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. 2008 Mar 28;9(5):480–485. doi: 10.1038/embor.2008.37

Figure 3.

Figure 3

Synoviolin induces ubiquitination of IRE1. (A) Different combinations of IRE1, HA-Ub and SYVN1 expression plasmids were co-transfected into human embryonic kidney 293 cells. IRE1 proteins in the lysates of transfected cells were immunoprecipitated with an IRE1 antibody. The conjugation of ubiquitin onto IRE1 was detected with an HA antibody (top panel). The same membrane was reprobed with an IRE1 antibody (middle panel). The protein expression of SYVN1 in the whole-cell lysates was detected with a SYVN1 antibody (bottom panel). (B) Synovial fibroblasts from normal mice were transfected with SYVN1 short interfering RNA expression plasmids (lane 1) or control plasmids (lane 2). Two days after transfection, GFP+ cells were sorted, reseeded and then transfected with HA-Ub expression plasmid. IRE1 ubiquitination in those transfected cells was determined as described in (A) (top panel). The expression levels of IRE1 (second panel), SYVN1 (third panel) and GFP (bottom panel) in the whole-cell lysates were analysed by western blotting. (C) IRE1 and HA-Ub expression plasmids were co-transfected with SYVN1 or its C307A (CA) mutant. IRE1 ubiquitination in the transfected cells was determined as described in (A). (D) IRE1 expression plasmids were co-transfected with SYVN1 or SYVN1/CA mutant. SYVN1 proteins in the lysates of transfected cells were immunoprecipitated with a Flag antibody and the interacting IRE1 was detected with an IRE1 antibody (top panel). The same membrane was reprobed with a SYVN1 antibody (middle panel). The expression levels of IRE1 in the whole-cell lysates were used as controls (bottom panel). GFP, green fluorescent protein; HA, haemagglutinin; IRE1, inositol-requiring enzyme 1; SYVN1, synoviolin; WT, wild type.