Fig. 3.

K117 site is essential for KRA-533 to bind and activate KRAS. (a) Thermal shift melting curve of purified KRAS protein (WT, S17A, K117A, or S17A/K117A (AA)) incubated with increasing concentrations of KRA-533. Melting temperature (Tm) values of DMSO control and 15 μM KRA-533 are shown. *P < 0.05, no significance → NS, by 2-tailed t test. (b) 600 nM purified KRAS protein (WT, S17A, K117A or AA) was incubated with 11 μM [γ-35S] GTPγS, purified GFP (RASGRP1, 180 nM) and GAP (RASA1, 180 nM) at 25 °C in the presence or absence of 15 μM KRA-533. The radioactivity remaining on the protein after intensive washing was quantified by liquid scintillation. Error bars represent ± SD. *P < 0.05 by 2-tailed t test. (c) A549 cells were transfected with GFP-tagged KRAS WT, S17A, K117A or AA, followed by treatment with KRA-533 (15 μM) for 48 h. KRAS·GTP (active KRAS) was pulled down by Raf-1-RBD. The GFP-tagged exogenous KRAS-GTP (GFP-KRAS-GTP) was analyzed by Western blot using anti-GFP antibody