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. 2012 Mar 19;196(6):789–800. doi: 10.1083/jcb.201105101

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© 2012 Sarkar and Zohn

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Figure 2. — Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1^W (W; wild type), Hectd1^O (O; opm; openmind), and Hectd1^X (X; XC gene trap) are mouse alleles; Myc-Hectd1^ANK, HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1^O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1^X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette (Zohn et al., 2007). HA-Hectd1^ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1^W and Hectd1^X but not Hectd1^O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1^W and Hectd1^X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1^X compared with Hectd1^W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1^+/+; O, Hectd1^opm/opm; X, Hectd1^X/X; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, immunoprecipitation; FK2, anti–mono- and polyubiquitinylated proteins; (Ub)_n, Ub_n; K63Ub, lysine 63 linked Ub_n chains.