Fig. 5: Perturbation of Ras/RAF-CRD interaction affects Ras functions and oncogenic properties.
From: Identification and characterization of binders to a cryptic and functional pocket in KRAS
A–D SW1990 cells stably engineered for doxycycline-inducible regulatable sh236 KRAS-specific shRNA expression and CMV-driven constitutive expression of the various flag-tagged KRASG12D alleles without (FKG12D) or with discrete mutations within the back-pocket (FKG12D/L23E or FKG12D/V45D or FKG12D/V45D/L23E) were treated for 72 h with doxycycline, lyzed and cell extracts further analyzed either by straight western-blot for the indicated read-outs (A and C), or by GST-cRaf-RBD pull-down to quantify GTP-loaded exogenously expressed KRAS binding (B, upper panel), or by anti-flag co-immuno-precipitation for endogenous proteins (D, upper panel). For (B, D), Input (lower panels) refer to straight western blots to assess expression levels across the conditions tested. E, F SW1990 cells stably engineered for doxycycline-inducible regulatable non targeting shNT control or sh236 KRAS-specific shRNA expression and Ubc-driven constitutive expression of the various flag-tagged KRASG12D and KRASG12D/V45D alleles were treated with doxycycline for either 14 days in a colony formation assay (left panel), or for 3 days only at which stage cells were lyzed and cell extracts analyzed by western-blot for the mentioned read-outs. Con.: control cells engineered from CMV-driven empty vector; SE: Short Exposure; LE: Long Exposure; a: endogenous KRASG12D; b: flag-tagged exogenously expressed KRAS. For (A–F), a minimum of three repeats has been performed with similar results; Source Data are provided as a Source Data file.
