Fig. 4.

SUP-36 protein levels are regulated by UBCH7/HHARI (UBC-18/ARI-1) and CDC34/CUL-1 (UBC-3/CUL-1). (A) Representative images for SUP-36::GFP expression levels measured in comma to twofold embryos under indicated RNAi conditions. (B) Quantification of embryos in the comma to twofold stage [L4440 vector, n = 27; ubc-18(RNAi), n = 45; ubc-3(RNAi), n = 67; ubc-18(RNAi);ubc-3(RNAi), n = 95]. Boxplots show quartiles, with mean indicated by the thick black line, and whiskers show 95% confidence intervals. Statistical significance was determined via Welch’s unequal variance t tests. Measurements for embryos in early embryogenesis and twofold to threefold stage are provided in Fig. S5A. (C) In vitro immunoprecipitation (IP) using recombinant T7-SUP-36 (IP: T7) and recombinant, untagged N8-CUL-1. Results were visualized by Western blot analysis with indicated antibodies. Replicates are shown in Fig. S5B. (D) In vitro ubiquitination of SUP-36. Each reaction contained T7-SUP-36 and other reaction components as indicated. All time points are post-ATP addition, except lane 1 (0 min). Longer time courses are shown in Fig. S6A. (E) The T7-SUP-36 in vitro ubiquitination assay was performed using Lys-less Ub, which is unable to form chains. Unmodified SUP-36 and single-ubiquitinated and double-ubiquitinated T7-SUP-36 are indicated by arrows. All time points are 10 min after ATP addition; longer time courses are shown in Fig. S6 A and B.