Supplementary Figure 8: USP14 binds to the 26S proteasome.

(a) Native PAGE assay of the USP14-proteasome interactions. The purified human proteasome (1.6 picomoles per reaction) was incubated for 20 minutes at 30°C with 16 picomoles of GST-USP14 (USP14G), untagged USP14, or GST. 64 picomoles UbAl were added where indicated. Samples were resolved on a 4% native PAGE gel and visualized by Suc-LLVY-AMC in-gel hydrolysis (upper panel). Binding of USP14G to the proteasome results in marked reduction of its electrophoretic mobility. The bottom panel shows Coomassie blue staining of the gel. Yeast CP was used as a marker. (b) A pull-down assay was performed in the presence and absence of UbAl. The presence of UbAl appears to enhance the interaction of USP14 with the proteasome. The binding of USP14G to proteasome is more obvious than that for untagged USP14.