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a Schematic illustration of the catalytic mechanisms employed by major E3 ligase families: RING, HECT, and RBR. Acceptor lysine (on E3 or on substrate) is shown in orange. b Schematic showing the experimental workflow of the E2 discharge assay using free amino acids. c E2 discharge assay using UbcH8∼ISG15 in the presence of various amino acids (50 mM) d E2 discharge assay using UbcH8∼ISG15 in the presence of increasing concentrations of L-lys as indicated. e E2 discharge assay using UbcH7∼Ub in the presence of various amino acids (50 mM f E2 discharge assay using <t>UbcH5A∼Ub</t> in the presence of various amino acids (50 mM)
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a Schematic illustration of the catalytic mechanisms employed by major E3 ligase families: RING, HECT, and RBR. Acceptor lysine (on E3 or on substrate) is shown in orange. b Schematic showing the experimental workflow of the E2 discharge assay using free amino acids. c E2 discharge assay using UbcH8∼ISG15 in the presence of various amino acids (50 mM) d E2 discharge assay using UbcH8∼ISG15 in the presence of increasing concentrations of L-lys as indicated. e E2 discharge assay using UbcH7∼Ub in the presence of various amino acids (50 mM f E2 discharge assay using UbcH5A∼Ub in the presence of various amino acids (50 mM)

Journal: bioRxiv

Article Title: ISGylation Mechanism Uncovers Conformational Specificity for HECT-family E3 ligase

doi: 10.1101/2025.09.26.678755

Figure Lengend Snippet: a Schematic illustration of the catalytic mechanisms employed by major E3 ligase families: RING, HECT, and RBR. Acceptor lysine (on E3 or on substrate) is shown in orange. b Schematic showing the experimental workflow of the E2 discharge assay using free amino acids. c E2 discharge assay using UbcH8∼ISG15 in the presence of various amino acids (50 mM) d E2 discharge assay using UbcH8∼ISG15 in the presence of increasing concentrations of L-lys as indicated. e E2 discharge assay using UbcH7∼Ub in the presence of various amino acids (50 mM f E2 discharge assay using UbcH5A∼Ub in the presence of various amino acids (50 mM)

Article Snippet: The following plasmids were obtained from Addgene: UBE1 (Addgene plasmid #34965; gift from Cynthia Wolberger), UbcH5A (Addgene plasmid #15782; gift from Wade Harper), and NEDD4L (Addgene plasmid #165103; gift from Arthur Haas).

Techniques: