Fig. 5

SNX27 facilitates TNFα-induced membrane-localized OTULIN. A and B SNX27 facilitates TNFα-induced membrane associated OTULIN. Membrane proteins of HeLa cells with SNX27 overexpression (A) or knockout (B) with TNFα stimulation were isolated by cell fractionation kit. Immunoblotting experiments were performed to check the membrane associated OTULIN and TNFR1. ATP1A1 was served as a membrane marker and loading control. The relative protein expression level that normalised to loading control was calculated by ImageJ and labelled below each blot. The value of time 0 of control cells was set as 1. Immunoblotting was performed at least twice, and one representative figure was shown. C SNX27 facilitates TNFα-induced membrane localization of OTULIN. MEF cells infected with Myc-OTULIN and GFP-SNX27 lentivirus were treated with TNFα for indicated time points and fixed for immunostaining. Myc antibody was used to detect the localization of OTULIN. Immunostaining was performed as described in "Methods". Scale bar represents 20 μm. One representative experiment was shown. D SNX27 depletion inhibits TNFα-induced membrane localization of OTULIN. Wild-type and SNX27 KO MEF cells were infected with Myc-OTULIN lentivirus and immunostaining assay was performed as Fig. 5C. Scale bar represents 20 μm. One representative experiment was shown. E Inhibition of SNX27 trafficking decrease TNFα-induced receptor associated OTULIN. HeLa cells were treated with cholera toxin for 24 h and then Flag-TNFα was added at indicated time points before harvest. Immunoprecipitation and immunoblotting were performed to detect TNFα receptor associated OTULIN. The relative enrichment of SNX27and OTULIN that normalised to immunoprecipitated TNFR1 was calculated by ImageJ and labelled below each blot. The value of time 0 without cholera toxin treatment was set as 1. Asterisks were used as indicated in Fig. 4B