Figure 3.

Assessment of cAMP response element-binding (CREB) stability and ubiquitin/proteasome involvement in GSKJ4-mediated CREB downregulation in U-937 cells. (A) Cells were treated for up 18 h with or without (NT) 10 μM GSKJ4 and grown in the presence of 20 μg/ml of protein synthesis inhibitor cycloheximide (CHX). Thereafter, CREB protein levels were determined by Western blotting (upper part) after 2, 4, 6, and 18 h of treatment. For each time point, the relative CREB/vinculin Ratio of three unrelated blot images were quantified by ImageJ software and reported in graph as average and SD measure (bottom part). (B) Proteasome inhibitor MG132 at 10 μM was administered, or not (NT), for 18 h to U-937 cells kept in the presence of 10 μM GSKJ4. CREB protein levels and the ubiquitin pattern were evaluated through Western blotting analysis in response to the above stimuli. Densitometric analyses of CREB/vinculin Ratio of three independent experiments were shown in chart and represented as average and SD values. *P < 0.05 by ANOVA and unpaired t-test.