts with CA2. A, Interaction between DCC1 and CA2 in the yeast two-hybrid assay. Activation was observed at 3 d on selection plates (synthetic dextrose [SD]-Leu-Trp-His-Ade) with X-α-gal. B, Interaction between DCC1 and CA2 in the pull-down assay. The 10% input and GST pull-down proteins were detected by immunoblotting using anti-His antibody (top row). CA2-GST and GST proteins were detected by immunoblotting using anti-GST antibody (bottom row). C, Interaction between DCC1 and CA2 in firefly luciferase complementation assays in transiently transfected leaf of N. benthamiana . D, Interaction between DCC1 and CA2 in coimmunoprecipitation assays. The 10% input and immunoprecipitated proteins with anti-GFP (GFP-IP) were detected by immunoblotting using an anti-MYC antibody (top row). The 10% input and DCC1-GFP proteins were detected by immunoblotting using an anti-GFP antibody (bottom row).

    Article Snippet: Finally, 10% input and pull-down proteins were separated by 12% SDS-PAGE and detected by immunoblotting using the antibodies anti-His, diluted 1:3,000 (Sigma; catalog no. H1029), or anti-GST, diluted 1:3,000 (Sigma; catalog no. G1160).

    Techniques: Y2H Assay, Activation Assay, Selection, Pull Down Assay, Luciferase, Transfection, Immunoprecipitation

    (A) Autoradiographic image of electrophoretically separated [γ- 32 P]ATP-labeled GST-pRB fusion protein. Synchronized HeLa cells were mock infected (lanes 1, 2, 4, 6, and 8) or infected with HSV-1(F) (lanes 3, 5, 7, and 9). The cells were harvested at the times indicated, solubilized, electrophoretically separated in denaturing gel, transferred to a nitrocellulose sheet, and reacted with polyclonal anti-cdk4. Immunoprecipitates were incubated with the substrate GST-pRB in a kinase buffer supplemented with [γ- 32 P]ATP. The reaction mixtures were subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and subjected to autoradiography. The radiolabeled product was collected on a phosphorimager. (B) Lanes 10 to 12, autoradiographic image of electrophoretically separated GST-pRB labeled with [γ- 32 P]ATP by cdk4 immune precipitated from synchronized HeLa cells harvested 12 h after mock infection (lane 10) or infection with HSV-1(F) (lane 11) or R7914 (lane 12). Experimental details were the same as for panel A. Lanes 13 to 15, autoradiographic image of electrophoretically separated histone H1 labeled with [γ- 32 P]ATP by cdk2 immune precipitated from synchronized HeLa cells 12 h after mock infection (lane 13) or infection with HSV-1(F) (lane 14) or with R7914 (lane 15). Experimental details were the same as for panel A.

    Journal: Journal of Virology

    Article Title: Role of Cyclin D3 in the Biology of Herpes Simplex Virus 1 ICP0

    doi: 10.1128/JVI.75.4.1888-1898.2001

    Figure Lengend Snippet: (A) Autoradiographic image of electrophoretically separated [γ- 32 P]ATP-labeled GST-pRB fusion protein. Synchronized HeLa cells were mock infected (lanes 1, 2, 4, 6, and 8) or infected with HSV-1(F) (lanes 3, 5, 7, and 9). The cells were harvested at the times indicated, solubilized, electrophoretically separated in denaturing gel, transferred to a nitrocellulose sheet, and reacted with polyclonal anti-cdk4. Immunoprecipitates were incubated with the substrate GST-pRB in a kinase buffer supplemented with [γ- 32 P]ATP. The reaction mixtures were subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and subjected to autoradiography. The radiolabeled product was collected on a phosphorimager. (B) Lanes 10 to 12, autoradiographic image of electrophoretically separated GST-pRB labeled with [γ- 32 P]ATP by cdk4 immune precipitated from synchronized HeLa cells harvested 12 h after mock infection (lane 10) or infection with HSV-1(F) (lane 11) or R7914 (lane 12). Experimental details were the same as for panel A. Lanes 13 to 15, autoradiographic image of electrophoretically separated histone H1 labeled with [γ- 32 P]ATP by cdk2 immune precipitated from synchronized HeLa cells 12 h after mock infection (lane 13) or infection with HSV-1(F) (lane 14) or with R7914 (lane 15). Experimental details were the same as for panel A.

    Article Snippet: Forty microliters-of complete kinase buffer was then added to each sample (2 μg of glutathione S -transferase [GST]-pRb [Santa Cruz Biotechnology], 10 μM ATP, and 20 μCi of [γ-32 P]ATP in incomplete kinase buffer), and samples were incubated at 30°C for 20 min.

    Techniques: Labeling, Infection, Incubation, Electrophoresis, Autoradiography

    Mena associates with activated Rac1, and loss of Mena in cardiomyocytes significantly increases Rac1 activity. A : cardiomyocytes were incubated with GST-Pak1-p21 binding domain (PBD) or glutathione- S -transferase (GST) alone. After pulldown, active Rac1

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Mena associates with Rac1 and modulates connexin 43 remodeling in cardiomyocytes

    doi: 10.1152/ajpheart.00749.2013

    Figure Lengend Snippet: Mena associates with activated Rac1, and loss of Mena in cardiomyocytes significantly increases Rac1 activity. A : cardiomyocytes were incubated with GST-Pak1-p21 binding domain (PBD) or glutathione- S -transferase (GST) alone. After pulldown, active Rac1

    Article Snippet: Active Rac1 was precipitated with the p21-binding domain of human p21-activated kinase 1 (Pak1) fused to glutathione- S -transferase (GST)-Pak1 (Thermo Fisher).

    Techniques: Activity Assay, Incubation, Binding Assay

    Silencing of integrin β1 inhibits IGFBP3-induced migration A. The activities of Cdc42, Rac1 and RhoA were detected in IGBBP3 knockdown cells (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). Equal amounts of input protein were subjected to Western blot using anti-Cdc42 (total Cdc42), anti-Rac1 (total Rac1), and anti-RhoA (total RhoA) antibodies. Equal amounts of protein were incubated with GST-PAK1 (detection of active Cdc42 or Rac1) and GST-Rhotekin (detection of active RhoA). Complexes were collected with gluthathione-Sepharose and resolved by Western blot. GTPγS was served as a positive control. Anti-GST antibodies were served as a loading control. B. The density of each band was measured by image J and normalized with the controls (LN1–1 pLKO-GFP). The activities of active small GTPase were conducted by dividing the density of active small GTPase to that of GST loading controls. The relative activities were obtained when the activity of active small GTPase in LN1–1 pLKO-GFP were set to 1. C. Representative data shows the relative migration activity of OEC-M1 cells with dominant-negative Cdc42 (Cdc42dn) and the corresponding controls (PB). The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB treated with treated with IGFBP3 and PB-Cdc42dn cells with/without IGFBP3 treatment with that in OEC-M1 PB cells. D. Representative data shows the relative migration activity of IGFBP3 expressing cells (OEC-M1 IGFBP3) and the corresponding controls (OEC-M1 PB) with anti-integrin β1 (200 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB and IGFBP3 cells treated with anti-integrin β1 or OEC-M1 IGFBP3 cells treated with IgG antibodies (200 ng/ml) to that in OEC-M1 PB cells treated with IgG antibodies. E. Immunoblot analysis of integrin β1 protein in OEC-M1 cells with ITGB1 shRNA expression (OEC-M1 ITGB1 sh3 and sh4) and vector controls (OEC-M1 pLKO-GFP). α-tubulin serves as an internal control. F. Representative data shows the relative migration activity of OEC-M1 pLKO-GFP, ITGB1 sh3 and sh4 cells upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 with ITGB1 knockdown and IGFBP3 treatment with that in the control cells. G. Representative data showed the relative migration activity of OEC-M1 treated with 10, 20 uM of PD98059 (PD) and dimethyl sulfoxide (DMSO) upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell/per field in OEC-M1 treated with PD98059 and IGFBP3 with that in the control cells. H. Immunoblot analysis revealed knockdown of ITGB1 inhibited IGFBP3-induced ERK phosphorylation at different time points in OEC-M1 cells (upper panel, 0, untreated; 10, 10 min; 30, 30 min for 100 ng/ml IGFBP3 treatment). The ratio of phosphorylated ERK/total ERK was obtained by dividing the intensity of phosphorylated ERK to that of total ERK. The relative expression was obtained when the ration of untreated cells were set to 1 (lower panel). Bar, SE; ** p

    Journal: Oncotarget

    Article Title: Insulin-like growth factor-independent insulin-like growth factor binding protein 3 promotes cell migration and lymph node metastasis of oral squamous cell carcinoma cells by requirement of integrin β1

    doi:

    Figure Lengend Snippet: Silencing of integrin β1 inhibits IGFBP3-induced migration A. The activities of Cdc42, Rac1 and RhoA were detected in IGBBP3 knockdown cells (IGFBP3 sh4 and sh5) and the corresponding controls (pLKO-GFP). Equal amounts of input protein were subjected to Western blot using anti-Cdc42 (total Cdc42), anti-Rac1 (total Rac1), and anti-RhoA (total RhoA) antibodies. Equal amounts of protein were incubated with GST-PAK1 (detection of active Cdc42 or Rac1) and GST-Rhotekin (detection of active RhoA). Complexes were collected with gluthathione-Sepharose and resolved by Western blot. GTPγS was served as a positive control. Anti-GST antibodies were served as a loading control. B. The density of each band was measured by image J and normalized with the controls (LN1–1 pLKO-GFP). The activities of active small GTPase were conducted by dividing the density of active small GTPase to that of GST loading controls. The relative activities were obtained when the activity of active small GTPase in LN1–1 pLKO-GFP were set to 1. C. Representative data shows the relative migration activity of OEC-M1 cells with dominant-negative Cdc42 (Cdc42dn) and the corresponding controls (PB). The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB treated with treated with IGFBP3 and PB-Cdc42dn cells with/without IGFBP3 treatment with that in OEC-M1 PB cells. D. Representative data shows the relative migration activity of IGFBP3 expressing cells (OEC-M1 IGFBP3) and the corresponding controls (OEC-M1 PB) with anti-integrin β1 (200 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 PB and IGFBP3 cells treated with anti-integrin β1 or OEC-M1 IGFBP3 cells treated with IgG antibodies (200 ng/ml) to that in OEC-M1 PB cells treated with IgG antibodies. E. Immunoblot analysis of integrin β1 protein in OEC-M1 cells with ITGB1 shRNA expression (OEC-M1 ITGB1 sh3 and sh4) and vector controls (OEC-M1 pLKO-GFP). α-tubulin serves as an internal control. F. Representative data shows the relative migration activity of OEC-M1 pLKO-GFP, ITGB1 sh3 and sh4 cells upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell /per field in OEC-M1 with ITGB1 knockdown and IGFBP3 treatment with that in the control cells. G. Representative data showed the relative migration activity of OEC-M1 treated with 10, 20 uM of PD98059 (PD) and dimethyl sulfoxide (DMSO) upon IGFBP3 (100 ng/ml) treatment. The relative migration activity was defined by normalizing the mean of migrated cell/per field in OEC-M1 treated with PD98059 and IGFBP3 with that in the control cells. H. Immunoblot analysis revealed knockdown of ITGB1 inhibited IGFBP3-induced ERK phosphorylation at different time points in OEC-M1 cells (upper panel, 0, untreated; 10, 10 min; 30, 30 min for 100 ng/ml IGFBP3 treatment). The ratio of phosphorylated ERK/total ERK was obtained by dividing the intensity of phosphorylated ERK to that of total ERK. The relative expression was obtained when the ration of untreated cells were set to 1 (lower panel). Bar, SE; ** p

    Article Snippet: The amount of total input and glutathione-S-transferase (GST)-Rhotekin-, GST-PAK1–bound small GTPase was determined by Western blot with anti-RhoA (Millipore), anti-Cdc42 (Millipore) and anti-Rac1 antibody (Novous Biologicals, Littleton, CO, USA), respectively.

    Techniques: Migration, Western Blot, Incubation, Positive Control, Activity Assay, Dominant Negative Mutation, Expressing, shRNA, Plasmid Preparation