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a Schematic of the CTLH E3 complex subunits organized by functional roles. b Assays testing MKLN1 ubiquitylation by the indicated CTLH E3 complexes. Ubiquitylation assays used N-terminally fluorescently labeled K0-ubiquitin (*UB K0 ) with all lysines mutated to arginines to visualize direct substrate modification without chain formation, with UBE2H as the E2. c In vitro ubiquitylation assays testing ZMYND19 ubiquitylation by the indicated CTLH E3 complexes. d Assays testing the effect of the GID4 substrate receptor on CTLH MKLN1 -dependent ZMYND19 ubiquitylation. e Assays testing UNG2 ubiquitylation by CTLH MKLN1 in the presence or absence of substrate adapter FAM72A. b – e All assays were performed for 15 min ( n ≥ 2 independent replicates). Source data are provided as a file.
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a Schematic of the CTLH E3 complex subunits organized by functional roles. b Assays testing MKLN1 ubiquitylation by the indicated CTLH E3 complexes. Ubiquitylation assays used N-terminally fluorescently labeled K0-ubiquitin (*UB K0 ) with all lysines mutated to arginines to visualize direct substrate modification without chain formation, with UBE2H as the E2. c In vitro ubiquitylation assays testing ZMYND19 ubiquitylation by the indicated CTLH E3 complexes. d Assays testing the effect of the GID4 substrate receptor on CTLH MKLN1 -dependent ZMYND19 ubiquitylation. e Assays testing UNG2 ubiquitylation by CTLH MKLN1 in the presence or absence of substrate adapter FAM72A. b – e All assays were performed for 15 min ( n ≥ 2 independent replicates). Source data are provided as a file.
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a Schematic of the CTLH E3 complex subunits organized by functional roles. b Assays testing MKLN1 ubiquitylation by the indicated CTLH E3 complexes. Ubiquitylation assays used N-terminally fluorescently labeled K0-ubiquitin (*UB K0 ) with all lysines mutated to arginines to visualize direct substrate modification without chain formation, with UBE2H as the E2. c In vitro ubiquitylation assays testing ZMYND19 ubiquitylation by the indicated CTLH E3 complexes. d Assays testing the effect of the GID4 substrate receptor on CTLH MKLN1 -dependent ZMYND19 ubiquitylation. e Assays testing UNG2 ubiquitylation by CTLH MKLN1 in the presence or absence of substrate adapter FAM72A. b – e All assays were performed for 15 min ( n ≥ 2 independent replicates). Source data are provided as a file.
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a Schematic of the CTLH E3 complex subunits organized by functional roles. b Assays testing MKLN1 ubiquitylation by the indicated CTLH E3 complexes. Ubiquitylation assays used N-terminally fluorescently labeled K0-ubiquitin (*UB K0 ) with all lysines mutated to arginines to visualize direct substrate modification without chain formation, with UBE2H as the E2. c In vitro ubiquitylation assays testing ZMYND19 ubiquitylation by the indicated CTLH E3 complexes. d Assays testing the effect of the GID4 substrate receptor on CTLH MKLN1 -dependent ZMYND19 ubiquitylation. e Assays testing UNG2 ubiquitylation by CTLH MKLN1 in the presence or absence of substrate adapter FAM72A. b – e All assays were performed for 15 min ( n ≥ 2 independent replicates). Source data are provided as a file.

Journal: Nature Communications

Article Title: Multimodal substrate recruitment enables CTLH MKLN1 E3 ligase to target acetylated N-, C-, and internal degrons

doi: 10.1038/s41467-026-78311-4

Figure Lengend Snippet: a Schematic of the CTLH E3 complex subunits organized by functional roles. b Assays testing MKLN1 ubiquitylation by the indicated CTLH E3 complexes. Ubiquitylation assays used N-terminally fluorescently labeled K0-ubiquitin (*UB K0 ) with all lysines mutated to arginines to visualize direct substrate modification without chain formation, with UBE2H as the E2. c In vitro ubiquitylation assays testing ZMYND19 ubiquitylation by the indicated CTLH E3 complexes. d Assays testing the effect of the GID4 substrate receptor on CTLH MKLN1 -dependent ZMYND19 ubiquitylation. e Assays testing UNG2 ubiquitylation by CTLH MKLN1 in the presence or absence of substrate adapter FAM72A. b – e All assays were performed for 15 min ( n ≥ 2 independent replicates). Source data are provided as a file.

Article Snippet: The following primary antibodies were used: Mouse monoclonal anti-ZMYND19 (Santa Cruz # sc-398514) Rabbit polyclonal anti-RANBP9 (Abnova # PAB16671 ), Mouse monoclonal anti-ARMC8 (Santa Cruz # sc-365307), Mouse monoclonal anti-MKLN1 (Santa Cruz # sc-398956), Rabbit polyclonal anti-RANBP9 (Proteintech # 17755-1-AP), Mouse monoclonal Anti-Ubiquitinylated proteins clone FK2 (Millipore # 04-263), Rabbit monoclonal β-Actin (13E5(Cell Signaling Technology # 4970)), Rabbit monoclonal Vinculin [EPR8185] (Abcam # ab129002), Mouse monoclonal UNG (OTI2C12) (Thermo Fisher Scientific # MA525686 ), Mouse monoclonal ANTI-FLAG M2 (Sigma-Aldrich # F1804), Mouse monoclonal HA-Tag (6E2) (Cell Signaling Technology # 2367), Rabbit Ponoclonal FAM72A (Thermo Fisher Scientific # PA583650 ).

Techniques: Functional Assay, Labeling, Ubiquitin Proteomics, Modification, In Vitro