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. 2004 Apr 29;23(10):2071–2082. doi: 10.1038/sj.emboj.7600218

Figure 3.

Figure 3

RSK isoforms are activated normally in PDK1(PHKI/PHKI) cells. (A) The indicated ES cell lines were grown to 80% confluence, serum starved for 3 h, incubated with or without 2 μM PD184352 for 1 h and then either left unstimulated or stimulated with 0.4 μg/ml TPA for 15 min. The cells were then lysed and the activity of RSK was measured after immunoprecipitation with an antibody that recognises all RSK isoforms. The results shown are mean±s.d. for three dishes of cells each assayed in duplicate. Lysates from the ES cells were also immunoblotted using the indicated antibodies. Thr360 is phosphorylated by ERK, Ser381 is phosphorylated by the C-terminal kinase domain of RSK (CT-KD) and Ser227 is the site phosphorylated by PDK1 (Frodin et al, 2000). (B) As above, except that the serum-starved cells were incubated with or without 2 μM PD184352 for 1 h or 100 nM wortmannin for 10 min and stimulated with 10% (v/v) serum for 15 min. Similar results were obtained in three different experiments.