Protein expression and pathway activation in HME16C cell lines. (A) Expression of HA-tagged H-RasV12, H-RasV12 effector domain mutants, and Rlf-CAAX in HME16C mammary epithelial cells. In this and subsequent figures, cells infected with the following vectors are indicated as follows: pLRT control (pLRT), H-RasV12 (V12), H-RasV12G37 (G37), H-RasV12S35 (S35), H-RasV12C40 (C40), and Rlf-CAAX (Rlf-CAAX). Cells were treated with 250 ng/mL doxycycline for 72 hours to induce gene expression, and anti-HA (HA tag) and anti-Ras (Ras) western blotting were performed. Erk activation was determined by western blotting for phosphorylated Erk (Phospho-Erk). The blot was subsequently stripped and probed with an anti-Erk2 antibody (Erk2). (B) Ral A activation was determined by a pull-down assay for GTP-bound, activated Ral A (GTP-Ral A). Total Ral A protein from lysates used in the Ral activation assay pull-down was determined by anti-Ral A western blotting (Ral A). (C) Akt activation in cell lysates was assessed by western blotting for phosphorylated Akt (Phospho-Akt). The membrane was stripped and probed with an anti-Akt antibody (Total Akt).