plasmid encoding full-length epha2 containing a c-terminal turbogfp tag  (OriGene)

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) Schematic representation of sample preparation and workflow for SiMPull-POP. 1-Membrane fractions containing EphA2-GFP were solubilized with the amphipathic copolymer DIBMA to generate DIBMALPs. 2-EphA2-GFP DIBMALPs were immobilized on a functionalized microscope slide displaying an EphA2 antibody. 3-DIBMALPs devoid of EphA2-GFP are washed away before imaging. (B) Representative single-molecule TIRF image in the presence (left) and absence (right) of EphA2 antibody. Each blue spot represents a DIBMALP containing EphA2-GFP. (C) Representative GFP photobleaching traces showing a stepwise decrease in GFP intensity over time; arrows represent individual photobleaching events. Photobleaching steps are used to infer EphA2 oligomerization status.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

Techniques: Sample Prep, Membrane, Microscopy, Imaging

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) Schematic of DIBMALP containing an EphA2-GFP monomer. (B) Step distribution of control DIBMALPs (black) or those formed from cells treated with EA1 (pink), MβCD (blue) or both (magenta). (C) Oligomeric distribution calculated from data in panel B. (D) Schematic representing DDM micelles containing EphA2-GFP. (E) Step distribution of DDM-solubilized EphA2-GFP in the same conditions as in DIBMALPs. (F) Oligomeric distribution of DDM-solubilized EphA2-GFP photobleaching data. p -values are from two-way ANOVA followed by Tukey multiple comparison test.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: Western blot analysis of EphA2 pS897 in HEK293T (A) , A375 (B) and A431 (C) cells. We show pS897 quantification (mean ± S.D) and representative blots. p -values from one-way ANOVA followed by Mann-Whitney U or t test. *, p ≤ 0.05; **, p ≤ 0.01.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

Techniques: Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) cAMP quantification in HEK293T cells transduced with Red Up cADDis cAMP biosensor ollowing treatment with MβCD. (B) Western blot analysis and quantification of PKA T197 phosphorylation in A375 cells following treatment with MβCD. (C-D) Western blot analysis and quantification of EphA2 S897 phosphorylation in A375 cells following treatment with forskolin and soproterenol, respectively. Data shown in panel B are normalized to the respective total PKA signal in Figure S15A. Data shown in panels C-D are normalized to the respective total EphA2 signal in Figure S15B-C. Quantitative comparisons between treatments were made with respect to normalized control conditions. Bar graphs show mean ± S.D., p -values in panels A-D are from an unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

Techniques: Transduction, Western Blot, Control

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) In the presence of normal levels of Chol (yellow oval), EphA2 (blue) can be found as a monomer and displays low Ser897 phosphorylation. We propose that in this state the kinase domain interacts with the SAM domain. (B) When Chol content is reduced, β-AR (purple) activity increases, promoting cAMP/PKA signaling that enhances Ser897 phosphorylation (red dot). This forces the kinase-SAM linker into an open conformation, which promotes higher-order oligomers independent from ligand stimulation.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

Techniques: Activity Assay