Figure 1

Ufm1 and Ufm1-conjugate levels are reduced in uba5^−/− fish at 6 dpf. (A) Schematic of Uba5 wild-type protein and mutation sites in zebrafish. The mutant strains were generated using CRISPR/Cas9 genome editing. The uba5^ex1s strain carries a 73-base pair insertion following the ATG, resulting in a frameshift and incorporation of a premature stop after 5 amino acids, whereas the uba5^ex3d strain carries a 24-base pair deletion that removes the ATP-binding domain. (B–G) Western blot for Ufm1 and Direct Blue staining for total protein (loading control) on protein lysates obtained at 6 dpf. Ufm1 and Ufm1-conjugate levels were normalized to that of the respective total protein levels. The changes in Ufm1 and Ufm1-conjugate levels in uba5 mutants were determined relative to the levels of wild-type siblings. Statistical significance was calculated using a one-way ANOVA with Dunnett’s post hoc multiple comparison correction test. Data are presented as mean ± SEM for three independent biological replicates, each consisting of a pooled sample of 20–25 embryos. UIS, Ufm1-interacting sequence; Ufc1, ubiquitin-fold modifier conjugating enzyme 1; Ufm1, ubiquitin-fold modifier 1.