FIG. 5.
TGF-β and RAS cooperatively down regulate RIN1 expression via Snail. (A) MCF10A and MDA-MB-231 cells were stimulated with TGF-β (5 ng/ml) for the indicated time or left unstimulated. Levels of endogenous RIN1 mRNA were determined in triplicate using quantitative real-time PCR and normalized to unstimulated cells. (B) Cells and conditions as in panel A were used, and changes in endogenous RIN1 protein were quantified by immunoprecipitation and immunoblotting and normalized to total TUBB (β-tubulin). (C) Immortalized human mammary epithelial cells (HMLE) and equivalent cells expressing HRASG12V (HMLER) were treated with TGF-β (5 ng/ml, 18 h). Endogenous RIN1 protein was immunoprecipitated and immunoblotted. Cell lysates were probed with antibodies to RAS and TUBB (β-tubulin). Normalization to TUBB levels was used to calculate change in RIN1.