Figure 3: The activity of WT H-Ras is dependent on oncogenic K-Ras.

(a–j) Cancer cell lines harbouring oncogenic K-Ras derived from pancreas (AsPC-1 (a,b), MIA PaCa-2 (c,d) and PL45 (e–h)) or colon (HCT-116 (i,j)) were infected with lentiviral tetracycline-inducible miR30-based scrambled shRNA (shCtrl) or K-Ras shRNA (shK-Ras). Following doxycycline treatment, cells were serum-starved for 20 h, and the levels of GTP-bound H-Ras (a-f,i,j) or N-Ras (g,h) were determined by the RBD pull-down assay as described in Methods. The efficiency and specificity of knockdown were analysed by immunoblotting with anti-K-, H- or N-Ras antibodies. Quantification of the levels of activated H-Ras (b,d,f,j) or activated N-Ras (h) was done by densitometry scanning and normalized to the levels of total H-Ras or N-Ras, respectively. Values are means±s.d. from three independent experiments presented as fold activation compared with shCtrl. (k) MIA PaCa-2 cells engineered to express shCtrl or shK-Ras as described in (a–j) were serum-starved for 20 h and then stimulated with 50 ng ml⁻¹ EGF for 10 min at 37 °C. Activation of EGFR signalling was determined by western blotting using anti-phospho-EGFR (Tyr1068) and anti-phospho-PLCγ1 (Tyr783) antibodies. (l) Quantification of the levels of activated EGFR in (k) was done by densitometry scanning and normalized to the levels of total EGFR. Values are means±s.d. from three independent experiments presented as fold activation compared with shCtrl (mock). (m) MIA PaCa-2 cells were either cultured in full-serum media (−) or starved by frequent replenishment of serum-free media (ss; changed every 2 h for a total 16 h). Cell lysates were probed with the specified antibodies. (n) The levels of GTP-bound H-Ras in (m) were determined by RBD pull-down and quantified by densitometry scanning and normalized to the total levels of H-Ras. Values are means±s.d. from three independent experiments presented as fold activation compared with full serum media (−). For all panels, tubulin was used as loading control. WCL, whole cell lysate; A.U., arbitrary units.