Addition of 4HT activates Raf-1 and ERK kinase activity. ▵Raf-1:ER cells were stimulated at 37°C in RPMI, 10% FCS and 100 nmol/L 4HT for the indicated time. The cells were lysed and analyzed for (A) Raf-1 activity by immunoprecipitation with an antiestrogen receptor antibody followed by an in vitro kinase assay by using GST-MEK1 as a substrate or (B) ERK activity by immunoprecipitation with an anti-ERK antibody, followed by an in vitro kinase assay by using MBP as a substrate. Positive control cells were stimulated with IL-3 for 10 minutes. Phosphorylated proteins were visualized after SDS-PAGE and autoradiography. A 15 minute and a 1-hour exposure are shown to clearly demonstrate the activity of ▵Raf-1:ER at both the 16 and 2 hour time points. The quantity of immunoprecipitated (IP) protein in each lane was assessed by immunoblotting (IB) with antibodies against GFP (GFP) or ERK (ERK).