Figure 1. In vitro phosphorylation of Nur77.

Recombinant GST–Nur77 (2 μg) was phosphorylated using 20 m-units of PKA, MSK1, PKB, RSK1 or RSK2 for 40 min. In parallel experiments, to confirm the activity of the kinase, 2 μg of CREB was phosphorylated by PKA or MSK1, 2 μg of LKB1 was phosphorylated by RSK1 or RSK2 and 2 μg of GSK3 was phosphorylated by PKB. Reactions were then run on SDS/polyacrylamide gels, and bands corresponding to Nur77, CREB, LKB1 and GSK3 were excised and the incorporation of ³²P was measured by Cerenkov counting and expressed as mol of ³²P incorporated per mol of protein. Error bars represent the S.D. for three points.