Skip to main content
. 2017 Nov 27;67(2):265–277. doi: 10.2337/db17-0321

Figure 3.

Figure 3

Clec16a possesses E3 ligase activity and directly ubiquitinates Nrdp1 via a non-K48 linkage. A: Representative α-Flag Western blot (WB) from purified Clec16a-6xHis-Flag protein after incubation in vitro with ATP, ubiquitin, E1, and 26 unique E2-conjugating enzymes at 37°C for 1 h. Arrows demarcate both the expected and novel high–molecular weight Clec16a proteins identified by Western blot. n = 3/group. B and C: Representative Western blot of in vitro ubiquitination assay of recombinant GST-Clec16a-Flag (B) or Clec16a-6xHis-Flag (C) protein after incubation with ATP, HA-ubiquitin (HA-Ub), and E1 in the presence or absence of E2 conjugation enzymes UbE2D3 and/or UbE2C at 37°C for 1 h. n = 3/group. D: Representative Western blot of in vitro ubiquitination assay of recombinant GST-Nrdp1-CSHQ after incubation with ATP, HA-ubiquitin, and E1 in the presence or absence of UbE2D3 and recombinant Clec16a-6xHis-Flag at 37°C for 1 h. n = 3/group. E: Representative WB of in vivo ubiquitination assay of overexpressed Myc-tagged Nrdp1 (Myc-Nrdp1) performed in HEK293T cells transfected with Flag–empty vector or Flag-Clec16a in the presence of HA-WT ubiquitin as well as ubiquitin mutants with all lysines converted to arginines (KO), ubiquitin only capable of K48-linked ubiquitination (K48), ubiquitin incapable of K48-linked ubiquitination (K48R), or ubiquitin only capable of K63-linked ubiquitination (K63) (as well as whole-cell lysate Myc-Nrdp1 and Flag-Clec16a levels). n = 3/group. IP, immunoprecipitation.