Fig. 3. Analysis of RhoA activity, pMBS^T853 and in vitro Rho-kinase activity after detachment. (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 then collected for RhoA activity assay. (B) Effect of Y-27632 on pMBS^T853 in attached and detached cells in the presence of serum. Levels of ppMLC^T18/S19 in the same samples are also shown. (C) pMBS^T853 was analysed in human fibroblasts either detached and incubated for 3 minutes in the presence of serum (Det) or attached in the presence of serum or after serum starvation for 30 minutes. (D) In vitro Rho-kinase activity was assayed. Radiolabeling of MLC was absent without HA-Rho kinase (lane 1, mock immunoprecipitation using Protein-A beads only) or when 10 µM Y-27632 was included in the kinase reaction (lane 4), confirming that the kinase reaction is specific to Rho kinase.