Figure 3
Involvement of DCC/caspase-3, APPL-1/Akt2 complexes and Akt phosphorylation. (a and b) Cells were pretreated with Ntn-1 (10 ng/ml) or DCC function-blocking antibody (2.5 μl/ml) for 30 min prior to 72 h incubation in hypoxic condition. Cell lysates were analyzed by western blotting with antibodies that recognize caspase-3 or APPL-1. Immunoprecipitation of anti-DCC was analyzed by western blotting with antibodies that recognize caspase-3 or APPL-1. (c) Cells were pretreated with Ntn-1 (10 ng/ml) for 30 min prior to 72 h incubation in hypoxic condition. Cell lysates were analyzed by western blotting with antibody that recognize Akt2. Immunoprecipitation of anti-APPL-1 was analyzed by western blotting with antibody that recognize Akt2. (d and e) Cells were pretreated with DCC function-blocking antibody (2.5 μl/ml), or combination of INs α6 and β4 (2.5 μl/ml) for 30 min prior to a 30-min Ntn-1 (10 ng/ml) treatment. And then, the cell incubated prior to 72 h in hypoxic condition. Total protein was extracted and blotted with phospho-Aktthr308, phospho-Aktser473, or Akt antibody. (a–e) Each of the examples is representative of four independent experiments. The right or lower part (a–e) depicting the bars denotes the mean±S.E. of four independent experiments for each condition determined from densitometry relative to β-actin or total Akt. *P<0.05 versus control, #P<0.05 versus hypoxia alone, **P<0.05 versus combination of hypoxia and Ntn-1. ROD, relative optical density
