Fig. 3

SNX27 inhibits LUBAC-mediated linear polyubiquitination. A SNX27 inhibits LUBAC-mediated linear polyubiquitin chain formation. HEK293T cells transfected with indicated plasmids were lysed and blotted with indicated antibodies. Linear polyubiquitin chain formation was detected using Flag antibody. INT-Flag-KO represent a lysine-deficient ubiquitin plasmid with an internal Flag tag [26]. Immunoblotting was performed at least twice, and one representative figure was shown. B Knockdown of SNX27 potentiates LUBAC mediated linear polyubiquitin chain formation. HEK293T cells stably expressing inducible shRNA for SNX27 were transfected with indicated plasmids, doxycycline was added for 36 h to induce knockdown of SNX27 after transfection. Immunoblotting was performed as A. C Knockout of SNX27 potentiates TNFα induced linear ubiquitination of NEMO. Cells were treated with TNFα at indicated time points and lysed in denature buffer with 1% SDS. Diluted cell lysates (0.1% SDS) were used for immunoprecipitation assay with NEMO antibody. Linear ubiquitin chain antibody was used to detect the linear ubiquitination of NEMO. Immunoblotting was performed as A