Sp1 mediates the recruitment of Brg-1 to the proximal region of the SPARC gene. 4T1 cells were transfected with mock (transfection reagent only), siRNA control (CTR), or siRNA specific for Sp1, and then cultured for 72 hrs prior to harvesting the cells. (A) Total cell extracts were prepared from transfected cells, and protein expression was analyzed by Western blotting using antibodies specific to Sp1, Brg-1 or p38. (B) Nuclear extracts were prepared and the binding reactions of Sp1, Brg-1 or p38 to the probe a were analyzed using the immobilized-template assay followed by Western blot analysis. (C) Cells were cross-linked and sonicated, and ChIP assay was performed. The precipitated DNAs and input DNA were quantified and then amplified using real-time qPCR. The occupancy level of Brg-1 at the SPARC promoter is calculated as described in Figure 1 D. The relative occupancy level of Brg-1 in mock-treated cells is set as 100%. Data represented as mean ± SEM (n = 3).