Figure 5. TGFβ induces occupancy of the Smad complex to the LPA₁ gene promoter.

A. Cell extracts from MDA-MB-231 and SKOV-3 cells treated with TGFβ (2.5 ng/ml) or vehicle for 1 h our were incubated with biotinylated DNA fragment containing the -401 TIE and strepatavidin beads. The DNA precipitates (DNAP) were subjected to western blot analysis for Smad3 and E2F4. Whole cell lysates were included as input. B. ChIP assays were performed to examine the binding of Smad3 and E2F4 to the -40 and -401 TIEs of the LPA₁ promoter and to the c-myc TIE (positive controls). The immunoprecipitation of Smad3 and E2F4 was verified by western blotting analysis of immunoprecipitates (IP) and whole cell lysates (WCL). The binding was quantitated by qPCR using SYBR Green and the specific primers listed in Table 1. The results were normalized to the Ct values of inputs and presented as percentages of inputs. ND: not detectable.