<p>(A) HeLa cells were transfected with RICTOR-directed siRNA or non-target siRNA and then stimulated with LPA (10 µM) for 20 minutes. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) and LPA-induced AKT Ser473 phosphorylation was determined by western blotting. A representative western blot is shown and the graph below provides quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 3). (B) HeLa cells transfected with ILK-directed siRNA and stimulated by LPA were analysed as described above. The graph below shows quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 2). (C) MCF7 cells transfected as indicated were stimulated with LPA (5 µM) for 5 min and analysed as described above. A representative western blot is presented and the graph below shows quantified levels of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 3, * represents <i>p</i><0.005). (D) MCF7 cells were transfected simultaneously with PAK1 and PAK2 siRNA or with non-target siRNA and then stimulated with LPA (5 µM) for 5 min. A representative western blot is shown. The graph below is a quantification of AKT pSer473 levels (mean ± s.e.m.; <i>n</i> = 3, ** represents <i>p</i><0.05). (E) HeLa cells were treated with increasing concentrations of the PAK inhibitor IPA3 or with DMSO and then stimulated with LPA (10 µM) for 20 min. The graph below is the quantification of AKT pSer473 levels after LPA-stimulation in cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in LPA-stimulated cells without the inhibitor (mean ± s.e.m.; <i>n</i> = 3, * represents <i>p</i><0.005).</p
