Cell culture and embryoid body (EB) formation
E14 ES cells (ATCC) were maintained on mouse embryo fibroblast (MEF) feeder cell layers in Dulbecco-modified Eagle medium containing 14% KnockOut Serum Replacement (Invirtogen), 1% fetal bovine serum, 100 units/ml LIF, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and 100 µM 2-mercaptoethanol. For EB formation assays, 2×105 cells were seeded into 35 mm low attachment sterile cell plate in 2 ml ES cell medium without LIF. Fresh medium was provided every two days.
Targeting vectors and generation of Trip12 mutant mice
The targeting vectors for Trip12 were constructed by inserting FLAG and HA epitope tag sequences in frame and Neo or IRES-hyg cassette into the exon 33 of the mouse Trip12 genome. Correctly targeted ES cell clone were injected into C57BL/6 blastocysts, producing chimeric mice. Highly chimeric males were mated to females, and then the progeny were intercrossed. All experiments were performed in accordance with the Declaration of Helsinki and were approved by University of Tsukuba Ethics Committee for Animal Experiments (approval ID:040042).
Cells were lysed in TNE buffer [10 mM Tris-HCl (pH 7.8), 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 µM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin]. The proteins were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore), immunoblotted with indicated antibodies. The antibodies employed in this study included mouse monoclonal antibodies specific for β-Actin (Sigma); rat monoclonal antibody for HA (Roche) and ARF (Calbiochem). Rabbit polyclonal antibody for BAF57 (GeneTex) and Trip12 (Bethyl Laboratories).
Embryos and cells were homogenized in 1 ml of sepasol (nacalai tesque) and total RNA was extracted according to the instruction manual. cDNA was synthesized from total RNA using RevatraAce reverse transcriptase (Toyobo) and random primers. Real-time PCRs were performed to amplify fragments representing for the indicated mRNA expression. We used primers 5′- CATCTGGAGCAGCATGGAGTC-3′ and 5′-GGGTACGACCGAAAGAGTTCG-3′ for p16, 5′-GTTCTTGGTCACTGTGAGGATTCAG-3′ and 5′-CCATCATCATCACCTGGTCCAG-3′ for ARF, 5′-TTGCACTCTGGTGTCTGAGC-3′ and 5′-TGCGCTTGGAGTGATAGAAA-3′ for p21, 5′-TTGGGTCTCAGGCAAACTCT-3′ and 5′-CTGTTGGCCCTTTTGTTTTG-3′ for p27, 5′-AGTACCTCAGCCTCCAGCAGAT-3′ and 5′-GCTTGCACTTCATCCTTTGGTT-3′ for Nanog, 5′-CACGAGTGGAAAGCAACTCA-3′ and 5′-TTCATGTCCTGGGACTCCTC-3′ for Oct-4, 5′-CATGAACAGGTTCATCTATG-3′ and 5′-GTTGTCAGCTTTGCAGCATG-3′ for AFP, 5′-TGTCTATGTTCGAGATTACAG-3′ and 5′-CATTGAGGTTTGAAATCGAC-3′ for Flk-1, 5′-GGTCATTGTGAATGTACCAC-3′ and 5′-CCTTTGTCCAGCTCATGGTG-3′ for NCAM,
Cells were treated with 100 µg/ml cycloheximide to prevent de novo protein synthesis, and lysed at various time points following cycloheximide treatment. The proteins were then separated by SDS-PAGE and detected by immunoblotting.
Whole mount immunostaining
Whole mount immunostaining of embryos were performed as described previously 
Microarray procedure and data analysis
The microarray procedure was performed as described in the Affymetrix GeneChip Expression Analysis manual (Affimetrix). Each array was analysed using GeneSpring (Agilent) program. Patterns were analysed to identify functional categories using Database for Annotation, Visualization and Integrated Discovery (DAVID) (http:// david.abcc.ncifcrf.gov) 
. We used the functional annotation tool program and reported only GOTERM_BP (Biological process) that had p
-values of <0.05.
All the data are representative of at least three different experiments. Statistical analysis was performed using t-test.