Figure 2.

JS-K inhibits ubiquitylation

(a) JS-K decreases accumulation of total ubiquitylated proteins. RPE cells were incubated with JS-K as indicated for 24 h and lysed in RIPA buffer as described previously (Yang et al., 2005). Total ubiquitylation in cells and β-Actin were immunoblotted using anti-ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-β-Actin antibodies. (b) JS-K increases cellular β-catenin levels. HEK293 cells were serum starved for 17 h. After this, the cells were exposed to 20 μM ALLN (Calbiochem) or 10 μM JS-K for 2 h. Cell lysates were prepared as described previously (Giarre et al., 1998). Samples were assessed by immunoblotting using β-catenin (Santa Cruz Biotechnology) and β-Actin antibodies. (c) JS-K increases Pdcd4 levels in cells. A549 cells were treated with 10 μM JS-K for 6 h. Cell lysates were immunoblotted as indicated (Jansen et al., 2005). (d) JS-K delays degradation of IL-1α-induced IκBα and phosphorylated IκBα. HeLa cells were incubated with 10 μM JS-K for 30 min prior to treatment with 10 ng/ml IL-1α (R&D systems, Minneapolis, MN) for 2 or 5 min. Cell lysates were immunoblotted using IκBα (Santa Cruz Biotechnology), phosphorylated IκBα (Cell Signaling Technology, Danvers, MA), and β-Actin antibodies.