Figure 4

Gel shift assay for SNP CysLTR2 c .-- 819 using nuclear extract from B cell lines. Putative transcription factor binding site of c.--819T (A) and G type (B) are indicated by underlines (TFSEARCH Searching Transcriptional Factor Binding Sites V1.3). Gel shift assays were performed using nuclear extracts of EBV-infected B cell lines and dsDNA probes possessing c.--819G or T (C). One million cells per milliliter of EBV-infected B cell lines established from asthmatics were cultured for 24 h with or without 10 ng/ml of PMA and 2 μM of ionomycin. The nuclear extracts of B cell lines were incubated with the ³²P-labeled probes, the sequences of which included each genotype of c.--819 (see Methods). To identify specific binding complexes, nuclear extract from B cell lines was preincubated with the unlabeled probe 10 min before the addition of the labeled probe. The open arrow shows the specific DNA--protein complexes and the filled arrow indicates the free unbound probe. Arrowheads denote nonspecific DNA--protein complexes. These data are representative of six independent experiments.