Fig. 3.

Identification of the ASE binding nuclear protein. (A) EMSAs with liver NEs prepared from mice at various ages. All lanes contain the ³²P-labeled ASE probe (approximately 40,000 cpm). Lane 1, without NEs; lanes 2–7, with NEs (80 μg) prepared from the liver tissues of 1-, 3-, 6-, 12-, 18-, and 21-month-old animals, respectively. Arrow: shifted band position. (B) Supershift EMSAs with antibodies specific to various Ets family proteins. All lanes contain the ³²P-labeled ASE probe (approximately 20,000 cpm). Lanes 1, 10, and 20, with no NEs; lanes 2–9, 12–19, and 21–26 with liver NEs (40 μg) prepared from 6-month-old mice. Lane 11, anti-Ets1 antibody with the ³²P-labeled probe with no NEs. Lanes 3–9, 13–19, and 22–26 are supershift EMSAs with various antibodies to specific proteins labeled at the top of lanes. Lanes without antibody are noted with (-) as shown at top. Arrow: shifted band position. (C) EMSAs and supershift EMSA with NEs prepared from 293T cells overexpressing mEts1. ³²P-labeled ASE probe and procedures are as described above differing only in NEs used. Lane 1, without NEs; lane 2, with 10 μg NEs from 6-month-old mouse liver; lane 3, with 10 μg NEs from wild-type 293T cells; lane 4, with 10 μg NEs from 293T cells overexpressing mEts1; lane 5, same as lane 4 with anti-Ets1 antibody. Arrow: shifted band position.