Paxillin proteasomal degradation is regulated by collagen. A) NBT-II cells plated for 18 h on plastic dishes were treated with the proteasome inhibitor lactacystin at 1 μM for 4 h. Cell lysates were immunoprecipitated with anti-paxillin antibody and immunoblot analysis was done with anti-ubiquitin (upper panel) or anti-paxillin antibody (lower panel). B) NBT-II cells were pulse-chased on plastic or collagen-coated dishes for 18 h and cell lysates were subjected to paxillin immunoprecipitation as described in Materials and Methods. When indicated, 1 μM lactacystin was added to the cells during the chase period. The lower panel is a quantification of the bands corresponding to 35S-labeled paxillin. The 35S-paxillin levels were calculated relatively to the density of the paxillin band immunoprecipitated from cells at the beginning of the radioactive chase (control lane). C) NBT-II cells cultured for the indicated times on plastic or collagen-coated dishes were trypsinized and counted using a hemocytometer. Values represent the mean of three independent experiments ± SEM.