Figure 6.

(A) pJNK is detected both in focal contacts and in the nucleus in cells plated on FN, but not in cells plated on collagen I. RSF plated on FN or collagen I for 10 h were fixed, permeabilized, and stained with antibodies specific for pJNK. Actin was detected by rhodamine-phalloidin. In cells on FN, strong staining for pJNK was detected in the nucleus and in focal contact sites at the ends of actin stress fibers (arrowheads). In cells plated on collagen I, stress fibers and focal contacts were poorly developed. Staining for pJNK was weak and diffuse in the cytoplasm and nucleus. As a control, the anti-pJNK antibody was added to RSF in the presence of 100 μg/ml of the pJNK peptide used to generate the antibody. No focal contact staining was detected (lower left panel). Antibodies that recognize all forms of JNK 1/2 showed a diffuse cytoplasmic staining pattern as well as faint focal contact staining (arrowheads, lower right panel). (B) Expression of GFP-FAT (upper panels) in RSF plated on FN in serum-free medium for 8–10 h (a time before appearance of apoptotic changes; see also Fig. 1), prevents pJNK location in focal contacts (arrows). Staining for pJNK was strong in focal contact sites (arrowheads) of adjacent cells that were not transfected. Expression of GFP-FRNK (middle panels) does not bar pJNK from focal contacts (arrows). pJNK staining in focal contacts of GFP-FAT–expressing cells is similar in intensity to that in focal contacts of adjacent nontransfected cells (arrowheads). Expression of GFP-FAK (lower panels) markedly increases pJNK staining in focal contacts (arrows) as well as in the cytoplasm, relative to adjacent untransfected cells (arrowheads). (C) Staining for pERK in both GFP-FAT (upper panels) and GFP-FRNK–expressing RSF (middle panels) is weak and not detected in focal contacts. pERK staining is strong in RSF expressing GFP-FAK (lower panels). However, focal contact staining is not detected.