Fig. 8. Ectopic Pax3 induces JNK activation and its accumulation in cytoplasmic multivesicular structures. Saos-2 cells were infected with control (Ad-ß-gal) (A,C) or Ad-Pax3^flag (B,D) adenoviruses. At three days postinfection cells were fixed with paraformaldehyde (A,B and insets) or paraformaldehyde-cytoskeletal fixation (C,D), and the distribution of (activated) phospho-JNK was determined by indirect fluorescence microscopy. Arrowheads indicate the localization of activated JNK (A,C) and exogenously expressed HA-tagged JNK (C, inset) to focal adhesions in control infected cells. Ad-Pax3^flag induces a large increase in activated JNK, which accumulates primarily in juxta-nuclear (B and arrow in D) or perinuclear (B, inset) multivesicular structures as well as at focal complexes (arrowheads in D). Ad-Pax3^flag also induces juxtanuclear vesicular accumulation of exogenous HA-tagged JNK (arrow in D, inset). Images in C, D and respective insets are confocal images. Bars, 20 µm. (E) Ectopic Pax3 induces increased JNK expression. Total extracts of control or Ad-Pax3^flag-infected Saos-2 cells were harvested postinfection at the indicated times and levels of total JNK proteins assessed by western blotting using an anti-JNK antibody. The same membrane was reprobed for actin as a loading control.