Structured Review

GE Healthcare gst syndapin i sh3 domain
<t>Syndapin</t> I depletion reduces the frequencies of mEPSCs. (A and F) Sample traces of whole-cell patch clamp recordings of mEPSCs from individual primary rat hippocampal neurons transfected at DIV 12 and analyzed 48 h later. (B) The frequency of mEPSCs was reduced in syndapin I RNAi neurons when compared with pRNAT and scrambled RNAi, respectively (B and D), whereas the mEPSC amplitudes did not differ (C and E). (F–J) Syndapin I RNAi rescue experiments with coexpression of mCherry–syndapin I and mutants thereof showing that both <t>SH3</t> domain protein interactions and F-BAR domain–mediated membrane interactions are crucial for syndapin I functions in postsynaptic neurotransmission. *, P
Gst Syndapin I Sh3 Domain, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gst syndapin i sh3 domain/product/GE Healthcare
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
gst syndapin i sh3 domain - by Bioz Stars, 2020-04
86/100 stars

Related Products / Commonly Used Together

full-length syndapin
gfp
xpress
gst
dna constructs plasmids

Images

1) Product Images from "ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function"

Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201307088

Syndapin I depletion reduces the frequencies of mEPSCs. (A and F) Sample traces of whole-cell patch clamp recordings of mEPSCs from individual primary rat hippocampal neurons transfected at DIV 12 and analyzed 48 h later. (B) The frequency of mEPSCs was reduced in syndapin I RNAi neurons when compared with pRNAT and scrambled RNAi, respectively (B and D), whereas the mEPSC amplitudes did not differ (C and E). (F–J) Syndapin I RNAi rescue experiments with coexpression of mCherry–syndapin I and mutants thereof showing that both SH3 domain protein interactions and F-BAR domain–mediated membrane interactions are crucial for syndapin I functions in postsynaptic neurotransmission. *, P
Figure Legend Snippet: Syndapin I depletion reduces the frequencies of mEPSCs. (A and F) Sample traces of whole-cell patch clamp recordings of mEPSCs from individual primary rat hippocampal neurons transfected at DIV 12 and analyzed 48 h later. (B) The frequency of mEPSCs was reduced in syndapin I RNAi neurons when compared with pRNAT and scrambled RNAi, respectively (B and D), whereas the mEPSC amplitudes did not differ (C and E). (F–J) Syndapin I RNAi rescue experiments with coexpression of mCherry–syndapin I and mutants thereof showing that both SH3 domain protein interactions and F-BAR domain–mediated membrane interactions are crucial for syndapin I functions in postsynaptic neurotransmission. *, P

Techniques Used: Patch Clamp, Transfection

Impaired spine and synapse formation upon syndapin I loss-of-function is caused by a loss of SH3 domain-dependent syndapin I functions in the postsynaptic compartment. (A and H) PM-mCherry signals of dendrites of neurons transfected as indicated at DIV 12 and fixed at DIV 14. Bars, 5 µm. (B–D) Quantitative analyses of general spine density (B) and of individual morphology groups (C and D) upon syndapin I RNAi. (E) Anti–PSD-95 (postsynaptic) and anti–synapsin 1 (presynaptic) immunolabeling along dendrites of transfected neurons. Bar, 5 µm. (F and G) Quantitation of PSD-95– (F) and synapsin 1–positive puncta (G) spatially overlapping with transfected neurons. (I–K) Quantitative analyses of general spine density (I) and of individual morphology groups (J and K) of syndapin I–depleted cells expressing Sdp I ΔSH3 compared with pRNAT control cells transfected in parallel. **, P
Figure Legend Snippet: Impaired spine and synapse formation upon syndapin I loss-of-function is caused by a loss of SH3 domain-dependent syndapin I functions in the postsynaptic compartment. (A and H) PM-mCherry signals of dendrites of neurons transfected as indicated at DIV 12 and fixed at DIV 14. Bars, 5 µm. (B–D) Quantitative analyses of general spine density (B) and of individual morphology groups (C and D) upon syndapin I RNAi. (E) Anti–PSD-95 (postsynaptic) and anti–synapsin 1 (presynaptic) immunolabeling along dendrites of transfected neurons. Bar, 5 µm. (F and G) Quantitation of PSD-95– (F) and synapsin 1–positive puncta (G) spatially overlapping with transfected neurons. (I–K) Quantitative analyses of general spine density (I) and of individual morphology groups (J and K) of syndapin I–depleted cells expressing Sdp I ΔSH3 compared with pRNAT control cells transfected in parallel. **, P

Techniques Used: Transfection, Immunolabeling, Quantitation Assay, Expressing

ProSAP1-mediated functions in spine head enlargement rely on complex formation with syndapin I. (A–G) Absence of ProSAP1-mediated spine head enlargement upon coexpression of the syndapin I SH3 domain blocking the syndapin I binding site of ProSAP1 (A and B), upon use of ProSAP1* (C–E), and upon concomitant syndapin I RNAi (F and G), respectively. (A, D, and F) Representative images of neurons transfected as indicated (cotransfected with PM-mCherry for morphological analysis). (B, E, and G) Quantification of head width of mushroom spines. (C) Coprecipitation studies with immobilized syndapin I and Abp1 SH3 domains and GFP-ProSAP1 versus GFP-ProSAP1* showing specific disruption of syndapin I interaction. (H–J) Quantitative analysis of head width of mushroom spines. Neither syndapin I RNAi nor overexpression of syndapin I modulate head sizes of mushroom spines. (J) ProSAP RNAi causes a decrease in head width not seen upon syndapin I RNAi and not rescued by syndapin I coexpression. *, P
Figure Legend Snippet: ProSAP1-mediated functions in spine head enlargement rely on complex formation with syndapin I. (A–G) Absence of ProSAP1-mediated spine head enlargement upon coexpression of the syndapin I SH3 domain blocking the syndapin I binding site of ProSAP1 (A and B), upon use of ProSAP1* (C–E), and upon concomitant syndapin I RNAi (F and G), respectively. (A, D, and F) Representative images of neurons transfected as indicated (cotransfected with PM-mCherry for morphological analysis). (B, E, and G) Quantification of head width of mushroom spines. (C) Coprecipitation studies with immobilized syndapin I and Abp1 SH3 domains and GFP-ProSAP1 versus GFP-ProSAP1* showing specific disruption of syndapin I interaction. (H–J) Quantitative analysis of head width of mushroom spines. Neither syndapin I RNAi nor overexpression of syndapin I modulate head sizes of mushroom spines. (J) ProSAP RNAi causes a decrease in head width not seen upon syndapin I RNAi and not rescued by syndapin I coexpression. *, P

Techniques Used: Blocking Assay, Binding Assay, Transfection, Over Expression

Identification of ProSAP1/Shank2 and ProSAP2/Shank3 as postsynaptically enriched Syndapin I interaction partners. (A) GST–syndapin I, II, and III specifically precipitate GFP-ProSAP1 expressed in HEK293 cells. (B) Coprecipitation analysis with GST–syndapin I and deletion mutants thereof. The SH3 domain is critical and sufficient for binding. A mutant SH3 domain (P434L; SH3*) did not bind. White lines indicate lanes omitted from blots (B and F). (C) Syndapin I SH3 precipitates GFP-ProSAP1 and GFP-ProSAP2 but not GFP-Shank1. (D) Alignment of +++APPPP motifs in ProSAP1 (NCBI Protein database accession no. NP_001004133 ), ProSAP2 (accession no. NP_067708 ), and Cobl (accession no. NP_766084 ; conserved amino acids are highlighted) and of corresponding residues in Shank1 (accession no. Q9WV48 ). (E) Scheme of rat ProSAP1b and deletion mutants used. Indicated are the N-terminal PDZ domain (medium grey), several proline-rich motifs (dark grey lines), and the C-terminal SAM (sterile alpha motif) domain (light grey). (F) GST–syndapin I precipitated GFP-ProSAP1 1–235 but none of the other ProSAP1 deletion mutants. (G) GFP fusion peptides encompassing the +++APPPP motifs of ProSAP1, ProSAP2, and Cobl associated with syndapin I SH3. (H and I) RKKAPPPPKR to GAGAAAAAAG mutation (amino acids 141–150 in ProSAP1; ProSAP1 1–235*) disrupted direct binding of ProSAP1 to syndapin I in both in vitro reconstitutions with purified proteins (H) and in coprecipitation analyses (I).
Figure Legend Snippet: Identification of ProSAP1/Shank2 and ProSAP2/Shank3 as postsynaptically enriched Syndapin I interaction partners. (A) GST–syndapin I, II, and III specifically precipitate GFP-ProSAP1 expressed in HEK293 cells. (B) Coprecipitation analysis with GST–syndapin I and deletion mutants thereof. The SH3 domain is critical and sufficient for binding. A mutant SH3 domain (P434L; SH3*) did not bind. White lines indicate lanes omitted from blots (B and F). (C) Syndapin I SH3 precipitates GFP-ProSAP1 and GFP-ProSAP2 but not GFP-Shank1. (D) Alignment of +++APPPP motifs in ProSAP1 (NCBI Protein database accession no. NP_001004133 ), ProSAP2 (accession no. NP_067708 ), and Cobl (accession no. NP_766084 ; conserved amino acids are highlighted) and of corresponding residues in Shank1 (accession no. Q9WV48 ). (E) Scheme of rat ProSAP1b and deletion mutants used. Indicated are the N-terminal PDZ domain (medium grey), several proline-rich motifs (dark grey lines), and the C-terminal SAM (sterile alpha motif) domain (light grey). (F) GST–syndapin I precipitated GFP-ProSAP1 1–235 but none of the other ProSAP1 deletion mutants. (G) GFP fusion peptides encompassing the +++APPPP motifs of ProSAP1, ProSAP2, and Cobl associated with syndapin I SH3. (H and I) RKKAPPPPKR to GAGAAAAAAG mutation (amino acids 141–150 in ProSAP1; ProSAP1 1–235*) disrupted direct binding of ProSAP1 to syndapin I in both in vitro reconstitutions with purified proteins (H) and in coprecipitation analyses (I).

Techniques Used: Binding Assay, Mutagenesis, In Vitro, Purification

Syndapin I interacts with ProSAP1 in vivo. (A) Specific coimmunoprecipitation of GFP-ProSAP1 with anti-FLAG antibodies immunoprecipitating FLAG–syndapin I. (B) Consistently, FLAG–syndapin I (arrowhead) was specifically coimmunoprecipitated with GFP-ProSAP1. (C) Immobilized GST–syndapin I SH3 specifically precipitated endogenous ProSAP1 from mouse brain cytosol (MBC). (D) Endogenous syndapin I was precipitated from rat brain preparations (RBC) with immobilized GST-ProSAP1 139–153 comprising the RKKAPPPP motif. (E–H) Syndapin I constitutively targeted to outer mitochondrial membranes recruited GFP-ProSAP1 (E) and GFP-ProSAP1 1–235 (F) in intact COS-7 cells, whereas Sdp I ΔSH3 did not (G and H). Bars, 10 µm. (I) Syndapin I and ProSAP1 immunolabeling of brain sections from adult mice. Colocalization in synapses of mossy fibers with dendrites of pyramidal cells in the stratum lucidum in the hippocampus CA3 is shown. Blue signal in merge, DAPI. Insets, 2.5-fold enlargements of the boxed areas. Bars, 25 µm. (J) Immunolabeling of neurons transfected with Xpress–syndapin I at DIV 12 and stained for syndapin I, ProSAP1, and the dendritic marker MAP2 at DIV 14. Insets, 1.5-fold enlargements of boxed areas. Bar, 10 µm. (K) Endogenous syndapin I colocalized with ProSAP1 and synapsin 1 (DIV 21). Insets, twofold enlargements of boxed areas. Bars: (main panels) 5 µm; (insets) 2 µm.
Figure Legend Snippet: Syndapin I interacts with ProSAP1 in vivo. (A) Specific coimmunoprecipitation of GFP-ProSAP1 with anti-FLAG antibodies immunoprecipitating FLAG–syndapin I. (B) Consistently, FLAG–syndapin I (arrowhead) was specifically coimmunoprecipitated with GFP-ProSAP1. (C) Immobilized GST–syndapin I SH3 specifically precipitated endogenous ProSAP1 from mouse brain cytosol (MBC). (D) Endogenous syndapin I was precipitated from rat brain preparations (RBC) with immobilized GST-ProSAP1 139–153 comprising the RKKAPPPP motif. (E–H) Syndapin I constitutively targeted to outer mitochondrial membranes recruited GFP-ProSAP1 (E) and GFP-ProSAP1 1–235 (F) in intact COS-7 cells, whereas Sdp I ΔSH3 did not (G and H). Bars, 10 µm. (I) Syndapin I and ProSAP1 immunolabeling of brain sections from adult mice. Colocalization in synapses of mossy fibers with dendrites of pyramidal cells in the stratum lucidum in the hippocampus CA3 is shown. Blue signal in merge, DAPI. Insets, 2.5-fold enlargements of the boxed areas. Bars, 25 µm. (J) Immunolabeling of neurons transfected with Xpress–syndapin I at DIV 12 and stained for syndapin I, ProSAP1, and the dendritic marker MAP2 at DIV 14. Insets, 1.5-fold enlargements of boxed areas. Bar, 10 µm. (K) Endogenous syndapin I colocalized with ProSAP1 and synapsin 1 (DIV 21). Insets, twofold enlargements of boxed areas. Bars: (main panels) 5 µm; (insets) 2 µm.

Techniques Used: In Vivo, Immunolabeling, Mouse Assay, Transfection, Staining, Marker

Related Articles

Construct:

Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function
Article Snippet: .. DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively. .. A vector expressing FLAG-mCherry (FLAGc) was generated by replacing GFP with mCherry in a pCMV-based FLAG-GFP expressing vector ( ).

Subcloning:

Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function
Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively. .. FLAGc-tagged syndapin I SH3 domain (FLAGc-Sdp I SH3) was generated by subcloning.

Plasmid Preparation:

Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function
Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively. .. A vector expressing FLAG-mCherry (FLAGc) was generated by replacing GFP with mCherry in a pCMV-based FLAG-GFP expressing vector ( ).

Expressing:

Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function
Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively. .. A vector expressing FLAG-mCherry (FLAGc) was generated by replacing GFP with mCherry in a pCMV-based FLAG-GFP expressing vector ( ).

Generated:

Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function
Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively. .. A vector expressing FLAG-mCherry (FLAGc) was generated by replacing GFP with mCherry in a pCMV-based FLAG-GFP expressing vector ( ).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    GE Healthcare gst syndapin i sh3 domain
    <t>Syndapin</t> I depletion reduces the frequencies of mEPSCs. (A and F) Sample traces of whole-cell patch clamp recordings of mEPSCs from individual primary rat hippocampal neurons transfected at DIV 12 and analyzed 48 h later. (B) The frequency of mEPSCs was reduced in syndapin I RNAi neurons when compared with pRNAT and scrambled RNAi, respectively (B and D), whereas the mEPSC amplitudes did not differ (C and E). (F–J) Syndapin I RNAi rescue experiments with coexpression of mCherry–syndapin I and mutants thereof showing that both <t>SH3</t> domain protein interactions and F-BAR domain–mediated membrane interactions are crucial for syndapin I functions in postsynaptic neurotransmission. *, P
    Gst Syndapin I Sh3 Domain, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gst syndapin i sh3 domain/product/GE Healthcare
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gst syndapin i sh3 domain - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    Image Search Results


    Syndapin I depletion reduces the frequencies of mEPSCs. (A and F) Sample traces of whole-cell patch clamp recordings of mEPSCs from individual primary rat hippocampal neurons transfected at DIV 12 and analyzed 48 h later. (B) The frequency of mEPSCs was reduced in syndapin I RNAi neurons when compared with pRNAT and scrambled RNAi, respectively (B and D), whereas the mEPSC amplitudes did not differ (C and E). (F–J) Syndapin I RNAi rescue experiments with coexpression of mCherry–syndapin I and mutants thereof showing that both SH3 domain protein interactions and F-BAR domain–mediated membrane interactions are crucial for syndapin I functions in postsynaptic neurotransmission. *, P

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Syndapin I depletion reduces the frequencies of mEPSCs. (A and F) Sample traces of whole-cell patch clamp recordings of mEPSCs from individual primary rat hippocampal neurons transfected at DIV 12 and analyzed 48 h later. (B) The frequency of mEPSCs was reduced in syndapin I RNAi neurons when compared with pRNAT and scrambled RNAi, respectively (B and D), whereas the mEPSC amplitudes did not differ (C and E). (F–J) Syndapin I RNAi rescue experiments with coexpression of mCherry–syndapin I and mutants thereof showing that both SH3 domain protein interactions and F-BAR domain–mediated membrane interactions are crucial for syndapin I functions in postsynaptic neurotransmission. *, P

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: Patch Clamp, Transfection

    Impaired spine and synapse formation upon syndapin I loss-of-function is caused by a loss of SH3 domain-dependent syndapin I functions in the postsynaptic compartment. (A and H) PM-mCherry signals of dendrites of neurons transfected as indicated at DIV 12 and fixed at DIV 14. Bars, 5 µm. (B–D) Quantitative analyses of general spine density (B) and of individual morphology groups (C and D) upon syndapin I RNAi. (E) Anti–PSD-95 (postsynaptic) and anti–synapsin 1 (presynaptic) immunolabeling along dendrites of transfected neurons. Bar, 5 µm. (F and G) Quantitation of PSD-95– (F) and synapsin 1–positive puncta (G) spatially overlapping with transfected neurons. (I–K) Quantitative analyses of general spine density (I) and of individual morphology groups (J and K) of syndapin I–depleted cells expressing Sdp I ΔSH3 compared with pRNAT control cells transfected in parallel. **, P

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Impaired spine and synapse formation upon syndapin I loss-of-function is caused by a loss of SH3 domain-dependent syndapin I functions in the postsynaptic compartment. (A and H) PM-mCherry signals of dendrites of neurons transfected as indicated at DIV 12 and fixed at DIV 14. Bars, 5 µm. (B–D) Quantitative analyses of general spine density (B) and of individual morphology groups (C and D) upon syndapin I RNAi. (E) Anti–PSD-95 (postsynaptic) and anti–synapsin 1 (presynaptic) immunolabeling along dendrites of transfected neurons. Bar, 5 µm. (F and G) Quantitation of PSD-95– (F) and synapsin 1–positive puncta (G) spatially overlapping with transfected neurons. (I–K) Quantitative analyses of general spine density (I) and of individual morphology groups (J and K) of syndapin I–depleted cells expressing Sdp I ΔSH3 compared with pRNAT control cells transfected in parallel. **, P

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: Transfection, Immunolabeling, Quantitation Assay, Expressing

    ProSAP1-mediated functions in spine head enlargement rely on complex formation with syndapin I. (A–G) Absence of ProSAP1-mediated spine head enlargement upon coexpression of the syndapin I SH3 domain blocking the syndapin I binding site of ProSAP1 (A and B), upon use of ProSAP1* (C–E), and upon concomitant syndapin I RNAi (F and G), respectively. (A, D, and F) Representative images of neurons transfected as indicated (cotransfected with PM-mCherry for morphological analysis). (B, E, and G) Quantification of head width of mushroom spines. (C) Coprecipitation studies with immobilized syndapin I and Abp1 SH3 domains and GFP-ProSAP1 versus GFP-ProSAP1* showing specific disruption of syndapin I interaction. (H–J) Quantitative analysis of head width of mushroom spines. Neither syndapin I RNAi nor overexpression of syndapin I modulate head sizes of mushroom spines. (J) ProSAP RNAi causes a decrease in head width not seen upon syndapin I RNAi and not rescued by syndapin I coexpression. *, P

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: ProSAP1-mediated functions in spine head enlargement rely on complex formation with syndapin I. (A–G) Absence of ProSAP1-mediated spine head enlargement upon coexpression of the syndapin I SH3 domain blocking the syndapin I binding site of ProSAP1 (A and B), upon use of ProSAP1* (C–E), and upon concomitant syndapin I RNAi (F and G), respectively. (A, D, and F) Representative images of neurons transfected as indicated (cotransfected with PM-mCherry for morphological analysis). (B, E, and G) Quantification of head width of mushroom spines. (C) Coprecipitation studies with immobilized syndapin I and Abp1 SH3 domains and GFP-ProSAP1 versus GFP-ProSAP1* showing specific disruption of syndapin I interaction. (H–J) Quantitative analysis of head width of mushroom spines. Neither syndapin I RNAi nor overexpression of syndapin I modulate head sizes of mushroom spines. (J) ProSAP RNAi causes a decrease in head width not seen upon syndapin I RNAi and not rescued by syndapin I coexpression. *, P

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: Blocking Assay, Binding Assay, Transfection, Over Expression

    Identification of ProSAP1/Shank2 and ProSAP2/Shank3 as postsynaptically enriched Syndapin I interaction partners. (A) GST–syndapin I, II, and III specifically precipitate GFP-ProSAP1 expressed in HEK293 cells. (B) Coprecipitation analysis with GST–syndapin I and deletion mutants thereof. The SH3 domain is critical and sufficient for binding. A mutant SH3 domain (P434L; SH3*) did not bind. White lines indicate lanes omitted from blots (B and F). (C) Syndapin I SH3 precipitates GFP-ProSAP1 and GFP-ProSAP2 but not GFP-Shank1. (D) Alignment of +++APPPP motifs in ProSAP1 (NCBI Protein database accession no. NP_001004133 ), ProSAP2 (accession no. NP_067708 ), and Cobl (accession no. NP_766084 ; conserved amino acids are highlighted) and of corresponding residues in Shank1 (accession no. Q9WV48 ). (E) Scheme of rat ProSAP1b and deletion mutants used. Indicated are the N-terminal PDZ domain (medium grey), several proline-rich motifs (dark grey lines), and the C-terminal SAM (sterile alpha motif) domain (light grey). (F) GST–syndapin I precipitated GFP-ProSAP1 1–235 but none of the other ProSAP1 deletion mutants. (G) GFP fusion peptides encompassing the +++APPPP motifs of ProSAP1, ProSAP2, and Cobl associated with syndapin I SH3. (H and I) RKKAPPPPKR to GAGAAAAAAG mutation (amino acids 141–150 in ProSAP1; ProSAP1 1–235*) disrupted direct binding of ProSAP1 to syndapin I in both in vitro reconstitutions with purified proteins (H) and in coprecipitation analyses (I).

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Identification of ProSAP1/Shank2 and ProSAP2/Shank3 as postsynaptically enriched Syndapin I interaction partners. (A) GST–syndapin I, II, and III specifically precipitate GFP-ProSAP1 expressed in HEK293 cells. (B) Coprecipitation analysis with GST–syndapin I and deletion mutants thereof. The SH3 domain is critical and sufficient for binding. A mutant SH3 domain (P434L; SH3*) did not bind. White lines indicate lanes omitted from blots (B and F). (C) Syndapin I SH3 precipitates GFP-ProSAP1 and GFP-ProSAP2 but not GFP-Shank1. (D) Alignment of +++APPPP motifs in ProSAP1 (NCBI Protein database accession no. NP_001004133 ), ProSAP2 (accession no. NP_067708 ), and Cobl (accession no. NP_766084 ; conserved amino acids are highlighted) and of corresponding residues in Shank1 (accession no. Q9WV48 ). (E) Scheme of rat ProSAP1b and deletion mutants used. Indicated are the N-terminal PDZ domain (medium grey), several proline-rich motifs (dark grey lines), and the C-terminal SAM (sterile alpha motif) domain (light grey). (F) GST–syndapin I precipitated GFP-ProSAP1 1–235 but none of the other ProSAP1 deletion mutants. (G) GFP fusion peptides encompassing the +++APPPP motifs of ProSAP1, ProSAP2, and Cobl associated with syndapin I SH3. (H and I) RKKAPPPPKR to GAGAAAAAAG mutation (amino acids 141–150 in ProSAP1; ProSAP1 1–235*) disrupted direct binding of ProSAP1 to syndapin I in both in vitro reconstitutions with purified proteins (H) and in coprecipitation analyses (I).

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: Binding Assay, Mutagenesis, In Vitro, Purification

    Syndapin I interacts with ProSAP1 in vivo. (A) Specific coimmunoprecipitation of GFP-ProSAP1 with anti-FLAG antibodies immunoprecipitating FLAG–syndapin I. (B) Consistently, FLAG–syndapin I (arrowhead) was specifically coimmunoprecipitated with GFP-ProSAP1. (C) Immobilized GST–syndapin I SH3 specifically precipitated endogenous ProSAP1 from mouse brain cytosol (MBC). (D) Endogenous syndapin I was precipitated from rat brain preparations (RBC) with immobilized GST-ProSAP1 139–153 comprising the RKKAPPPP motif. (E–H) Syndapin I constitutively targeted to outer mitochondrial membranes recruited GFP-ProSAP1 (E) and GFP-ProSAP1 1–235 (F) in intact COS-7 cells, whereas Sdp I ΔSH3 did not (G and H). Bars, 10 µm. (I) Syndapin I and ProSAP1 immunolabeling of brain sections from adult mice. Colocalization in synapses of mossy fibers with dendrites of pyramidal cells in the stratum lucidum in the hippocampus CA3 is shown. Blue signal in merge, DAPI. Insets, 2.5-fold enlargements of the boxed areas. Bars, 25 µm. (J) Immunolabeling of neurons transfected with Xpress–syndapin I at DIV 12 and stained for syndapin I, ProSAP1, and the dendritic marker MAP2 at DIV 14. Insets, 1.5-fold enlargements of boxed areas. Bar, 10 µm. (K) Endogenous syndapin I colocalized with ProSAP1 and synapsin 1 (DIV 21). Insets, twofold enlargements of boxed areas. Bars: (main panels) 5 µm; (insets) 2 µm.

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Syndapin I interacts with ProSAP1 in vivo. (A) Specific coimmunoprecipitation of GFP-ProSAP1 with anti-FLAG antibodies immunoprecipitating FLAG–syndapin I. (B) Consistently, FLAG–syndapin I (arrowhead) was specifically coimmunoprecipitated with GFP-ProSAP1. (C) Immobilized GST–syndapin I SH3 specifically precipitated endogenous ProSAP1 from mouse brain cytosol (MBC). (D) Endogenous syndapin I was precipitated from rat brain preparations (RBC) with immobilized GST-ProSAP1 139–153 comprising the RKKAPPPP motif. (E–H) Syndapin I constitutively targeted to outer mitochondrial membranes recruited GFP-ProSAP1 (E) and GFP-ProSAP1 1–235 (F) in intact COS-7 cells, whereas Sdp I ΔSH3 did not (G and H). Bars, 10 µm. (I) Syndapin I and ProSAP1 immunolabeling of brain sections from adult mice. Colocalization in synapses of mossy fibers with dendrites of pyramidal cells in the stratum lucidum in the hippocampus CA3 is shown. Blue signal in merge, DAPI. Insets, 2.5-fold enlargements of the boxed areas. Bars, 25 µm. (J) Immunolabeling of neurons transfected with Xpress–syndapin I at DIV 12 and stained for syndapin I, ProSAP1, and the dendritic marker MAP2 at DIV 14. Insets, 1.5-fold enlargements of boxed areas. Bar, 10 µm. (K) Endogenous syndapin I colocalized with ProSAP1 and synapsin 1 (DIV 21). Insets, twofold enlargements of boxed areas. Bars: (main panels) 5 µm; (insets) 2 µm.

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: In Vivo, Immunolabeling, Mouse Assay, Transfection, Staining, Marker