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. 2023 Oct 23;14:6718. doi: 10.1038/s41467-023-42216-3

Fig. 4. CLEC2D promotes Smurf1-mediated degradation of MyD88 through K48-linked ubiquitination.

Fig. 4

a Immunoblot analysis of curdlan (20 μg/well)-induced ubiquitination of MyD88 in wild-type and Clec2d-deficient BMDCs with magnetic beads-coupled Tandem Ubiquitin Binding Entities (TUBEs). Three times experiments were repeated independently with similar results. b, c Immunoblot analysis of the degradation of MyD88 expressed in HEK293T cells with co-expression of MyD88-his, Clec2d-Flag and Ub-HA (b) or Ub-K48-HA and Ub-K63-HA (c) in the presence of MG132 (20 μM). Three times experiments were repeated independently with similar results. d–e Immunoblotting and quantification analysis of the degradation of MyD88 expressed in HEK293T cells with co-expression of MyD88-His, Clec2d-Flag and Ub-HA (d) or Ub-K48-HA and Ub-K63-HA (e). f Immunoblotting and quantification analysis of the degradation of MyD88 in RAW264.7 cells stably expressing mouse Clec2d or mock, which were pretreated with Smurf1 inhibitor (Smurf1-IN-A01, 10 μM), SPOP inhibitor (SPOP-IN-6b dihydrochloride, 10 μM) for 30 min and then stimulated with Curdlan (20 μg/well) for indicated time. Data were presented as mean ± SEM; n = 3 (e, f), n = 4 (d) biologically independent samples. Data were analyzed by one-way ANOVA adjusted for multiple comparisons in (e, f), Data were analyzed by unpaired two-sided Student’s t-test in (d). Source data are provided as a Source Data file.