Fig. 2. Serum or LPA in medium affects ROCK-mediated MLC phosphorylation, which is closely related to PMA-induced apoptosis. PMA, specific ROCK inhibitor (Y27632; 20 µM), 10% (v/v) heat-inactivated FBS or LPA (10 µM) was added to the medium separately or in different combinations as indicated. (A) After 12 hours, viability was determined using the Trypan Blue exclusion method. Results are the means±s.d. from three independent experiments. (B) In parallel, cells treated with PMA for 8 hours were harvested for immunoblotting with antibodies specific for phosphoERK1/2, phosphoMLC and MLC.