Fig. 5. Cell suspension or cytochalasin D treatment inhibit HEF1 degradation. MG-63 cells on FN-coated dishes were serum-starved for 6 hours and then treated with TGF-ß1 (2.5 ng/ml) for 12 hours to induce HEF1 expression. TGF-ß1-containing medium was removed and cell layers were washed twice with serum-free medium. (A) Cell layers were then treated with 20 µg/ml cycloheximide in the absence (adhesion) or presence (adhesion + CD) of 2 µM cytochalasin D for indicated times, or detached and plated onto agar-coated dishes in the presence 20 µg/ml cycloheximide (suspension). Times indicate hours after addition of cytochalasin D or placement in suspension. Aliquots of cell lysates containing equal amounts of protein were processed for immunoblotting with anti-HEF1 antibody. Membranes were stripped and reprobed for p130 Cas, which served as a loading control. (B) The levels of total HEF1 as shown in A were analyzed by densitometry and the time at 0 hours was set as 100%.