Figure 8. Synergy between sHB-EGF and Kras^G12D occurred at the level of proliferation, protein phosphorylation, and Kras expression.

Pancreas samples were compared at postnatal day 1, immediately prior to morphological change. P values are relative to normal controls. A. Percent of cyclinD1-positive acinar cells according to genotype. B. Percent of mitotic acinar cells as measured by immunolabeling for phospho-histone H3. C. Quantitative comparison of RNA from sHB-EGF alone or from Kras^G12D; sHB-EGF mice revealed little change in Cdkn1a or Cdkn1b but a 2.8-fold decrease in Cdkn2a and a 2.1-fold decrease in Cdkn2d when Kras^G12D was combined with sHB-EGF overexpression. D. Comparison of phosphorylation levels in sHB-EGF mice and Kras^G12D; sHB-EGF mice. Numbers indicate fold difference of intensities between genotypes. Intensities for Erk1/2 were quantified on a lighter exposure. Specific phosphorylation sites detected were: Mek1/2, S218/S222, S222/S226; Erk1/2,T202/Y204, T185/Y187; Akt, S473, T308; Ampk1a,T174; p70S6K, T229; Gsk3α/β, S21/S9; Hck, Y411. E. Total protein lysates were analyzed for Kras protein levels. While Kras^G12D expression alone or sHB-EGF overexpression alone had no effect on total Kras levels, together they increased the amount of Kras protein relative to β actin. N, normal pancreas; K, Kras^G12D alone; H, sHB-EGF alone; HK, both Kras^G12D and sHB-EGF expression; arrow, expected Kras band size; asterisk, location of nonspecific band. Protein size standards are indicated on right.