pad-wnt-3a Search Results


wnt3a constructs  (Addgene inc)
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Addgene inc wnt3a constructs
a , Predicted Wnt7a structure in AlphaFold (blue) with the EBP region highlighted in orange and the three positively charged motifs (red) within the EBP. Note that EBP is a solvent-exposed region. b, Wnt7a-ESP*Scramble mutant maintains the dilysine motif and exhibits no impairment in EV secretion. c, Isothermal calorimetry measurements for COPB2 1-304 binding to potential dilysine/arginine motifs within the EBP. Wild-type COPB2 1-304 binds to the LKIKKP sub-region. d-f, views of the KxKx motif of Wnt7a bound to COPB2 1-304 . d , Top view of the WD-repeat domain of COPB 1-304 (green) with the LKIKKP peptide (orange) in ribbon representation. e, Close-up view of the LKIKKP peptide with a difference electron density map calculated by omitting the peptide and contoured at 3σ (blue mesh). COPB2 surface is colored by electrostatic potential ranging from −5kT/e (red) to 5kT/e (blue). f, Lateral view of the binding motif with hydrogen bonds and distances. g, Structure-based point mutations confirm the molecular recognition of the KxKx motif in ITC assays. h , Double lysine mutation of K253 and K255 by alanine disrupts Wnt7a-EVs secretion. i , Replacement of Wnt7a-EBP by either Wnt10a-EBP or Wnt16 EBP containing KR and RR (right panel). Replacement with Wnt10a-EBP or Wnt16 EBP rescues Wnt7a-EVs secretion. j , EV secretion analysis of Wnt10b after EBP removal or double arginine mutation within its EBP (right panel). Double arginine mutation disrupts secretion of Wnt10b on EVs to the same extent as removal of the entire Wnt10b EBP sequence. k , Secretion analysis of <t>Wnt3a</t> after EBP removal or mutation of the entire positively charged motifs RPR, KHR, KH within its EBP (right panel). Only concomitant mutation of RPR and KHR motifs disrupts secretion of Wnt3a on EVS to the same extent as removal of the entire Wnt3a EBP sequence. Experiments are representative of three independent biological replicates performed in HEK293T cells transfected with different Wnt-HA tagged truncates.
Wnt3a Constructs, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a , Predicted Wnt7a structure in AlphaFold (blue) with the EBP region highlighted in orange and the three positively charged motifs (red) within the EBP. Note that EBP is a solvent-exposed region. b, Wnt7a-ESP*Scramble mutant maintains the dilysine motif and exhibits no impairment in EV secretion. c, Isothermal calorimetry measurements for COPB2 1-304 binding to potential dilysine/arginine motifs within the EBP. Wild-type COPB2 1-304 binds to the LKIKKP sub-region. d-f, views of the KxKx motif of Wnt7a bound to COPB2 1-304 . d , Top view of the WD-repeat domain of COPB 1-304 (green) with the LKIKKP peptide (orange) in ribbon representation. e, Close-up view of the LKIKKP peptide with a difference electron density map calculated by omitting the peptide and contoured at 3σ (blue mesh). COPB2 surface is colored by electrostatic potential ranging from −5kT/e (red) to 5kT/e (blue). f, Lateral view of the binding motif with hydrogen bonds and distances. g, Structure-based point mutations confirm the molecular recognition of the KxKx motif in ITC assays. h , Double lysine mutation of K253 and K255 by alanine disrupts Wnt7a-EVs secretion. i , Replacement of Wnt7a-EBP by either Wnt10a-EBP or Wnt16 EBP containing KR and RR (right panel). Replacement with Wnt10a-EBP or Wnt16 EBP rescues Wnt7a-EVs secretion. j , EV secretion analysis of Wnt10b after EBP removal or double arginine mutation within its EBP (right panel). Double arginine mutation disrupts secretion of Wnt10b on EVs to the same extent as removal of the entire Wnt10b EBP sequence. k , Secretion analysis of Wnt3a after EBP removal or mutation of the entire positively charged motifs RPR, KHR, KH within its EBP (right panel). Only concomitant mutation of RPR and KHR motifs disrupts secretion of Wnt3a on EVS to the same extent as removal of the entire Wnt3a EBP sequence. Experiments are representative of three independent biological replicates performed in HEK293T cells transfected with different Wnt-HA tagged truncates.

Journal: bioRxiv

Article Title: Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation

doi: 10.1101/2023.05.30.542914

Figure Lengend Snippet: a , Predicted Wnt7a structure in AlphaFold (blue) with the EBP region highlighted in orange and the three positively charged motifs (red) within the EBP. Note that EBP is a solvent-exposed region. b, Wnt7a-ESP*Scramble mutant maintains the dilysine motif and exhibits no impairment in EV secretion. c, Isothermal calorimetry measurements for COPB2 1-304 binding to potential dilysine/arginine motifs within the EBP. Wild-type COPB2 1-304 binds to the LKIKKP sub-region. d-f, views of the KxKx motif of Wnt7a bound to COPB2 1-304 . d , Top view of the WD-repeat domain of COPB 1-304 (green) with the LKIKKP peptide (orange) in ribbon representation. e, Close-up view of the LKIKKP peptide with a difference electron density map calculated by omitting the peptide and contoured at 3σ (blue mesh). COPB2 surface is colored by electrostatic potential ranging from −5kT/e (red) to 5kT/e (blue). f, Lateral view of the binding motif with hydrogen bonds and distances. g, Structure-based point mutations confirm the molecular recognition of the KxKx motif in ITC assays. h , Double lysine mutation of K253 and K255 by alanine disrupts Wnt7a-EVs secretion. i , Replacement of Wnt7a-EBP by either Wnt10a-EBP or Wnt16 EBP containing KR and RR (right panel). Replacement with Wnt10a-EBP or Wnt16 EBP rescues Wnt7a-EVs secretion. j , EV secretion analysis of Wnt10b after EBP removal or double arginine mutation within its EBP (right panel). Double arginine mutation disrupts secretion of Wnt10b on EVs to the same extent as removal of the entire Wnt10b EBP sequence. k , Secretion analysis of Wnt3a after EBP removal or mutation of the entire positively charged motifs RPR, KHR, KH within its EBP (right panel). Only concomitant mutation of RPR and KHR motifs disrupts secretion of Wnt3a on EVS to the same extent as removal of the entire Wnt3a EBP sequence. Experiments are representative of three independent biological replicates performed in HEK293T cells transfected with different Wnt-HA tagged truncates.

Article Snippet: Wnt10b and Wnt3a constructs used in this publication were a gift from Marian Waterman, David Virshup and Xi He from the plasmid kit (Addgene kit # 1000000022).

Techniques: Solvent, Mutagenesis, Binding Assay, Sequencing, Transfection