Fig. 1. Increased phosphorylation of 46 kDa Jnk in the apical cytoplasmic compartment of E18.5 129/Sv-Rac1Leu61 intestinal epithelium. (A) Replicate immunoblots of small intestinal extracts (50 µg protein/lane) from E18.5 Rac1Leu61 and normal chimeric mice were probed with antibodies that recognize Rac1, p-Jnk, the 46 kDa or 55 kDa forms of Jnk (designated Jnk-Ab1 and Jnk-Ab2, respectively), and p-c-Jun. (B-S) Sections of E18.5 jejunum from Rac1Leu61 (B-I; L-S) and normal (J,K) chimeric mice. Each antibody staining (C,E,G,I,K,M,O,Q,S) is paired with X-Gal staining (B,D,F,H,J,L,N,P,R) of the same section to show the epithelial cell genotype. At E18.5, the proliferative zone lies in the intervillus region (below broken line) positioned between rudimentary villi (above broken line). Sections from Rac1Leu61 (C,E) and normal chimeric jejunums (K) were stained with rabbit anti-p-Jnk, Cy3-tagged sheep anti-mouse Ig (red) and bis-benzimide (dark blue). 129/Sv epithelial cells in the Rac1Leu61 chimera show markedly enhanced p-Jnk staining localized principally to the apical cytoplasm (arrows). (G) High-power view of 129/Sv villus epithelial cells from a Rac1Leu61 chimera, stained with rabbit anti-Rac1, donkey anti-rabbit Ig and bis-benzamide. Although most of the immunoreactive protein is cytoplasmic, a small fraction localizes to the apical cell surface (arrow). (I) Section from a Rac1Leu61 chimera stained with the same reagents as in C,E, except that the p- Jnk mAb was pre-incubated with phosphorylated Jnk peptide. (M) Section stained with Jnk-Ab1 that recognizes total cellular 46 kDa Jnk, Cy3-tagged donkey anti-rabbit Ig, and bis-benzimide. Jnk (red) localizes to the apical cytoplasm, cell-cell and cell-substrate borders of both 129/Sv-Rac1Leu61 and B6-ROSA26 villus and intervillus epithelial cells. (O) Section incubated with Jnk-Ab2 that recognizes total cellular 55 kDa Jnk, Cy3-labeled donkey anti-rabbit Ig and bis-benzimide. 55 kDa Jnk localizes to the cytoplasm and nuclei of both 129/Sv-Rac1Leu61 and B6-ROSA26 villus and intervillus epithelial cells. (Q) Section stained with mouse anti-p-c-Jun, Cy3-sheep anti-mouse Ig and bis-benzimide. The nuclear staining for p-c-Jun is not appreciably different between the 129/Sv-Rac1Leu61 and B6-ROSA26 epithelial populations (compare B6-ROSA26 cells indicated by arrows to 129/Sv-Rac1Leu61cells indicated by a box). (S) Section from a Rac1Leu61 chimera stained with the same reagents as in Q, except that the p-c-Jun mAb was pre-incubated with phosphorylated c-Jun peptide. Scale bars: B,C,H-S, 25 µm; D-G, 10 µm.