L744832 blocks UCN-01-induced MEK/ERK activation while promoting activation of SEK/JNK and p34cdc2 upstream of mitochondrial dysfunction. (A) U937 and Jurkat cells were exposed to 10 μM L744832 in either the presence or absence of UCN-01 (U937, 100 nM; Jurkat, 150 nM) for 18 hours, after which Western blot analysis was performed to monitor phosphorylation of the p42/44MAPK-related proteins (eg, MEK, ERK, CREB, and p90RSK), stress-related (SAPK/JNK) proteins (eg, SEK1 and JNK1), and cdc2/Cdk1. (B) The phosphorylation of ERK1/2, JNK1, and cdc2 was also monitored in HL-60 and Raji cells after treatment with 10 μM L744832 with or without UCN-01 (HL-60, 100 nM; Raji, 150 nM) for 18 hours. (C-D) Bcl-2/U937 and pCEP/U937 cells (C) or Bcl-xL/U937 and pcDNA3.1/U937 cells (D) were exposed to 10 μM L744832 with or without 100 nM UCN-01 for 18 hours, after which cells were lysed and subjected to Western blot analysis to assess phosphorylation statuses of ERK1/2, JNK1, and cdc2. In each panel, 30 μg protein was loaded in each lane; blots were subsequently stripped and reprobed for expression of β-actin to ensure equivalent loading and transfer of protein. The results are representative of 3 separate experiments.