Fig. 2: Itch is a E3 ligase for degradation of IκB through Cat D inhibition. | Cell Death & Disease

Fig. 2: Itch is a E3 ligase for degradation of IκB through Cat D inhibition.

From: Itch and autophagy-mediated NF-κB activation contributes to inhibition of cathepsin D-induced sensitizing effect on anticancer drugs

Fig. 2

A Caki cells were transduced with lentivirus containing either shRNA targeting Itch or a non-target sequence; alternatively, they were transiently transfected with Cont siRNA or β-TrCP siRNA and treated with 2 μM Pep A for 24 h. B Caki cells were pretreated with 0.5–1 μM clomipramine for 30 min, and they were then treated with 2 μM Pep A for 24 h. C Binding of IκB and Itch or β-TrCP was analyzed via the IP assay. D Caki cells were transiently co-transfected with Cont siRNA or Itch siRNA and HA-K63-Ub, and they were pretreated with 5 nM bafilomycin A1 for 30 min. They were then treated with 2 μM Pep A for 24 h. The ubiquitination of endogenous IκB was analyzed via the DUB assay. E, F Caki cells were transiently transfected with Itch WT and mutant (E) or Cont siRNA and two different Itch siRNA (F), and they were then treated with 20 μg/mL CHX and 2 μM Pep A for the indicated time points. Band intensity of Bcl-xL was analyzed using ImageJ. Protein expression was measured via western blotting (A–F).

Back to article page