RalGDS knockdown does not affect growth factor activation of another PDK1 substrate, PKCδ. (A) MCF10A cells stably expressing RalGDS shRNA or empty vector were serum starved for 6 h and pretreated with either LY294002 for 1 h or control medium, and then cells were stimulated with EGF (5 ng/ml) for the indicated times. Cell lysates were then assayed for pPKCδ (Thr505) and total PKCδ. pPKCδ band intensities were quantified with NIH ImageJ 1.34s, and the data represent the averages of data from three independent experiments. (B) MCF10A cells pretreated as for panel A were stimulated with insulin (1 μM) for the indicated times, and cell lysates were assayed for pPKCδ (Thr505) and total PKCδ. pPKCδ band intensities were quantified with NIH ImageJ 1.34s, and the data represent the averages of data from three independent experiments.