Figure 1.

Mutation of Lys‐6 to Arg enhances stability of ECE1c. (A) DLD‐1 clone cells expressing Flag‐tagged ECE1c^WT or ECE1c^K6R proteins were incubated with 20 μg·mL⁻¹ cycloheximide (CHX) in the absence (for 36 h) or presence (for 12 h) of 25 μm silmitasertib. ECE1c proteins were detected by western blot with an anti‐Flag antibody, using β‐actin as loading control. Representative blots are shown (upper). Relative levels (%) of Flag‐ECE1c proteins from three independent experiments were calculated (lower). (B) DLD‐1 clones described in A were grown in medium without FBS for 48 h. Culture supernatants were used to measure ET‐1 levels using ELISA according to manufacturer’s instructions (Thermo Fisher). Data represent average ± SEM (n = 3). ANOVA and Tukey tests were used. *P ≤ 0.05, **P ≤ 0.01.