Figure 2.

Ubiquitin ligase activity of SMURF2 positively regulates KRAS protein stability. (A) HEK293 cells were transfected with KRAS in the presence or absence of SMURF2 (SM2) CA. Twelve hours post-transfection, indicated cells were treated with either 5 mM 3-MA or 2 µM MG132 for 4 hours. Cell lysates were then subjected to immunoblot analysis with the indicated antibodies. (B) HEK293 cells were transfected either with G12V mutant KRAS in the presence or absence of SM2-CA. Twelve hours post-transfection, cells were treated with cycloheximide (50 µg/ml), and cell lysates were harvested at the indicated time points and analyzed by immunoblot analysis with the antibodies mentioned. (C) Graphical representation of the quantification of KRAS protein levels shown in B to determine protein half-life. Relative KRAS levels were determined by densitometric scanning of the representative immunoblot considering 0 hour band intensity as 1 (arbitrary units). (D) Endogenous KRAS half-life was determined for H2347 and H441 cells on Smurf2 knockdown. Indicated cells were transfected with Smurf2 siRNA, and 48 hours post-transfection, cells were treated with cycloheximide (50 µg/ml), harvested at indicated times, and analyzed by immunoblot analysis. (E) Protein half-lives were determined as explained in C. Each value represents protein intensity average ± SD from three independent experiments and plotted on a log-linear scale.