Structured Review

Millipore protein a beads slurry
NS1 interacts with the structural proteins. ( A )  Capsid ,  prM and Envelope proteins co-immunoprecipitate with NS1 . Naïve VeroE6 (CTRL), VeroE6_NS1 WT  (WT) or VeroE6_NS1 HA  (HA) cells were infected with 1 MOI of DVR2A ΔNS1  TCPs. Forty-height hours post-infection, cell lysates clarified by centrifugation were used for immunoprecipitation with HA-affinity agarose beads and eluates (IP) or whole cell lysates (Input) analyzed by western-blotting using antibodies specified on the right of each panel. Numbers on the left refer to molecular weight standards expressed in kDa; black arrowhead on the right indicates the ΔNS1 protein expressed by the DVR2A ΔNS1  genome. A representative experiment of four independent repetitions is shown. ( B )  Interaction between NS1 and prM/E in DENV-2 wild-type virus-infected cells . VeroE6 cells were mock infected or infected with DENV-2 at an MOI of 1. Forty-eight hours later clarified cell lysates were used for immunoprecipitation using a NS1-specific rabbit polyclonal antiserum or the corresponding pre-immune (PIS) antiserum and protein A-Sepharose beads. After extensive washing, eluted protein complexes were analyzed by western-blotting using polyclonal anti-NS1 and anti-prM or mouse monoclonal anti-E or anti-C specific antibodies as specified on the right of each panel. DENV proteins are indicated with arrowheads, asterisks refer to the immunoglobulin heavy chain.
Protein A Beads Slurry, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/protein a beads slurry/product/Millipore
Average 99 stars, based on 4 article reviews
Price from $9.99 to $1999.99
protein a beads slurry - by Bioz Stars, 2022-10
99/100 stars

Images

1) Product Images from "Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins"

Article Title: Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005277

NS1 interacts with the structural proteins. ( A )  Capsid ,  prM and Envelope proteins co-immunoprecipitate with NS1 . Naïve VeroE6 (CTRL), VeroE6_NS1 WT  (WT) or VeroE6_NS1 HA  (HA) cells were infected with 1 MOI of DVR2A ΔNS1  TCPs. Forty-height hours post-infection, cell lysates clarified by centrifugation were used for immunoprecipitation with HA-affinity agarose beads and eluates (IP) or whole cell lysates (Input) analyzed by western-blotting using antibodies specified on the right of each panel. Numbers on the left refer to molecular weight standards expressed in kDa; black arrowhead on the right indicates the ΔNS1 protein expressed by the DVR2A ΔNS1  genome. A representative experiment of four independent repetitions is shown. ( B )  Interaction between NS1 and prM/E in DENV-2 wild-type virus-infected cells . VeroE6 cells were mock infected or infected with DENV-2 at an MOI of 1. Forty-eight hours later clarified cell lysates were used for immunoprecipitation using a NS1-specific rabbit polyclonal antiserum or the corresponding pre-immune (PIS) antiserum and protein A-Sepharose beads. After extensive washing, eluted protein complexes were analyzed by western-blotting using polyclonal anti-NS1 and anti-prM or mouse monoclonal anti-E or anti-C specific antibodies as specified on the right of each panel. DENV proteins are indicated with arrowheads, asterisks refer to the immunoglobulin heavy chain.
Figure Legend Snippet: NS1 interacts with the structural proteins. ( A ) Capsid , prM and Envelope proteins co-immunoprecipitate with NS1 . Naïve VeroE6 (CTRL), VeroE6_NS1 WT (WT) or VeroE6_NS1 HA (HA) cells were infected with 1 MOI of DVR2A ΔNS1 TCPs. Forty-height hours post-infection, cell lysates clarified by centrifugation were used for immunoprecipitation with HA-affinity agarose beads and eluates (IP) or whole cell lysates (Input) analyzed by western-blotting using antibodies specified on the right of each panel. Numbers on the left refer to molecular weight standards expressed in kDa; black arrowhead on the right indicates the ΔNS1 protein expressed by the DVR2A ΔNS1 genome. A representative experiment of four independent repetitions is shown. ( B ) Interaction between NS1 and prM/E in DENV-2 wild-type virus-infected cells . VeroE6 cells were mock infected or infected with DENV-2 at an MOI of 1. Forty-eight hours later clarified cell lysates were used for immunoprecipitation using a NS1-specific rabbit polyclonal antiserum or the corresponding pre-immune (PIS) antiserum and protein A-Sepharose beads. After extensive washing, eluted protein complexes were analyzed by western-blotting using polyclonal anti-NS1 and anti-prM or mouse monoclonal anti-E or anti-C specific antibodies as specified on the right of each panel. DENV proteins are indicated with arrowheads, asterisks refer to the immunoglobulin heavy chain.

Techniques Used: Infection, Centrifugation, Immunoprecipitation, Western Blot, Molecular Weight

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore protein a beads slurry
    NS1 interacts with the structural proteins. ( A )  Capsid ,  prM and Envelope proteins co-immunoprecipitate with NS1 . Naïve VeroE6 (CTRL), VeroE6_NS1 WT  (WT) or VeroE6_NS1 HA  (HA) cells were infected with 1 MOI of DVR2A ΔNS1  TCPs. Forty-height hours post-infection, cell lysates clarified by centrifugation were used for immunoprecipitation with HA-affinity agarose beads and eluates (IP) or whole cell lysates (Input) analyzed by western-blotting using antibodies specified on the right of each panel. Numbers on the left refer to molecular weight standards expressed in kDa; black arrowhead on the right indicates the ΔNS1 protein expressed by the DVR2A ΔNS1  genome. A representative experiment of four independent repetitions is shown. ( B )  Interaction between NS1 and prM/E in DENV-2 wild-type virus-infected cells . VeroE6 cells were mock infected or infected with DENV-2 at an MOI of 1. Forty-eight hours later clarified cell lysates were used for immunoprecipitation using a NS1-specific rabbit polyclonal antiserum or the corresponding pre-immune (PIS) antiserum and protein A-Sepharose beads. After extensive washing, eluted protein complexes were analyzed by western-blotting using polyclonal anti-NS1 and anti-prM or mouse monoclonal anti-E or anti-C specific antibodies as specified on the right of each panel. DENV proteins are indicated with arrowheads, asterisks refer to the immunoglobulin heavy chain.
    Protein A Beads Slurry, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein a beads slurry/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein a beads slurry - by Bioz Stars, 2022-10
    99/100 stars
      Buy from Supplier

    94
    Millipore protein g coated agarose bead slurry
    Characterization of armed CRAds.  A, B,  MDA-MB-231 cells were infected with Ad5-Δ24-sOPG-Fc, Ad5-Δ24-sOPG-Fc-RGD or Adwt300. At the indicated times, cellular RNA was subjected to QRT-PCR to detect expression of:  A,  the sOPG-Fc gene (cells infected with Ad5-Δ24-sOPG-Fc or Ad5-Δ24-sOPG-Fc-RGD) or the 14.7k gene (cells infected with Adwt300); and  B,  the ADP gene. The copy number was normalized to total cellular RNA.  C,  Secretion of sOPG-Fc. MDA-MB-231 cells were infected with Ad5-Δ24, Ad5-Δ24RGD, Ad5-Δ24-sOPG-Fc or Ad5-Δ24-sOPG-Fc-RGD. At the indicated times, medium was harvested, diluted 1:300 and expression of sOPG-Fc was detected in an ELISA.  D , sOPG-Fc binds RANKL. Medium from MDA-MB-231 cells 48 h postinfection was incubated in the presence (+) or absence (−) of sRANKL and pulled down with protein G-agarose. Proteins were released and subjected to immunoblot using anti-RANKL primary antibody. C: sRANKL.
    Protein G Coated Agarose Bead Slurry, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein g coated agarose bead slurry/product/Millipore
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein g coated agarose bead slurry - by Bioz Stars, 2022-10
    94/100 stars
      Buy from Supplier

    96
    Millipore protein a sepharose bead slurry
    For3p, bud6p, tea1p, and tip1p coimmunoprecipitate. (a) For3p-myc and bud6p-HA coimmunoprecipitate. Extracts expressing (1) for3p-myc (BFY19), (2) for3p-myc bud6p-HA (BFY168), or (3) bud6p-HA (FC592) were immunoprecipitated using protein <t>A–Sepharose</t> beads complexed with either α-HA or α-myc antibodies and were Western blotted. (b) For3p-TAP immunoprecipitates tea1p. Extracts expressing (1) for3-myc (BFY19) or (2) for3p-TAP (BFY198) were immunoprecipitated with mouse α-rabbit Dynal magnetic beads complexed with rabbit IgG, and were Western blotted with α-myc antibodies (the HRP anti–rabbit secondary recognizes both the α-myc antibody and the TAP tag) or anti-tea1p antibodies. (c) Tea1p-HA immunoprecipitates for3p-myc and tip1p. Extracts expressing tea1p-HA for3p-myc (BFY190, lanes 1 and 2) or for3p-myc (BFY19, lanes 3 and 4) were immunoprecipitated with sheep α-mouse Dynal magnetic beads complexed with mouse anti-HA antibodies, washed in buffer containing either 250 mM NaCl (lanes 1 and 3) or 150 mM NaCl (lanes 2 and 4; see Materials and methods), and immunoblotted. White lines indicate that intervening lanes have been spliced out.
    Protein A Sepharose Bead Slurry, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein a sepharose bead slurry/product/Millipore
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein a sepharose bead slurry - by Bioz Stars, 2022-10
    96/100 stars
      Buy from Supplier

    99
    Millipore protein g sepharose 4b ff beads
    A region within the SOCS3 SH2 domain is necessary and sufficient for cavin-1 interaction. a Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding Flag-SOCS3 and myc-tagged cavin-1 as indicated were processed by immunoprecipitation (IP) with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. b Protein-equalised soluble cell extracts from WT and SOCS3-null AS-M.5 cells treated with 50 μM Fsk and 6 μM MG132 for 4 h were processed by IP with anti-cavin-1 antibody prior to SDS-PAGE and immunoblotting with the indicated antibodies. As loss of SOCS3 in AS-M.5 cells reduces cavin-1 protein levels ( a ), twice the amount of protein was used as input for SOCS3-null cells to compensate. NS = non-specific band. c Upper: Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either Flag-tagged WT SOCS3 or the indicated truncation mutants and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the same samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. Lower: Schematic of the SOCS3 truncation constructs used, KIR = kinase inhibitory region, ESS = extended SH2 subdomain, PEST = PEST (ProGluSerThr) motif. d Upper: Protein-equalised soluble cell extracts from HEK293 cells co-transfected with expression constructs encoding either full-length SOCS3 (WT), a truncated ΔSH2 SOCS3domain (ΔSH2), or SOCS box domain (SB) fused to GFP and myc-tagged cavin-1 as indicated were processed by IP with anti-GFP antibody and protein <t>G-Sepharose</t> beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the same samples used in the IP were also fractionated by SDS-PAGE and for immunoblotting in parallel. Lower: Schematic of the SOCS3-GFP fusions used
    Protein G Sepharose 4b Ff Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein g sepharose 4b ff beads/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein g sepharose 4b ff beads - by Bioz Stars, 2022-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    NS1 interacts with the structural proteins. ( A )  Capsid ,  prM and Envelope proteins co-immunoprecipitate with NS1 . Naïve VeroE6 (CTRL), VeroE6_NS1 WT  (WT) or VeroE6_NS1 HA  (HA) cells were infected with 1 MOI of DVR2A ΔNS1  TCPs. Forty-height hours post-infection, cell lysates clarified by centrifugation were used for immunoprecipitation with HA-affinity agarose beads and eluates (IP) or whole cell lysates (Input) analyzed by western-blotting using antibodies specified on the right of each panel. Numbers on the left refer to molecular weight standards expressed in kDa; black arrowhead on the right indicates the ΔNS1 protein expressed by the DVR2A ΔNS1  genome. A representative experiment of four independent repetitions is shown. ( B )  Interaction between NS1 and prM/E in DENV-2 wild-type virus-infected cells . VeroE6 cells were mock infected or infected with DENV-2 at an MOI of 1. Forty-eight hours later clarified cell lysates were used for immunoprecipitation using a NS1-specific rabbit polyclonal antiserum or the corresponding pre-immune (PIS) antiserum and protein A-Sepharose beads. After extensive washing, eluted protein complexes were analyzed by western-blotting using polyclonal anti-NS1 and anti-prM or mouse monoclonal anti-E or anti-C specific antibodies as specified on the right of each panel. DENV proteins are indicated with arrowheads, asterisks refer to the immunoglobulin heavy chain.

    Journal: PLoS Pathogens

    Article Title: Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins

    doi: 10.1371/journal.ppat.1005277

    Figure Lengend Snippet: NS1 interacts with the structural proteins. ( A ) Capsid , prM and Envelope proteins co-immunoprecipitate with NS1 . Naïve VeroE6 (CTRL), VeroE6_NS1 WT (WT) or VeroE6_NS1 HA (HA) cells were infected with 1 MOI of DVR2A ΔNS1 TCPs. Forty-height hours post-infection, cell lysates clarified by centrifugation were used for immunoprecipitation with HA-affinity agarose beads and eluates (IP) or whole cell lysates (Input) analyzed by western-blotting using antibodies specified on the right of each panel. Numbers on the left refer to molecular weight standards expressed in kDa; black arrowhead on the right indicates the ΔNS1 protein expressed by the DVR2A ΔNS1 genome. A representative experiment of four independent repetitions is shown. ( B ) Interaction between NS1 and prM/E in DENV-2 wild-type virus-infected cells . VeroE6 cells were mock infected or infected with DENV-2 at an MOI of 1. Forty-eight hours later clarified cell lysates were used for immunoprecipitation using a NS1-specific rabbit polyclonal antiserum or the corresponding pre-immune (PIS) antiserum and protein A-Sepharose beads. After extensive washing, eluted protein complexes were analyzed by western-blotting using polyclonal anti-NS1 and anti-prM or mouse monoclonal anti-E or anti-C specific antibodies as specified on the right of each panel. DENV proteins are indicated with arrowheads, asterisks refer to the immunoglobulin heavy chain.

    Article Snippet: Samples were incubated with 40 μl of protein A beads slurry (Sigma Aldrich, St. Louis, USA) for 1 hour at 4°C, washed three times with 1 ml of lysis buffer and protein A-bound complexes were transferred into a fresh tube.

    Techniques: Infection, Centrifugation, Immunoprecipitation, Western Blot, Molecular Weight

    Characterization of armed CRAds.  A, B,  MDA-MB-231 cells were infected with Ad5-Δ24-sOPG-Fc, Ad5-Δ24-sOPG-Fc-RGD or Adwt300. At the indicated times, cellular RNA was subjected to QRT-PCR to detect expression of:  A,  the sOPG-Fc gene (cells infected with Ad5-Δ24-sOPG-Fc or Ad5-Δ24-sOPG-Fc-RGD) or the 14.7k gene (cells infected with Adwt300); and  B,  the ADP gene. The copy number was normalized to total cellular RNA.  C,  Secretion of sOPG-Fc. MDA-MB-231 cells were infected with Ad5-Δ24, Ad5-Δ24RGD, Ad5-Δ24-sOPG-Fc or Ad5-Δ24-sOPG-Fc-RGD. At the indicated times, medium was harvested, diluted 1:300 and expression of sOPG-Fc was detected in an ELISA.  D , sOPG-Fc binds RANKL. Medium from MDA-MB-231 cells 48 h postinfection was incubated in the presence (+) or absence (−) of sRANKL and pulled down with protein G-agarose. Proteins were released and subjected to immunoblot using anti-RANKL primary antibody. C: sRANKL.

    Journal: Cancer gene therapy

    Article Title: Arming a Replicating Adenovirus with Osteoprotegerin Reduces the Tumor Burden in a Murine Model of Osteolytic Bone Metastases of Breast Cancer

    doi: 10.1038/cgt.2010.47

    Figure Lengend Snippet: Characterization of armed CRAds. A, B, MDA-MB-231 cells were infected with Ad5-Δ24-sOPG-Fc, Ad5-Δ24-sOPG-Fc-RGD or Adwt300. At the indicated times, cellular RNA was subjected to QRT-PCR to detect expression of: A, the sOPG-Fc gene (cells infected with Ad5-Δ24-sOPG-Fc or Ad5-Δ24-sOPG-Fc-RGD) or the 14.7k gene (cells infected with Adwt300); and B, the ADP gene. The copy number was normalized to total cellular RNA. C, Secretion of sOPG-Fc. MDA-MB-231 cells were infected with Ad5-Δ24, Ad5-Δ24RGD, Ad5-Δ24-sOPG-Fc or Ad5-Δ24-sOPG-Fc-RGD. At the indicated times, medium was harvested, diluted 1:300 and expression of sOPG-Fc was detected in an ELISA. D , sOPG-Fc binds RANKL. Medium from MDA-MB-231 cells 48 h postinfection was incubated in the presence (+) or absence (−) of sRANKL and pulled down with protein G-agarose. Proteins were released and subjected to immunoblot using anti-RANKL primary antibody. C: sRANKL.

    Article Snippet: Tubes were incubated overnight at 4 °C before adding 30 μl of a protein G-coated agarose bead slurry (EZview Red Protein G Affinity Gel; Sigma-Aldrich, St. Louis, MO).

    Techniques: Multiple Displacement Amplification, Infection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Incubation

    For3p, bud6p, tea1p, and tip1p coimmunoprecipitate. (a) For3p-myc and bud6p-HA coimmunoprecipitate. Extracts expressing (1) for3p-myc (BFY19), (2) for3p-myc bud6p-HA (BFY168), or (3) bud6p-HA (FC592) were immunoprecipitated using protein A–Sepharose beads complexed with either α-HA or α-myc antibodies and were Western blotted. (b) For3p-TAP immunoprecipitates tea1p. Extracts expressing (1) for3-myc (BFY19) or (2) for3p-TAP (BFY198) were immunoprecipitated with mouse α-rabbit Dynal magnetic beads complexed with rabbit IgG, and were Western blotted with α-myc antibodies (the HRP anti–rabbit secondary recognizes both the α-myc antibody and the TAP tag) or anti-tea1p antibodies. (c) Tea1p-HA immunoprecipitates for3p-myc and tip1p. Extracts expressing tea1p-HA for3p-myc (BFY190, lanes 1 and 2) or for3p-myc (BFY19, lanes 3 and 4) were immunoprecipitated with sheep α-mouse Dynal magnetic beads complexed with mouse anti-HA antibodies, washed in buffer containing either 250 mM NaCl (lanes 1 and 3) or 150 mM NaCl (lanes 2 and 4; see Materials and methods), and immunoblotted. White lines indicate that intervening lanes have been spliced out.

    Journal: The Journal of Cell Biology

    Article Title: Regulation of a formin complex by the microtubule plus end protein tea1p

    doi: 10.1083/jcb.200403090

    Figure Lengend Snippet: For3p, bud6p, tea1p, and tip1p coimmunoprecipitate. (a) For3p-myc and bud6p-HA coimmunoprecipitate. Extracts expressing (1) for3p-myc (BFY19), (2) for3p-myc bud6p-HA (BFY168), or (3) bud6p-HA (FC592) were immunoprecipitated using protein A–Sepharose beads complexed with either α-HA or α-myc antibodies and were Western blotted. (b) For3p-TAP immunoprecipitates tea1p. Extracts expressing (1) for3-myc (BFY19) or (2) for3p-TAP (BFY198) were immunoprecipitated with mouse α-rabbit Dynal magnetic beads complexed with rabbit IgG, and were Western blotted with α-myc antibodies (the HRP anti–rabbit secondary recognizes both the α-myc antibody and the TAP tag) or anti-tea1p antibodies. (c) Tea1p-HA immunoprecipitates for3p-myc and tip1p. Extracts expressing tea1p-HA for3p-myc (BFY190, lanes 1 and 2) or for3p-myc (BFY19, lanes 3 and 4) were immunoprecipitated with sheep α-mouse Dynal magnetic beads complexed with mouse anti-HA antibodies, washed in buffer containing either 250 mM NaCl (lanes 1 and 3) or 150 mM NaCl (lanes 2 and 4; see Materials and methods), and immunoblotted. White lines indicate that intervening lanes have been spliced out.

    Article Snippet: For immunoprecipitations, 50 μl soluble yeast extract was added to 25 μl protein A–Sepharose bead slurry (Sigma-Aldrich; a), 25 μl Dynal mouse anti–rabbit magnetic bead slurry (Dynal Corp.; c), or 25 μl Dynal sheep anti–mouse magnetic bead slurry (Dynal Corp.; ).

    Techniques: Expressing, Immunoprecipitation, Western Blot, Magnetic Beads

    A region within the SOCS3 SH2 domain is necessary and sufficient for cavin-1 interaction. a Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding Flag-SOCS3 and myc-tagged cavin-1 as indicated were processed by immunoprecipitation (IP) with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. b Protein-equalised soluble cell extracts from WT and SOCS3-null AS-M.5 cells treated with 50 μM Fsk and 6 μM MG132 for 4 h were processed by IP with anti-cavin-1 antibody prior to SDS-PAGE and immunoblotting with the indicated antibodies. As loss of SOCS3 in AS-M.5 cells reduces cavin-1 protein levels ( a ), twice the amount of protein was used as input for SOCS3-null cells to compensate. NS = non-specific band. c Upper: Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either Flag-tagged WT SOCS3 or the indicated truncation mutants and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the same samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. Lower: Schematic of the SOCS3 truncation constructs used, KIR = kinase inhibitory region, ESS = extended SH2 subdomain, PEST = PEST (ProGluSerThr) motif. d Upper: Protein-equalised soluble cell extracts from HEK293 cells co-transfected with expression constructs encoding either full-length SOCS3 (WT), a truncated ΔSH2 SOCS3domain (ΔSH2), or SOCS box domain (SB) fused to GFP and myc-tagged cavin-1 as indicated were processed by IP with anti-GFP antibody and protein G-Sepharose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the same samples used in the IP were also fractionated by SDS-PAGE and for immunoblotting in parallel. Lower: Schematic of the SOCS3-GFP fusions used

    Journal: Nature Communications

    Article Title: Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability

    doi: 10.1038/s41467-017-02585-y

    Figure Lengend Snippet: A region within the SOCS3 SH2 domain is necessary and sufficient for cavin-1 interaction. a Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding Flag-SOCS3 and myc-tagged cavin-1 as indicated were processed by immunoprecipitation (IP) with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. b Protein-equalised soluble cell extracts from WT and SOCS3-null AS-M.5 cells treated with 50 μM Fsk and 6 μM MG132 for 4 h were processed by IP with anti-cavin-1 antibody prior to SDS-PAGE and immunoblotting with the indicated antibodies. As loss of SOCS3 in AS-M.5 cells reduces cavin-1 protein levels ( a ), twice the amount of protein was used as input for SOCS3-null cells to compensate. NS = non-specific band. c Upper: Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either Flag-tagged WT SOCS3 or the indicated truncation mutants and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the same samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. Lower: Schematic of the SOCS3 truncation constructs used, KIR = kinase inhibitory region, ESS = extended SH2 subdomain, PEST = PEST (ProGluSerThr) motif. d Upper: Protein-equalised soluble cell extracts from HEK293 cells co-transfected with expression constructs encoding either full-length SOCS3 (WT), a truncated ΔSH2 SOCS3domain (ΔSH2), or SOCS box domain (SB) fused to GFP and myc-tagged cavin-1 as indicated were processed by IP with anti-GFP antibody and protein G-Sepharose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the same samples used in the IP were also fractionated by SDS-PAGE and for immunoblotting in parallel. Lower: Schematic of the SOCS3-GFP fusions used

    Article Snippet: Non-specifically binding proteins were removed from soluble fractions by a 1 h pre-clearing step using 40 μl of 50% (v/v) slurry of protein G-Sepharose 4B FF beads (Sigma) re-suspended in 100 μl 2% (w/v) IgG-free bovine serum albumin (BSA).

    Techniques: Transfection, Expressing, Construct, Immunoprecipitation, SDS Page