Figure 4

CaM/PI3K/Akt pathway is involved in the SP6616-mediated β-cell protection. (a) INS-832/13 cells were incubated with SP6616 (1, 5, 10 μM) in the presence or absence of STZ (0.4 mM) for 24 h, and the cell lysate was analyzed by western blot using p-Akt and Akt antibodies. (b) Relative protein levels of p-Akt/Akt in a. (c) INS-832/13 cells were incubated with SP6616 (10 μM) and STZ (0.4 mM) for 20 h in the presence or absence of wortmmanin (2 μM) for another 4 h, and then cell lysate was analyzed by western blot using p-Akt and Akt antibodies. (d) Relative protein levels of p-Akt/Akt in c. (e) INS-832/13 cells were transfected with Kv2.1 N or EGFP, and incubated with STZ (0.4 mM) in the presence or absence of SP6616 (10 μM), and then the cell lysate was analyzed by western blot using p-Akt and Akt antibodies. (f) Relative protein levels of p-Akt/Akt in e. (g) INS-832/13 cells were incubated with SP6616 (1, 5, 10 μM) in the presence or absence of STZ (0.4 mM) for 24 h, and the cell lysate was analyzed by western blot using the corresponding antibodies. (h) Relative protein levels of p-Foxo1/Foxo1 in g. (i) Relative protein levels of p-Bad/Bad in g. (j) Relative protein levels of XIAP/GAPDH in g. (k) INS-832/13 cells were incubated with SP6616 (10 μM) and STZ (0.4 mM) for 20 h in the presence or absence of wortmmanin (2 μM) for another 4 h, and then cell lysate was analyzed by western blot using the corresponding antibodies. (l) Relative protein levels of p-Foxo1/Foxo1 in k. (m) Relative protein levels of p-Bad/Bad in k. (n) Relative protein levels of XIAP/GAPDH in k. (o) INS-832/13 cells were pre-incubated with SP6616 (10 μM) and STZ (0.4 mM) for 23 h and then with or without stimulation of CPZ (50 μM) for 1 h, finally the cell lysate was analyzed by western blot using p-Akt and Akt antibodies. (p) Relative protein levels of p-Akt/Akt in o. All data were obtained from three independent experiments and presented as means±S.E.M. (*P<0.05, **P<0.01, ***P<0.001; ns, no significance)