Journal: Molecular Cell

Article Title: Reconstitution of the destruction complex defines roles of AXIN polymers and APC in β-catenin capture, phosphorylation, and ubiquitylation

doi: 10.1016/j.molcel.2021.07.013

Figure Lengend Snippet: Reconstitution of the biochemical activity of the DC (A) Schematic representation of the DC and accessory components for biochemical assays. (B) Purified DC components analyzed using SDS-PAGE and Coomassie staining. kd, kinase-dead. (C) Purified ubiquitylation machinery components. (D) In vitro DC activity assay (reaction time 60 min). Top panel: dual-channel fluorimetry of SDS-PAGE gel with β-catenin in red and ubiquitin in green. Middle and bottom panels: separated channels. Asterisks in the bottom panel mark ubiquitylated APC variants. See Figure S2 for additional time points and Figure S3 for analyses of β-catenin phosphorylation. (E) Quantification of APC-dependent β-catenin poly-ubiquitylation. See B and S2C for quantification details. Data are means from three independent experiments ± SEM. Statistical significance was tested using one-way ANOVA with the Bonferroni test for multiple comparisons. Asterisks above bars (without brackets) refer to comparisons to the no-APC condition (black bar). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. See for details of statistical analyses. (F) In vitro DC activity assay (as in D, but at 30 min time point) ± GSK3 or CK1 inhibitors at the indicated concentrations. The asterisk in the bottom panel marks ubiquitylated APC. See A and S4B for additional time points and analyses of β-catenin phosphorylation. (G) Quantification of APC-dependent β-catenin poly-ubiquitylation ± GSK3 inhibitor, on the basis of data shown in (F) (n = 1 representative experiment). See Figure S4 C for CK1 inhibitor quantification.

Article Snippet: cDNA constructs of DC components and the SCF β-TrCP E3 ligase complex were codon-optimized for E.coli expression by GenScript and were subcloned into the pLIB vector ( ) under control of the polyhedron gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV).

Techniques: Activity Assay, Purification, SDS Page, Staining, In Vitro, Ubiquitin Proteomics, Phospho-proteomics

Journal: Molecular Cell

Article Title: Reconstitution of the destruction complex defines roles of AXIN polymers and APC in β-catenin capture, phosphorylation, and ubiquitylation

doi: 10.1016/j.molcel.2021.07.013

Figure Lengend Snippet: Interaction of APC with the SCF β-TrCP E3 ubiquitin ligase complex The indicated proteins and complexes were resolved by size exclusion chromatography and fractions analyzed using SDS-PAGE and Coomassie (input) or silver staining (SEC fractions). The top and bottom parts of the gels were separated for silver staining of SKP1 and RBX1 to avoid overstaining of the other components. See Figure S8 for purification of the E3 complexes and analyses of β-catenin and AXIN1 interactions with SCF β-TrCP .

Article Snippet: cDNA constructs of DC components and the SCF β-TrCP E3 ligase complex were codon-optimized for E.coli expression by GenScript and were subcloned into the pLIB vector ( ) under control of the polyhedron gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV).

Techniques: Ubiquitin Proteomics, Size-exclusion Chromatography, SDS Page, Silver Staining, Purification