Analysis of eIF-2α and phospho eIF-2α protein levels. Western blot from osteosarcoma cells (A) and 4 representative osteosarcoma tissues (C); Quantitation of densitometry signals from osteosarcoma cells (B) and osteosarcoma patient tissues (D). The cytoplasmic extracts prepared from Vehicle control (Veh) and (10 μM) 2-ME-treated cells, and from normal (N) and osteosarcoma tumor (T) tissues were analyzed by western blot hybridization using anti-eIF2α, anti-phophoeIF2α and anti-GADPH antibodies. The bands were analyzed by quantitative densitometry. After western blot analysis, the signals from the corresponding bands were quantitated and the ratio of peIF-2α over eIF-2α was determined after normalization to GAPDH.