Figure 1
Development and validation of the mouse model expressing a catalytically inactive MYSM1. (A) Domain structure of the mouse MYSM1 protein indicating the mutation in the MPN catalytic domain predicted to render the protein catalytically inactive. (B) Catalytic activity assay of recombinant mouse MYSM1 against ubiquitin-Rhodamine substrate demonstrates that the D660N mutation results in a full loss of the DUB catalytic activity. (C) Sanger sequencing of the Mysm1 locus in the genomic DNA of wild type control and Mysm1DN/+ heterozygous mice, indicating the DNA sequences and the corresponding amino acid sequences of the wild type and mutant proteins. (D) High embryonic lethality of Mysm1DN/DN mice: offspring genotypes obtained from the mating of two Mysm1+/DN heterozygous parents show strong deviation from expected Mendelian ratios. (E) Length and weight of the age- and sex- matched mice of Mysm1+/+, Mysm1−/− and Mysm1DN/DN genotypes; bars represent means ± SEM, statistical analysis with ANOVA comparing each group to the control, *p < 0.05, **p < 0.01, ***p < 0.001, NS—not significant. (F) Representative image of the age- and sex- matched mice of Mysm1+/+, Mysm1−/− and Mysm1DN/DN genotypes, showing reduced body size and tail dysmorphology. (G) Western blot of mouse bone marrow cell lysates showing comparable MYSM1 protein levels in Mysm1+/+ and Mysm1DN/DN samples. (H) Western blot of splenocyte lysates from tamoxifen-treated CreERT2 transgenic mice of Mysm1fl/+, Mysm1fl/fl, and Mysm1fl/DN genotypes, showing strong depletion of MYSM1 protein in the Mysm1Δ/Δ cells, and comparable MYSM1 protein levels between Mysm1Δ/+ and Mysm1Δ/DN samples. β-actin is used as a loading control in (G,H).
