Effect of MPA treatment on MEK-Erk pathway. (A) 32D cells were treated with 2 μM MPA alone or MPA plus 100 μM guanosine (Gua), and cells were harvested at indicated time points. Western blot analysis was performed using antibodies specific for phospho-MEK, phospho-Erk, and total MEK and Erk protein. (B) 32D cells were treated with 2 μM MPA for the indicated time points. Total lysate (800 μg) was used for immunoprecipitation with MEK-specific antibody, followed by an MEK kinase activity assay using Erk and MBP substrates as described in “Materials and methods.” Data are from 1 of 2 representative experiments, each performed in triplicate. (C) FL5.12 cells were treated as 32D cells in panel A and were harvested at indicated times. Total cell lysate (3 μg) was used for MEK and Erk analysis.