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. 2019 Dec 31;30:101422. doi: 10.1016/j.redox.2019.101422

Fig. 5.

Fig. 5

Cat K decreases Raptor protein stability. (A) Caki cells were treated with 2 μM ODN for the indicated time points. (B) Caki cells were transfected with pRK5 (Vec) or pRK5-myc-Raptor (Raptor) and treated with 2 μM ODN for 12 h. (C) Caki cells were transfected with pRK5 (Vec) or pRK5-myc-Raptor (Raptor) and treated with 2 μM ODN and 50 ng/mL TRAIL for 24 h. (D) Flow cytometry and western blotting analysis in Caki cells transfected with pRK5 (Vec) or pRK5-Flag-PRAS40 (PRAS40 WT) and treated with 2 μM ODN and 50 ng/mL TRAIL for 24 h. (E) Caki cells were treated with 2 μM ODN for the indicated time. mRNA levels were assessed by reverse transcription PCR. (F) Caki cells were pretreated with 0.5 μM MG132 for 30 min and then treated with 2 μM ODN for 24 h. (G) Caki cells were treated with 20 μg/mL CHX in the presence or absence of 2 μM ODN for the indicated time. (H) To analyze the ubiquitination of endogenous Raptor, Caki cells were transfected with HA-ubiquitin (HA-Ub) and treated with 2 μM ODN in the presence of 0.5 μM MG132 for 12 h. Immunoprecipitation was performed using an anti-Raptor antibody. (I) Indicated cells were treated with the various concentrations of ODN for 24 h. (J) The indicated cancer cell lines were transfected with control siRNA (siCont) or Cat K siRNA (siCat K) for 24 h. Apoptosis and protein expression were measured by flow cytometry and western blotting, respectively. The values in the graphs (C, D and G) represent the mean ± SD of three independent experiments. *p < 0.01 compared to the combinations of ODN and TRAIL in Caki/Vec. #p < 0.01 compared to the CHX.