FIGURE 2:

Serine-to-alanine substitutions in the P-motif promotes stabilization and cleavage. (a) Schematic showing the position of the P-motif with the wt sequence and mutants expanded below. Serines that were substituted by alanines are numbered and indicated by underlining. (b) Comparison of expression in liver cells (Huh 7) of wt CREB-H and the various P-motif mutants (3S+DSG, DSG, 3S). Labeling is as for Figure 1. Bottom, actin as a sample loading control. (c) Same analysis as in b, showing a longer exposure of the cleaved products. (d) Quantitative estimates of protein abundance. Blots were scanned in the appropriate channel using a LI-COR Odyssey laser scanning system, background subtracted, volume integrated, and normalized for loading using actin abundance in the separate channel. Fold increases in overall levels of the mutant proteins are compared with wt levels, which were standardized to 1. (e) Soluble extracts of cells transfected with the wt or the 3S+DSG mutant were fractionated and equal cell equivalents of the nuclear fractions analyzed by Western blotting for cleaved CREB-H. Lamin a/c, which fractionated exclusively to the nuclear fraction, was used as a loading control.