Fig. 5. The time course of nuclear p42/p44 MAPKs activation correlates with the phosphorylation of a nuclear p42/p44 MAPKs substrate: HIF-1. Quiescent Raf-1:ER cells were either left untreated (normoxia, lane 1) or placed in hypoxic conditions for 5 hours (lanes 2-8). Before the end of the hypoxic period, Raf-1:ER cells were stimulated with 100 nM estradiol for the indicated times. After 180 minutes of estradiol treatment the cells were treated for 30 minutes with the tyrosine phosphatase inhibitor bpV(phen) 1 mM (lane 8). Immunoblotting was performed using an anti-HIF-1 antiserum (upper blot), the monoclonal antibody anti-activated p42/p44 MAPKs (middle blot) or an anti-MKP1 antiserum (lower blot).