(a-d) MIA PaCa-2 cells engineered as in Figure 2a to express inducible shCtrl, shSos, *SosWT, and *SosRA/LE (a, b) or *SosF929A (c, d) as indicated were cultured in 0.5% serum in the presence of doxycycline for the time indicated. Efficiency of knock-down and ectopic expression of Sos were confirmed by western blotting. Tubulin was used as a loading control. Cell densities were determined by Syto60 staining and quantified (b, d) as described in Methods. Values are means +/- SD of technical triplicates presented as fold activation compared to shCtrl. The experiment shown is representative of three independent experiments. (e, f) Oncogenic K-Ras activity does not depend on the expression of Sos. (e) The levels of K-Ras-GTP in serum-deprived MIA PaCa-2 cells harboring the indicated constructs were determined by the RBD pull down assay. Tubulin was used as a loading control. WCL, whole cell lysate. (f) The levels of activated Ras were quantified by densitometry scanning and normalized to the total levels of K-Ras. Values are means +/- SD from three independent experiments presented as fold activation compared to shCtrl. A.U., arbitrary units.