Figure 2.

Clec16a promotes the ubiquitination of Nrdp1. A: Representative Western blot (WB) of in vivo ubiquitination assay of overexpressed HA-Nrdp1 performed in HEK293T cells transfected with Myc-tagged ubiquitin (Myc-Ub), Flag–empty vector, Flag-Clec16a, or Flag-USP8 (as well as whole-cell lysate HA-Nrdp1, Flag-Clec16a, and Flag-USP8 levels) treated with DMSO (-) or 20 μmol/L MG132 (+) overnight. n = 3/group. B: Representative Western blot of in vivo ubiquitination assay of endogenous Nrdp1 performed in 293T cells transfected with Myc-ubiquitin and Flag–empty vector or Flag-Clec16a (as well as whole-cell lysate Nrdp1 and Flag-Clec16a levels). n = 3/group. C: Representative WB of in vivo endogenous ubiquitination assay of endogenous Nrdp1 ubiquitination performed in primary dermal fibroblasts derived from WT and Clec16a-deficient mice (as well as whole-cell lysate Nrdp1 levels). n = 3/group. D: Representative Western blot of in vivo ubiquitination assay of overexpressed HA-WT or C34S H36Q mutant (CSHQ) Nrdp1 performed in HEK293T cells transfected with Myc-ubiquitin and Flag–empty vector or Flag-Clec16a (as well as whole-cell lysate HA-Nrdp1 and Flag-Clec16a levels). n = 3/group. E: Representative Western blot of in vivo ubiquitination assay of overexpressed HA-Nrdp1 performed in HEK293T cells transfected with Myc-ubiquitin and Flag–empty vector or Flag-Clec16a (as well as whole-cell lysate HA-Nrdp1 and Flag-Clec16a levels) treated with DMSO (−) or 10 μmol/L lenalidomide (+) for 24 h. n = 3/group. F: GSIS after static incubations in 1.67 mmol/L and 16.7 mmol/L glucose performed in isolated MIP-CreERT2 control or β-Clec16a^KO islets pretreated with vehicle (DMSO) or 10 μmol/L lenalidomide for 24 h (hr) (n = 4/group). *P < 0.05 nonpaired Student t test. IP, immunoprecipitation.