Fig. 3: OTUD3 was the DUB that maintained IRP2 stability.
From: Deubiquitylase OTUD3 prevents Parkinson’s disease through stabilizing iron regulatory protein 2
a The indicated OTU subfamily of DUBs was each transfected into HEK293T cells, and then the protein level of IRP2 were detected. b Increasing amount of OTUD3 WT or C76A were transfected into HEK293T cells and IRP2 expression was detected. c OTUD3 was depleted by siRNA in HEK293T cells, IRP2 levels were analyzed. d siRNA-resistant (si-res) OTUD3 WT or C76A was introduced into HEK293T cells together with OTUD3 siRNA. IRP2 levels were measured. e, f HEK293T cells transfected with the indicated plasmids or siRNA were treated with cycloheximide (10 µg/ml). Quantification of IRP2 levels relative to GAPDH was shown. Results were shown as mean ± s.e.m. n = 3 independent experiments, two-way ANOVA test. g HEK293T cell lysates were subject to immunoprecipitation with control IgG or anti-OTUD3 antibodies. The immunoprecipitates were then blotted. h The interaction of OTUD3 and IRP2 was assessed by GST pull-down assay. i Overview of OTUD3 and IRP2 structures. j Co-IP assays were performed to map the domain of IRP2 required for interaction with OTUD3. k Co-IP assays were performed to map the domain of IRP2 required for interaction with OTUD3-OTU. All panels are representative results of three or more independent experiments. The statistics data and uncropped western blots can be found in Supplemental Material.
