mFox-2A localizes in the nucleus and specifically binds to UGCAUG sequence in an gel mobility shift assay. (A) MELCs transfected either with mFox-2A/EGFP or EGFP vector alone were fixed and stained for endogenous SC35 and for nucleic acids (DNA), then visualized for the presence of EGFP, SC35, and DAPI. Green represents EGFP epitopes; red, SC35 epitopes; yellow, the colocalization of mFox-2A and SC35; and blue, DNA stained with DAPI. TxRed indicates EGFP-transfected cells were stained with a secondary mouse antibody conjugated with Texas Red but without the primary antibody and served as a negative control. (B) TER119⁺ fetal liver cells stained for endogenous Fox-2 and DNA. Green indicates Fox-2; blue, DNA. (C) Left panel shows purified mFox-2A. mFox-2A expressed as GST fusion proteins were cleaved with PreScission protease (Amersham Pharmacia) and purified to homogeneity. Right panel shows gel mobility shift assay using 3 copies of sequences between the first and second UGCAUG repeats. ³²P-labeled wild-type RNA (3WT; lanes 1-4) or mutant RNA (3MU; lane 5) were incubated with increasing amounts of mFox-2A in the presence of nonspecific competitor tRNA. A 10-fold excess (compared with the ³²P-labeled probe) of unlabeled 3WT RNA (3WT; lane 4) was added as a competitor.