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Figure 1. Pirh2 and KLHDC10 directly bind to Ala-tails (A) Domain arrangement of human Pirh2: NTM, N-terminal module; RING, really interesting new gene domain; CTD, C-terminal domain. (B) In vitro GST pull-down assay using the specified GST-Pirh2 truncation constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (C) <t>AlphaScreen</t> assay to assess Pirh2-Ala-tail binding. Reactants were GST fusions with Pirh2 fragments containing both the NTM and RING domains (Pirh21–195) or the NTM alone (Pirh21–137) and a biotinylated Ala-tail peptide (MDELYKAAAAAA). Proximity-induced fluorescence signal (see STAR Methods) was monitored in the presence of increasing amounts of a competing, non-biotinylated Ala-tail peptide to determine IC50 values from the dose-response curve. Each data point is a technical triplicate, and error bars represent ± SD. The y axis presents the normalized AlphaScreen signal as arbitrary fluorescence units (AFUs). (D) Domain arrangement of human KLHDC10. (E) In vitro GST pull-down assay using the GST-KLHDC10 and GST-KLHDC2 constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (F) AlphaScreen assay to assess KLHDC10-Ala-tail binding. As in (C) but using GST-KLHDC10 and GST-KLHDC10b1-88–442 instead. See also Figures S1, S2, S10, and S11.
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Figure 1. Pirh2 and KLHDC10 directly bind to Ala-tails (A) Domain arrangement of human Pirh2: NTM, N-terminal module; RING, really interesting new gene domain; CTD, C-terminal domain. (B) In vitro GST pull-down assay using the specified GST-Pirh2 truncation constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (C) <t>AlphaScreen</t> assay to assess Pirh2-Ala-tail binding. Reactants were GST fusions with Pirh2 fragments containing both the NTM and RING domains (Pirh21–195) or the NTM alone (Pirh21–137) and a biotinylated Ala-tail peptide (MDELYKAAAAAA). Proximity-induced fluorescence signal (see STAR Methods) was monitored in the presence of increasing amounts of a competing, non-biotinylated Ala-tail peptide to determine IC50 values from the dose-response curve. Each data point is a technical triplicate, and error bars represent ± SD. The y axis presents the normalized AlphaScreen signal as arbitrary fluorescence units (AFUs). (D) Domain arrangement of human KLHDC10. (E) In vitro GST pull-down assay using the GST-KLHDC10 and GST-KLHDC2 constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (F) AlphaScreen assay to assess KLHDC10-Ala-tail binding. As in (C) but using GST-KLHDC10 and GST-KLHDC10b1-88–442 instead. See also Figures S1, S2, S10, and S11.
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Figure 1. Pirh2 and KLHDC10 directly bind to Ala-tails (A) Domain arrangement of human Pirh2: NTM, N-terminal module; RING, really interesting new gene domain; CTD, C-terminal domain. (B) In vitro GST pull-down assay using the specified GST-Pirh2 truncation constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (C) <t>AlphaScreen</t> assay to assess Pirh2-Ala-tail binding. Reactants were GST fusions with Pirh2 fragments containing both the NTM and RING domains (Pirh21–195) or the NTM alone (Pirh21–137) and a biotinylated Ala-tail peptide (MDELYKAAAAAA). Proximity-induced fluorescence signal (see STAR Methods) was monitored in the presence of increasing amounts of a competing, non-biotinylated Ala-tail peptide to determine IC50 values from the dose-response curve. Each data point is a technical triplicate, and error bars represent ± SD. The y axis presents the normalized AlphaScreen signal as arbitrary fluorescence units (AFUs). (D) Domain arrangement of human KLHDC10. (E) In vitro GST pull-down assay using the GST-KLHDC10 and GST-KLHDC2 constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (F) AlphaScreen assay to assess KLHDC10-Ala-tail binding. As in (C) but using GST-KLHDC10 and GST-KLHDC10b1-88–442 instead. See also Figures S1, S2, S10, and S11.
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Figure 1. Pirh2 and KLHDC10 directly bind to Ala-tails (A) Domain arrangement of human Pirh2: NTM, N-terminal module; RING, really interesting new gene domain; CTD, C-terminal domain. (B) In vitro GST pull-down assay using the specified GST-Pirh2 truncation constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (C) <t>AlphaScreen</t> assay to assess Pirh2-Ala-tail binding. Reactants were GST fusions with Pirh2 fragments containing both the NTM and RING domains (Pirh21–195) or the NTM alone (Pirh21–137) and a biotinylated Ala-tail peptide (MDELYKAAAAAA). Proximity-induced fluorescence signal (see STAR Methods) was monitored in the presence of increasing amounts of a competing, non-biotinylated Ala-tail peptide to determine IC50 values from the dose-response curve. Each data point is a technical triplicate, and error bars represent ± SD. The y axis presents the normalized AlphaScreen signal as arbitrary fluorescence units (AFUs). (D) Domain arrangement of human KLHDC10. (E) In vitro GST pull-down assay using the GST-KLHDC10 and GST-KLHDC2 constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (F) AlphaScreen assay to assess KLHDC10-Ala-tail binding. As in (C) but using GST-KLHDC10 and GST-KLHDC10b1-88–442 instead. See also Figures S1, S2, S10, and S11.
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Figure 1. Pirh2 and KLHDC10 directly bind to Ala-tails (A) Domain arrangement of human Pirh2: NTM, N-terminal module; RING, really interesting new gene domain; CTD, C-terminal domain. (B) In vitro GST pull-down assay using the specified GST-Pirh2 truncation constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (C) AlphaScreen assay to assess Pirh2-Ala-tail binding. Reactants were GST fusions with Pirh2 fragments containing both the NTM and RING domains (Pirh21–195) or the NTM alone (Pirh21–137) and a biotinylated Ala-tail peptide (MDELYKAAAAAA). Proximity-induced fluorescence signal (see STAR Methods) was monitored in the presence of increasing amounts of a competing, non-biotinylated Ala-tail peptide to determine IC50 values from the dose-response curve. Each data point is a technical triplicate, and error bars represent ± SD. The y axis presents the normalized AlphaScreen signal as arbitrary fluorescence units (AFUs). (D) Domain arrangement of human KLHDC10. (E) In vitro GST pull-down assay using the GST-KLHDC10 and GST-KLHDC2 constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (F) AlphaScreen assay to assess KLHDC10-Ala-tail binding. As in (C) but using GST-KLHDC10 and GST-KLHDC10b1-88–442 instead. See also Figures S1, S2, S10, and S11.

Journal: Cell reports

Article Title: Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.

doi: 10.1016/j.celrep.2023.113100

Figure Lengend Snippet: Figure 1. Pirh2 and KLHDC10 directly bind to Ala-tails (A) Domain arrangement of human Pirh2: NTM, N-terminal module; RING, really interesting new gene domain; CTD, C-terminal domain. (B) In vitro GST pull-down assay using the specified GST-Pirh2 truncation constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (C) AlphaScreen assay to assess Pirh2-Ala-tail binding. Reactants were GST fusions with Pirh2 fragments containing both the NTM and RING domains (Pirh21–195) or the NTM alone (Pirh21–137) and a biotinylated Ala-tail peptide (MDELYKAAAAAA). Proximity-induced fluorescence signal (see STAR Methods) was monitored in the presence of increasing amounts of a competing, non-biotinylated Ala-tail peptide to determine IC50 values from the dose-response curve. Each data point is a technical triplicate, and error bars represent ± SD. The y axis presents the normalized AlphaScreen signal as arbitrary fluorescence units (AFUs). (D) Domain arrangement of human KLHDC10. (E) In vitro GST pull-down assay using the GST-KLHDC10 and GST-KLHDC2 constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (F) AlphaScreen assay to assess KLHDC10-Ala-tail binding. As in (C) but using GST-KLHDC10 and GST-KLHDC10b1-88–442 instead. See also Figures S1, S2, S10, and S11.

Article Snippet: AlphaScreen signal was measured using PHERAstar microplate reader (BMG Labtech) at RT.

Techniques: In Vitro, Pull Down Assay, Construct, Western Blot, Amplified Luminescent Proximity Homogenous Assay, Binding Assay