Figure 6: KLF-1 is preferentially monoubiquitylated by WWP-1.
(a) In vivo ubiquitylation assay similar to Fig. 5d except 0.1 mM Bortezomib was added to the cells. Denatured lysates were immunoprecipitated with anti-FLAG beads and analysed by immunoblot with the indicated ubiquitin antibodies. The asterisks (*) indicate nonspecific bands present in all samples. Bottom panel shows that similar amounts of KLF-1 were immunoprecipitated. (b) In vitro ubiquitylation assay using 35S-labelled KLF-1 incubated with bacterially expressed recombinant E1, UBC-18 and GST–WWP-1. Recombinant WT ubiquitin or the indicated ubiquitin mutants were added to the reaction where indicated. Uncropped images from the autoradiography scan and immunoblots are shown in Supplementary Fig. 10.
