Fig. 1. PAK4 expression and activity in MDCK cells. (A) Analysis of endogenous PAK isoform expression in MDCK cell lysates. A western blot of MDCK cell lysates was probed with antibodies against PAK4 (lane 1) and PAK1, -2 and -3 (lane 2). (B) Kinase activity of PAK4. MDCK cells were transfected with HA-PAK4wt (Wt), HA-PAK4 () and HA-PAK4GBD (GBD) and incubated for 24 hours in 0.2% FCS. PAK4 was immunoprecipitated from cell lysates with anti-HA mAb and its kinase activity assayed in the presence of Histone H1 and [-³²P]ATP. Substrate phosphorylation was analysed after SDS-PAGE and autoradiography. Phosphorylation of Histone (arrow) and autophosphorylation (arrowhead) is indicated. Retained cell lysates were subjected to western blotting with the anti-HA mAb to show expression levels of exogenous PAK4 proteins. Autoradiographs and western blots were quantified using Kinetic Imaging software and the level of kinase activity normalised to protein expression levels. The results shown are the means±s.e.m. of three independent experiments.