s-FKN stimulates with distinct time courses ERKs, JNK1, and p38 activities in MonoMac6 cells.

MonoMac6 cells were incubated with 60 nM s-FKN for the indicated times and extracted proteins from lysates were resolved by SDS-PAGE and electrophoretically transferred to Immobilon-P membrane before being detected by immunoblotting with antiactive ERKs (A) or p38 (C) antibodies. JNK1 activity (B) was performed, from cell lysates, by in vitro kinase assay using GST-ATF2 as substrate after immunoprecipitation (IP) with specific antibodies as described in “Materials and methods. Amounts of ERKs, JNK1, or p38 in immunoprecipitates were assessed by Western blotting (WB) analysis with the appropriate antibodies. The results are representative of 3 experiments.