Figure 1.

SMURF2 ubiquitin ligase activity controls mutant KRAS steady-state levels. (A) H358 (KRAS^G12C), H441 (KRAS^G12V), and H2347 (KRAS^WT) human lung adenocarcinoma cells were transfected with either control (C) or Smurf2 (S) siRNA. Forty-eight hours posttransfection, cell lysates were subjected to immunoblot analysis using specified antibodies. (B) Quantification of KRAS steady-state level in H358, H441, and H2347 cells on C or S siRNA. Immunoblot obtained from A were scanned and quantified using ImageJ software, and KRAS steady-state levels were normalized using GAPDH as loading control. Relative KRAS protein levels were obtained, and mean ± SEM values were calculated from three independent experiments; * denotes significant difference from control at P < .05; **, denotes significant difference from control at P < .005; NS, not significant. (C) H441 cells were transfected with Smurf2 si-R (SM2-siR) construct before siRNA-mediated Smurf2 knockdown. Cell lysates were prepared 24 hours post siRNA transfection and immunoblotted using indicated antibodies. (D) HEK293 cells were co-transfected with Myc-tagged KRAS [either wild type or various mutants (G12D, V, C, or S)] in the presence or absence of FLAG-tagged SMURF2 [either wild type or C716A (CA) mutants]. Twelve hours post-transfection, cell lysates were prepared and subjected to immunoblot analysis using indicated antibodies.