Fig. 3

Itch-dependent K63-linked ubiquitylation of SuFu leads to Gli3R formation. a Gli3/SuFu proteins interaction by NanoBiT technology. Itch^−/− MEFs were transfected with indicated plasmids. *P < 0.05, Gli3+SuFu WT versus Gli3; **P < 0.05, Gli3+SuFu WT+Itch versus Gli3+SuFu WT; ***P < 0.05, Gli3+SuFuK321/457R versus Gli3+SuFu WT. b, c Association between endogenous Gli3 and Flag-SuFu WT or Flag-SuFuK321/457R assayed by determining the amount of SuFu that co-precipitated with anti-Gli3 antibody or control goat antisera (IgG) from MEFs lysates (b). The ratio of the SuFu signal to the Gli3FL signal from b was plotted (c). *P < 0.05. d–g Gli3/SuFu, interaction, assessed as in (b), in WT MEFs transfected with siItch or siCTR (d) or in Itch^−/− MEFs transfected with the indicated plasmids (f). The ratio of the SuFu signal to the Gli3FL signal from (d) and (f) was plotted (respectively (e) and (g)). *P < 0.05. h WT MEFs were co-transfected with indicated plasmids in the presence or absence of Itch. Cell lysates were immunoprecipitated with anti-Flag agarose beads (1st IP). After two elutions with Flag peptide, cell lysates were re-immunoprecipitated with anti-HA agarose beads (2nd IP), followed by immunoblotting as indicated. i WT MEFs were co-transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting as indicated. j Gli3FL and Gli3R protein levels in SuFu^−/− MEFs before and after expression of SuFu WT or SuFuK321/457R. k Gli3 half-life in SuFu^−/− MEFs after cycloheximide treatment. Gli3FL and Gli3R protein levels were analysed by immunoprecipitation from whole-cell lysates. The graph shows densitometric analysis. *P < 0.05, SuFu WT versus empty vector; **P < 0.05, SuFuK321/457R versus SuFu WT. l Subcellular fractions generated from WT MEFs transfected with Flag-SuFu WT or Flag-K321/457R. Lamin B and Tubulin were used as nuclear and cytoplasmic markers, respectively. m Gli3FL and Gli3R protein levels in WT MEFs transfected with siCTR or siItch. n Gli3 half-life in Itch^−/− MEFs transfected as indicated and then treated with cycloheximide for the indicated times. The graph shows densitometric analysis. *P < 0.05, Itch versus pcDNA; **P < 0.05, C830A versus Itch. o The graphs show the mRNA levels of the indicated Hh target genes in SuFu^−/− MEFs transfected with Gli3 alone or in combination with Flag-SuFu WT or Flag-K321/457R. *P < 0.05, Gli3+SuFu WT versus Gli3; **P < 0.05, Gli3+SuFuK321/457R versus Gli3+SuFu WT. *Non-specific band. Each experiment was performed three times independently. Error bars indicate SD. P-values were determined using Student’s t-test