Fig. 5. Forskolin stimulation of CD8 cells results in RhoA phosphorylation at a serine residue. (A) For each experimental condition, confluent monolayers of CD8 cells were metabolically labeled with [³²P] orthophosphate (250 µCi/ml), for 3 hours. Cells were lysed and RhoA was immunoprecipitated with 10 µl agarose-conjugated RhoA and detected by autoradiography. Experimental conditions included: control condition, stimulation with forskolin (10^–4 M for 15 minutes at 37°C); incubation with H89 (30 µM for 30 minutes at 37°C) followed by stimulation with forskolin. Equal amounts of RhoA were precipitated in each condition (I.P. RhoA). Forskolin stimulation increased the phosphorylated state of RhoA nearly 2.5-fold and pretreatment with H89 abolished the forskolin effect. A densitometric profile (mean values±s.e., n=3) of bands corresponding to phosphorylated RhoA is shown on the right. (B) Cell lysate from basal and forskolin-stimulated CD8 cells incubated with 30 µl monoclonal agarose conjugated anti-phosphoserine. Immunocomplexes were mixed with 20 µl of Laemmli buffer and blotted with anti-RhoA antibodies. Forskolin treatment resulted in a nearly two-fold increase in serine-phosphorylated RhoA. A densitometric profile (mean values±s.e., n=3) of bands corresponding to serine phosphorylated RhoA is shown on the right.