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Fig. 2. Identification of FILIA. (A) SDS-PAGE of zona-free ovulated eggs (2000) isolated from wild-type or Mater^tm/tm mice. After fixation and staining, a protein (50 kDa) diminished in Mater^tm/tm eggs was designated FILIA and excised from the gel for analysis by mass spectrometry. Molecular masses (kDa) are indicated on the left. (B) RT-PCR of total RNA isolated from kidney, ovary, uterus, testis, brain, heart, lung, liver, spleen, stomach, small intestines, muscle and water using primers specific for Filia (top) or actin (bottom) to control for RNA integrity. Size (bp) is indicated on the left. (C) In situ hybridization of fixed, paraffin-embedded wild-type ovarian (2-week-old) sections probed with DIG-labeled antisense (a) and sense (b) synthetic Filia oligonucleotide probes. (D) Total RNA (0.6 µg) was isolated from ovaries of wild-type, Mater^tm/+ and Mater^tm/tm mice, separated by electrophoresis, transferred to a nylon membrane and probed with ³²P-labeled FILIA cDNA. Molecular masses (kb) are indicated on the left. The two isoforms, FILIA 1.6 and FILIA 1.2, are indicated on the right. Scale bar: 50 µm. -, water; Br, brain; Hrt, heart; Ki, kidney; Li, liver; Lu, lung; Mu, muscle; Ov, ovary; Si, small intestines; Sp, spleen; St, stomach; Te, testis; Ut, uterus.

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