FIGURE 6:

The selective GSK-3 inhibitor CHIR99021 induces cleavage of CREB-H and inhibits phosphorylation at serine 73. (a) Transfected COS-1 cells expressing the precursor form of CREB-H under control of a TK promoter were untreated (–) or treated (+) with 10 µM CHIR99021 (6 h) after an incubation of 16 h in 5% serum. Equal cell equivalents were analyzed by Western blot with the anti-SV5 antibody. FL indicates the precursor form of CREB-H and Cl the cleavage product. (b, c) Cells expressing CREB-H, as described, were either untreated (–) or treated with CHIR99021 (10 μM, CHIR), LY294002 (10 μM, LY), wortmannin (0.2 µM, Wort), TBCA (10 μM), or SB216763 (20 μM) for 6 h. Extracts were analyzed by Western blot using anti-SV5 antibody and simultaneously with anti-pS73 antibody. Image in b shows the precursor form of the protein; c shows higher-sensitivity scans for detection of the cleavage product.