Na k atpase α1

These assays were performed as described (Yang et al., 2007 (link)) using antibodies against GAPDH, β-actin, and caspase-3 (Cell Signaling Technology Inc., MA, USA); and Na⁺/K⁺-ATPase α1 (Abcam Inc. Cambridge, UK). Tylophorine and Reevesiosides A-I were prepared as described (Chang et al., 2013 (link), Yang et al., 2010 (link)). DMSO (D1435, ≧ 99.5%), digoxin (D6003, ≧ 95%, HPLC), digitoxin (D5878, ≧ 92%, HPLC), digitoxigenin (D9404, 99%, TLC), ouabain (O3125, ≧ 95%, HPLC), dihydroouabain (D0670, ≧ 98.5%, TLC), oleandrin (O9640, ≧ 98%, HPLC), N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (Db cAMP) (D0627, ≧ 96%, HPLC), and aldosterone (A9477, ≧ 95%, HPLC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Western blots were performed as described (Yang et al., 2007 (link)) with the following antibodies: p-Akt (S473), p-Akt (T308), Akt, p-p70S6K (T389), p70S6K, RSK1, p-RSK2 (S227), RSK2, p-PDK1 (S241), PDK1, PI3K p110α, and GAPDH (Cell Signaling Technology Inc., MA, USA); Na⁺/K⁺-ATPase α1 (Abcam Inc. Cambridge, UK); and an antibody against TGEV N protein, as described (Yang et al., 2007 (link)). Ouabain (O3125, ≧95%, HPLC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). p-RSK1 (T359/S363) antibody, BX795, rottlerin, GF109203X, rapamycin, and triciribine were purchased from Merck Millipore Calbiochem (Billerica, MA). LY294002, PP2, KX2-391 and SU6656 were from Selleckchem (Houston, TX, USA). Farnesyl thiosalicylic acid (FTA, a Rac1 inhibitor) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Wortmannin was purchased from Life Technologies (San Diego, CA, USA).