Total foxo3

Asialo-rhuEPO^P was purified from transgenic tobacco line A56-5 [33] (link) using two-step procedure as described previously [35] (link). The number of asialo-rhuEPO^P units was calculated from protein concentration as described by Erbayraktar et al. [26] (link). Cell culture grade mammalian cell-produced sialylated EPO (rhuEPO^M) was purchased from R & D Systems (Minneapolis, MN, USA). Claycomb medium, FBS, norephinephrine, STS and HOECHST 33342 stain were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Lactate dehydrogenase (LDH) assay kit was purchased from Roche (Indianapolis, IN, USA). M-PER mammalian protein extraction reagent, Halt™ protease and phosphatase inhibitor cocktail were obtained from Thermo Fisher Scientific (Grand Island, NY, USA). Bradford reagent was purchased from Bio-Rad laboratories (Hercules, CA, USA). Antibodies for detection of Bcl-2, Bax, p-Bad/Bad, cytochrome c (cyt c), caspase-3, Mst1, total FOXO3, p-Ser253-FOXO3, p-JAK2/JAK2, p-Akt/Akt, p62, Beclin1, LC3B, tubulin and VDAC were purchased from Cell Signaling (Boston, MA, USA) except anti-p-Ser207 FOXO3 (Zhenjiang Hope Biotechnology Co, Zhenjiang, China) and anti-β-actin (Sigma-Aldrich). West Pico chemiluminescent substrate for western blotting was purchased from Thermo Fisher Scientific.