Sp1 and Brg-1 are bound to the SPARC promoter in tumor cell lines. (A) Nucleotide sequence (from - 201 to +19) indicating the position of six GGAGG boxes (underlined) in the mouse SPARC promoter. (B) Nuclear extracts were prepared from 4T1, 168FARN and 67NR cells. Proteins which bind to probe a (spanning GGAGG-rich nucleotides -130/-56) or probe b (spanning nucleotides -50/+19) were isolated from the nuclear extracts using the immobilized-template assay and then subjected to Western blot analysis with antibodies against Brg-1, Sp1 or p38. p38 and the probe b were used to serve as a non-specific binding control (right panel). Unpurified total nuclear extracts were also subjected to Western blot analysis and probed with the same antibodies (left panel). (C) Cross-linked and sonicated chromatin samples were prepared from 4T1, 168FARN and 67NR cells. ChIP assays were performed using antibodies against Sp1, Brg-1 or non-specific rabbit IgG as a control. Immunoprecipitated DNA and serially diluted input genomic DNA was amplified with primers specific to the SPARC promoter. PCR products were then analyzed using 2.0% agarose gels and stained with ethidium bromide. The illustrated results are representative of three independent experiments. (D) The ChIP DNA and input genomic DNA from (C) were quantified and amplified by real-time qPCR as described in the Materials and Methods section. The occupancy level of Brg-1 or Sp1 at the SPARC promoter is represented as the ratio of signal from IP samples versus that of the input minus background of IgG control. The relative occupancy level of Brg-1 or Sp1 in 4T1 cells is set as 1. Data represented as mean ± SEM (n = 3).