biotin peg3 propionic acid Search Results


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  • 96
    Vector Laboratories vectabond
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    Thermo Fisher bodipyfl c5 hexadecanoyl phosphatidylcholine
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    Millipore n hydroxysuccinimide nhs
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    Millipore protease cocktail inhibitors
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    InvivoGen 5 ppp dsrna
    ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or <t>biotinylated-5'ppp-dsRNA</t> at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P
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    Creative PEGWorks methoxypoly
    ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or <t>biotinylated-5'ppp-dsRNA</t> at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P
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    Nektar mpeg spa
    ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or <t>biotinylated-5'ppp-dsRNA</t> at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P
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    Millipore tris
    ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or <t>biotinylated-5'ppp-dsRNA</t> at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P
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    Laysan Bio mpeg spa
    ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or <t>biotinylated-5'ppp-dsRNA</t> at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P
    Mpeg Spa, supplied by Laysan Bio, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nektar hcl h2 n peg cooh
    ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or <t>biotinylated-5'ppp-dsRNA</t> at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P
    Hcl H2 N Peg Cooh, supplied by Nektar, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore porcine intestinal heparin
    ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or <t>biotinylated-5'ppp-dsRNA</t> at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P
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    Thermo Fisher neutravidin
    ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or <t>biotinylated-5'ppp-dsRNA</t> at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P
    Neutravidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or biotinylated-5'ppp-dsRNA at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P

    Journal: PLoS Pathogens

    Article Title: ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response

    doi: 10.1371/journal.ppat.1008457

    Figure Lengend Snippet: ZFYVE1 competes with MDA5 for viral RNA binding. (A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or biotinylated-5'ppp-dsRNA at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. * P

    Article Snippet: Reagents, antibodies, cells and viruses Trizol (Takara Bio), SYBR Green (Bio-Rad), RNase I (Ambion), Flag antibody-conjugated beads (Bimake), dual-specific luciferase assay kit (Promega), polybrene (Millipore), poly(I:C)-HMW (Invivogen), poly(I:C)-LMW (Invivogen), 5’ppp-dsRNA (Invivogen), DNAase I, 3 × Flag peptide (Sigma), Z-link Psoralen-PEG3-Biotin (Thermo), type II collagenase (Worthington), first-strand cDNA synthesis kit (Fermentas), GammaBind G Plus-Sepharose (Amersham Biosciences), and ELISA kits for murine IFN-β and TNF (BioLegend); mouse monoclonal antibodies for Flag and β-actin (Sigma), HA (Origene), p-IRF3, p65 and p-p65 (Cell Signaling Technology), IRF3 (Santa Cruz Biotechnology), p-TBK1 and TBK1 (Abcam), and ZFYVE1 (ABclonal); Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen); and HEK293 (American Type Culture Collection) and THP1 cells (American type culture collection) were purchased from the indicated companies.

    Techniques: RNA Binding Assay, Binding Assay, Transfection, Incubation, Western Blot, Expressing, Plasmid Preparation, Infection, Cell Culture, Immunoprecipitation, Lysis, Real-time Polymerase Chain Reaction, Inhibition, In Vitro, Over Expression, Activation Assay, Mutagenesis, Luciferase