Fig. 3

USP13 deubiquitinates HMGB1. A, B HEK293T cells were co-transfected with Myc-HMGB1, HA-Ub, and Flag-USP13 plasmids (A, N = 2) and with Myc-HMGB1, HA-Ub, Flag-USP13 wildtype (WT), ZnF or USP domain plasmids (B, N = 2) for 48 h. The cells were treated with 10 μM MG132 for the last 18 h before harvesting the cells. WCLs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. C HEK293T cells were co-transfected with indicated plasmids for 48 h and treated with MG132 for the last 18 h. Flag-USP13 WT and inactive USP13C345A/M664/739E (USP13 Mut) plasmids were used (N = 2). Data represents one of two similar independent experiments in A–C, respectively. D HEK293T cells were co-transfected with indicated plasmids for 48 h and treated with MG132 for the last 18 h. shUSP13 was used to knock down USP13 and scramble plasmid was used as a negative control. E The Myc-HMGB1 plasmid was overexpressed in HEK293T cells for 48 h, and the cells were treated with 10 μM Spautin-1 for the last 18 h; subsequently, IP and IB were performed. F HEK293T cells were co-transfected with the indicated plasmids for 48 h, and then treated with MG132 for the last 18 h, and IP and IB were performed to observe Ub-K48 modification. G HEK293T cells were co-transfected with Myc-HMGB1 and HA-Ub for 48 h and treated with MG132 for the last 18 h. WCL were immunoprecipitated with anti-Myc antibody and subjected to LC–MS/MS analyses