Causative mutations in MommeD30 , MommeD18 , MommeD8 , MommeD28 and MommeD34 . (a) MommeD30 carries a 1 bp deletion in Wiz that leads to a frame-shift at amino acid 553. Western blot analysis of embryo heads at 12.5 days post-coitum (dpc) shows reduced levels of Wiz protein in WizMommeD30 heterozygotes and no Wiz protein in homozygotes. Wiz protein was detected at approximately 120 and approximately 130 kDa. Each track represents a different animal. Tubulin was used as a loading control. (b) MommeD18 harbors a point mutation in Rif1 that introduces a premature stop codon at amino acid 1,669. Western analysis of testes tissue shows that RifMommeD18 heterozygotes have a reduced dosage of Rif1. No Rif1 protein was detected in a homozygote. Each track represents a different animal. Rif1 protein was detected at approximately 260 kDa and Tubulin was used as a loading control. (c) ENU point mutations in MommeD8, MommeD28 and MommeD34 occur in conserved regions of the Rlf protein. Asterisks indicate mutation. (d) Western blotting of Rlf in embryo head lysates from 14.5 dpc Rlf+/+, RlfMommeD8/D8, RlfD28/D28 and RlfD34/D34 revealed greatly reduced amounts of Rlf protein. Each track represents a different animal. Rlf protein was detected at approximately 280 kDa. Tubulin was used as a loading control. (e) Total, cytoplasmic and nuclear fractions of HeLa cells were isolated and protein concentration quantified. Equal amounts of each fraction were immunoblotted with anti-Rlf, GAPDH (cytoplasmic marker) and SMARCA5 (nuclear marker) antibodies, revealing nuclear localization of RLF. Rlf protein was detected at approximately 280 kDa, GAPDH protein at 37 kDa and SMARCA5 at approximately 120 kDa. (f) Coat color of offspring carrying the Avy allele produced from a RlfMommeD8 heterozygous female crossed to a pseudoagouti Avy male. RlfMommeD8 heterozygous offspring showed a shift in coat color towards pseudoagouti compared to wild-type littermates.