Fig. 1.

RHBDF1 facilitates ligand-dependent EGFR transactivation by modulating TGFα secretion. (A) RHBDF shRNA (shRHB)-transfected MDA-MB-231 cells treated with EGF (10 ng/mL) or TGFα (10 ng/mL) for 5 min, or with LPA (10 nM) or S1P (10 nM) for 30 min, and subjected to western blotting analysis; shScr, scrambled RNA control. (B) Western blotting analysis of similarly treated RHBDF1-overexpressing MDA-MB-231 cells (RHB) and empty vector-transfected control (Vector). (C) Analysis of S1P (10 nM)-triggered EGFR phosphorylation in shScr- and shRHB-treated MCF-10A, MDA-MB-468, MCF7, and T-47D cells. (D) Cultured shScr- and shRHB-treated MDA-MB-231 cells were treated with S1P (10 nM) for 30 min and maintained in serum-free media for 2 h. The collected conditioned media were added to 24 h pre-starved parental MDA-MB-231 cells. Cell lysates were subjected to immunoblot analysis to detect EGFR phosphorylation levels. Changes in the concentration of TGFα (E), HB-EGF (F) or AREG (G) in conditioned media of shScr- or shRHB-treated MDA-MB-231 cells as a function of time of S1P (10 nM) stimulation. Bar graphs represent densitometric analysis of the results of independent western blotting experiments. In all panels, data are means ± SEM (n = 3); *p < 0.05, **p < 0.01 and *** p < 0.001.