LIL-Stat transcription factor is tyrosine phosphorylated and recognized by anti-Stat1N antibody. (A) Five micrograms of nuclear extract derived from control B1 cells was supershifted with antibodies against phospho-tyrosine residue (lane 2), Stat1 N-terminal region (lane 4), Stat1 C-terminal region (lane 6), and against Stat3 (lane 7). Quantification of LIL-Stat binding activity was performed by phosphoimager analysis. (B) Functionality of Stat1 and Stat3 antibodies was shown as follows: IFN-γ–treated B1 nuclear extracts were band-shifted with ³²P-labeled Stat1 oligonucleotide from the FcγR1 promoter (lane 9) and supershifted with the antibodies against the N- (lane 10) and C- (lane 11) terminal Stat1 domains. Five micrograms of nuclear proteins derived from control B1 cells was band-shifted with the ³²P-labeled Stat3 oligonucleotide from the c-fos promoter (lane 12) and supershifted with the Stat3 anitbody (lane 13). (C) Band-shift assays of control B1 nuclear extracts with the oligonucleotides for Stat1 (GAS), Stat3 (hSIE), and LIL-Stat (LILRE) showed differential binding to the Stat1, Stat3, and LIL-Stat oligonucleotide (lanes 14, 17, and 20, respectively). Lanes 15, 18, and 21 represent the respective oligonucleotides alone, whereas cold competition is represented in lanes 16, 19, and 22. (D) Band-shift assay of control B1 nuclear extract with the oligonucleotide for LIL-Stat (lane 23). Cold competition is performed with 100-fold molar excess of unlabeled oligonucleotide LILRE (lane 24), GAS (lane 25), and hSIE (lane 26).