Figure 2.

Calcimycin-induced activation of MEK1/2 and RAF1 but not p38 and JNK2 is important for calcimycin-induced apoptosis. (A) Calcimycin-induced activation of MEK1/2 is blocked by DNM RAF1. N/N1003A cells transfected with pCMV-Neo vector or pCMV-DNMRAF1 were grown to 100% confluence and subsequently subjected to treatment with 5 μM calcimycin for 0, 1, 5, or 10 h in MEM. Then, total proteins were isolated, and resolved in 10% SDS-PAGE and analyzed with antibodies against either total MEK1/2 (T-MEK1/2) or phospho-MEK1/2 (p-MEK1/2, activated form) as described previously (Li et al., 2003; Liu et al., 2004). (B) Calcimycin-induced activation of RAF1 is blocked by DNM RAS. N/N1003A cells transfected with pCMV-Neo vector or pCMV-DNMRAS were processed as described in Figure 2A. Western blots were analyzed with antibodies against either total RAF-1 (T-RAF-1) or phospho-RAF-1 (p-RAF-1, activated form) as described Figure 2A. (C) Analysis of p38 kinase activation with Western blot analysis. N/N1003A cells were grown to 100% confluence and then either pretreated with 2 μM PD169316 (right, IC₅₀ = 89 nM) or 0.01% DMSO (left) for 6 h followed by treatment with 5 μM calcimycin for 0, 1, 5, or 10 h in MEM. Then total proteins were isolated, resolved in 10% SDS-PAGE, and analyzed with antibodies against either total p38 (T-p38) or phospho-p38 kinase (p-p38, activated form) as described previously (Li et al., 2003). (D) Analysis of JNK2 kinase activation with Western blot analysis. N/N1003A cells were grown to 100% confluence and then either pretreated with 1 μM SP600125 (right, IC₅₀ = 40 nM) or 0.01% DMSO (left) for 6 h followed by treatment with 5 μM calcimycin for 0, 1, 5, or 10 h in MEM. Then, total proteins were isolated, resolved in 10% SDS-PAGE and analyzed with antibodies against either total JNK1/2 (T-JNK1/2) or phospho-JNK1/2 (p-JNK1/2, activated form) as described previously (Li et al., 2003; Feng et al., 2004; Wang et al., 2005).