A and C, RT-PCR was performed on CPAE cell poly-A+ RNA which was reversely transcribed in cDNA and subsequently amplified with primers specific for RhoA (A) or Rho kinase (C) as described in Methods. PCR products were analysed on a 1 % agarose gel. The specificity of the PCR products was verified by subcloning and sequence analysis. Lanes ‘+’ and ‘-’ refer to PCR reactions where the reverse transcription step was, respectively, included or excluded. Lane M is a DNA size standard. B and D, for Western blotting, 50 μg of total cellular protein was separated on SDS-PAGE (7.5 % acrylamide for Rho kinase or 10 % acrylamide for RhoA) and electroblotted on a PVDF membrane. Blots were probed with a monoclonal antibody against RhoA (B) or a polyclonal antiserum against Rho kinase (D). Lanes ‘+’ and ‘-’ correspond to blots where the primary antibody was, respectively, included or excluded. The migration pattern of protein molecular mass standards is shown on the left. The duplet in B is unlikely to represent different Rho isoforms since the monoclonal antibody is specific for RhoA. The position of the lower band is consistent with the molecular mass of RhoA (21.8 kDa); the upper band may represent a post-translational modification. The lower band in D migrating at ≈100 kDa probably represents a degradation product.