Figure 5: WWP-1 associates with and ubiquitylates KLF-1.

(a) HA-WWP-1 and FLAG-KLF-1 were expressed in HEK293T cells. Lysates were immunoprecipitated with FLAG antibodies and probed with antibodies against HA (top panel) or FLAG (bottom panel). Molecular size in kilodaltons (kDa) is indicated on the left. (b) GST pull-down assay in which GST–WWP-1 or GST alone bound to glutathione beads was incubated with in vitro-translated ³⁵S-labelled KLF-1 (left panel) or PHA-4 (right panel). (c) GST pull-down with ³⁵S-labelled KLF-1 and a GST fusion protein containing only the WW domains of WWP-1. (d) In vivo ubiquitylation of KLF-1 by WWP-1. HEK293T cells were transfected with HA-tagged ubiquitin and FLAG-KLF-1 and/or HA-WWP-1. Denatured lysates were immunoprecipitated with anti-FLAG beads and analysed by immunoblot with an ubiquitin antibody. Similar banding pattern was seen when probed with an anti-HA antibody (data not shown). Bottom panel shows that similar amounts of KLF-1 were immunoprecipitated. (e) In vitro ubiquitylation assay using ³⁵S-labelled KLF-1 incubated with bacterially expressed recombinant E1, UBC-18 and GST–WWP-1. Recombinant ubiquitin was added to the reaction where indicated. Uncropped images from the autoradiography scans and immunoblots are shown in Supplementary Fig. 9.