Figure 6

RGS and RGS17 inhibit AKT activation. (A) SKOV-3 cells transfected with control (NC) or RGS10 (10) siRNA constructs were serum starved and stimulated with vehicle or LPA at the indicated concentrations for 5 minutes. Control wells were pre-incubated with LY294002, a PI3K inhibitor, for 1 hour. Cell lysates were analyzed with SDS-PAGE and immunoblotting with anti-phospho AKT antibodies (top) to measure AKT activation. Phospho-AKT blots were re-probed with GAPDH antibodies as loading controls (bottom). Activation of AKT was quantified by densitometry of bands and normalized to GAPDH controls. (B) SKOV-3 cells were transfected with either empty vector (V), plasmid encoding HA-tagged RGS10 (10), or plasmid encoding HA-tagged RGS17 (17). Cells were serum-starved overnight and treated with vehicle or LPA for 5 minutes at 37°C. Control cells were also treated with 10 μM LY294002 (LY) to block activation. Cell lysates were analyzed with SDS-PAGE and immunoblotting with anti-phospho AKT antibodies (top left) to measure AKT activation, or HA antibodies (top right) to confirm transfection. Phospho-AKT blots were re-probed with GAPDH antibodies as loading controls (bottom). Activation of AKT was quantified by densitometry of bands and normalized to GAPDH controls.