Structured Review

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: 94/100, based on 3 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 94 stars, based on 3 article reviews
Price from $9.99 to $1999.99
protein g sepharose 4b ff beads - by Bioz Stars, 2022-11
94/100 stars

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1) Product Images from "Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability"

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

Journal: Nature Communications

doi: 10.1038/s41467-017-02585-y

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
Figure Legend 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

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

2) Product Images from "Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability"

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

Journal: Nature Communications

doi: 10.1038/s41467-017-02585-y

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
Figure Legend 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

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

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    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: 94/100, 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 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein g sepharose 4b ff beads - by Bioz Stars, 2022-11
    94/100 stars
      Buy from Supplier

    97
    Millipore bsa
    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
    Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsa/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsa - by Bioz Stars, 2022-11
    97/100 stars
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    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

    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