Figure 3

AB215 induced IDs cooperatively inhibit E2 activating P-ERK1/2. MCF7 cells were reverse transfected with siRNA in serum free/phenol red free RPMI1640 and plated in phenol red free RPMI1640 supplemented with 10% heat inactivated charcoal-stripped FBS. Control was transfected with non-sense siRNA. After 24 hours of transfection, cells were treated with or without 10nM E2 along with 500 ng/ml BMP2 or AB215. After 48 hours of exposure, RNA was extracted for qRT-PCR analysis or harvested for western blot analysis. A) KD efficiency of each IDs using siRNA. mRNA level of each IDs were measured using qRT-PCR to confirm the effectiveness of mRNA knocked down. Data are shown as means + SD in quadruplicates, n = 3. B) KD samples are analyzed for western blot to detect phosphorylation of ERK1/2. The figure shown is done in two separate blots (Control, ID1, ID2-KDs in one blot and ID3, ID4-KDs in another blot). (* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001).