Fig. 2.

Itch ubiquitylates SuFu through K63 linkage. a HEK293T cells were co-transfected with HA-Ub in the presence or absence of Flag-Itch or Flag-C830A. Cell lysates were immunoprecipitated with an anti-SuFu antibody, followed by immunoblotting with an anti-HA antibody to detect ubiquitylated forms. b Itch^−/− MEFs were transfected with HA-Ub in the presence or absence of Flag-Itch or Flag-C830A. The assay was carried out as described in a. Wild-type (WT) MEFs were used as control to evaluate the basal ubiquitylation of endogenous SuFu. c, d In vitro translated ³⁵S-labelled SuFu WT (c, d) or SuFu K-less (d) was incubated alone or in combination with GST-Itch for the indicated times. The ubiquitylated forms were detected by fluorography. e Schematic representation of SuFu protein showing its lysine residues involved in Itch-dependent ubiquitylation. f, g Flag-SuFu WT or Flag-SuFu mutants were co-transfected in HEK293T cells with HA-Ub in the presence or absence of Myc-Itch. The assay was carried out as described in a. h HEK293T cells were transfected with HA-Ub and Flag-SuFu in the presence or absence of Myc-Itch. Transfected cells were treated with MG132 (50 µM for 4 h) to enrich for ubiquitylated proteins. The assay was carried out as described in a. i HEK293T cells were transfected with Flag-SuFu in the presence or absence of increasing amount of Myc-Itch. Total protein levels were analysed by immunoblotting. j HEK293T cells were transfected in the presence or absence of increasing amount of Myc-Itch. Total protein levels were analysed by immunoblotting. k Immunoblotting analysis of SuFu and Itch proteins in HEK293T cells transfected with control (siCTR) or Itch siRNAs (siItch). β-Actin is shown as a control for loading (*non-specific bands). l SuFu protein levels in WT MEF or Itch^−/− MEF cells treated with cycloheximide (CHX, 100 µg/ml) at different time points. m Purified recombinant proteins wild-type Ub or Ub mutants K48 only (K48O), K48R, K63 only (K63O), K63R, or K-less were incubated with GST-Itch and in vitro translated ³⁵S-labelled SuFu for the indicated times. Ubiquitylated SuFu was detected by fluorography