Figure 2.
JSP-1 did not inactivate MAP kinases. Flag-tagged Jnk (A) or Flag-tagged p38 (B) was coexpressed with a control vector (lane 1) or an active mutant form of Cdc42 (V12) in the absence (lane 2) or presence (lane 3) of JSP-1. Cells were serum starved overnight before harvesting. (C) Myc-tagged Erk was coexpressed with a control vector (lanes 1 and 2) or with JSP-1 (lane 3). Cells were serum starved overnight and harvested without stimulation (lane 1) or following stimulation with epidermal growth factor for 25 minutes before harvesting (lanes 2 and 3). The kinase activity of Jnk, p38, and Erk was determined by immunoprecipitation of the kinases and immune-complex assays with GST-Jun, GST-ATF2, or myelin basic protein (A Upper, B Upper, and C Upper, respectively) as substrate. The level of Jnk, p38, and Erk in each reaction was determined by immunoblotting with the anti-Flag antibody (A Lower and B Lower) or the anti-Myc antibody, 9E10 (C Lower).