FIG. 5.

Effect of selective protein kinase inhibitors on Gro/TLE hyperphosphorylation induced by Hes1. (A) ROS17/2.8 cells were transfected with Hes1 and cultured for 24 h. During the last 2 h, cells were cultured in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of the protein kinase CK2 inhibitor, chrysin, followed by cell lysis and Western blotting with either panTLE (lanes 1 and 2) or anti-FLAG (lanes 3 and 4) antibodies (Ab). The presence of chrysin had two effects: it abolished the hyperphosphorylated form induced by Hes1 (long arrow), and it increased the amount of Gro/TLE in the cell extract. (B to E) ROS17/2.8 cells were not transfected (lanes 1 and 3) or transfected (lanes 2 and 4 to 6) with Hes1 and then incubated for the last 2 h in the absence (lanes 1 and 2) or presence (lanes 3 to 6) of the indicated amounts of either the protein kinase CK2 inhibitor DRB (B and C) or the PI 3-kinase inhibitor LY294002 (LY., D and E). Lysates were then collected and analyzed by Western blotting with either panTLE (B and D) or anti-FLAG (C and E) antibodies. DRB effectively reduced Hes1-induced hyperphosphorylation of Gro/TLEs without affecting the expression or mobility of Hes1, while LY294002 had no effect on the Hes1-induced phosphorylation of Gro/TLEs.