Fig. 4.

SNX27 facilitates TNFα-induced receptor associated OTULIN. A TNFα stimulation potentiates the interaction of SNX27 and OTULIN. HeLa cells transfected with control vector or Flag-OTULIN virus were treated with TNFα and immunoprecipitated using Flag-resin. SNX27 antibody was used to detect the interaction. The relative enrichment of SNX27 that normalised to its corresponding input was quantified by ImageJ and labelled below each blot. The value of time 0 of OTULIN overexpression cells was set as 1. Immunoblotting was performed at least twice, and one representative figure was shown. B SNX27 facilitates TNFα-induced receptor complex associated OTULIN. HeLa cells were transfected with control vector, OTULIN and/or SNX27 viruses. Cells were treated with Flag-tagged TNFα for indicate time points before harvest. Immunoprecipitation and immunoblotting were performed as A. Asterisks indicate the amount of Flag-TNFα in control condition was much higher than in stimulation conditions because most Flag-TNFα was washed away in stimulation conditions. The relative enrichment of SNX27 and OTULIN that normalised to immunoprecipitated TNFR1 was quantified by ImageJ and labelled below each blot. The value of time 0 of SNX27 and OTULIN overexpressed cells was set as 1. C Depletion of SNX27 impairs TNFα-induced receptor complex associated OTULIN. HeLa parental and SNX27 knockout cells were treated with Flag-tagged TNFα for indicate time points. Immunoprecipitation and immunoblotting were performed as Fig. 4A. The relative enrichment of SNX27 and OTULIN that normalised to immunoprecipitated TNFR1 was quantified by ImageJ and labelled below each blot. The value of time 0 of each immunoprecipitated protein was set as 1. Asterisks were used as indicated in B