Figure 3. TGFβ regulates c-Jun in a p38-dependent manner in PC-3U cells. (A and B) Cell lysates derived from PC-3U cells, treated or transfected as indicated, were subjected to immunoblotting for p-Ser63-c-Jun, total c-Jun, p-p38, p-GSK-3β Ser9, total GSK-3β and HA (p38). Actin served as internal control. (C) PC-3U cells transiently transfected with HA-p38 wild-type or HA-p38DN were subjected to a non-radioactive in vitro kinase assay using recombinant c-Jun, and cell lysates were subsequently subjected to immunoprecipitation (IP) with HA-antibodies. The product was immunoblotted with p-Ser63-c-Jun antibodies. (D) Inhibitor of p38 (SB203580) were added 1 h before treatment of cells. (E) PC-3U cells transiently transfected with wild-type or Ser9Ala mutant GSK-3β were treated with or without TGFβ, as indicated. The cells were then lysed and subjected to immunoblotting using p-Ser63-c-Jun and GSK-3β antisera. β-tubulin served as internal control for equal loading of proteins.