Raf:ER activates multiple downstream effectors. Raf:ER three-dimensional cultures were grown for 10 days, treated with diluent or 100 nM 4-hydroxytamoxifen (4-HT) and grown for an additional 48 hours. (a) Acini were immunostained with α-c-Fos (red) and counterstained with Hoechst (blue). Bar = 150 μm. (b) Acini were immunostained with α-Bim antibody (red) and counterstained with Hoechst (blue). Bar = 30 μm. (c) The lysates of acini were immunoblotted with α-Bim antibody (upper panel) or α-α-tubulin (bottom panel) antibodies. The Bim signal intensity from the 4-HT-treated acini normalized to the tubulin signal intensity and divided by the normalized Bim signal intensity from the ethanol-treated acini is shown.