Specific association of SCL, E2A, and Sp1 with the c-kitpromoter in vivo.

Exponentially growing TF-1 cells were fixed in 1% formaldehyde and sonicated. Fragmented chromatin extracts were then subjected to immunoprecipitation with α-SCL, α-E2A, α-Sp1, and isotype-matched control antisera (α-PU.1 and α-HA). Precipitated chromatin was heated overnight at 65°C to reverse cross-linking, and DNA molecules were purified and subjected to PCR analysis to test for the presence of the c-kit promoter (−180 to −35) or the indicated controlc-kit locus regions (−2468 to −2149 and +13 532 to +13 781). Input chromatin represents 1.25% of the amount used in each immunoprecipitation, and 3 4-fold serial dilutions of the immunoprecipitated samples were done prior to amplification. The PCR products were analyzed by agarose gel electrophoresis, transferred onto Biodyne B membrane, and hybridized with internal oligonucleotide probes.