Fig. 2. Effect of detachment and Y-27632 on ppMLC^T18/S19 and pMLC^S19. (A) Cells were detached with trypsin-EDTA for about 5 minutes at room temperature and the serum-containing medium was then added. The cell suspension was either directly transferred to 40 ml ice-cold PBS to stop the reaction (after 0.5 minutes) or incubated at 37°C for 3 minutes or 10 minutes before adding 40 ml ice-cold PBS. Cells were pelleted at 4°C and subjected to ppMLC^T18/S19 analysis. Attached cells (Att) were included for comparison. (B) The pMLC^S19 levels were analysed in human fibroblasts either detached and incubated for 3 minutes in the presence of serum or attached in the presence of serum or after serum starvation for 30 minutes. The ppMLC^T18/S19 from the same samples is also shown. (C) Response of pMLC^S19 and ppMLC^T18/S19 to the treatment with 10 µM Y-27632 for the indicated times. The results from at least three independent experiments are summarized and presented (right) as mean±s.e.m. relative to the time 0. (D) Immunofluorescence staining of focal adhesions (vinculin) and actin stress fibers after Y-27632 treatment.