Fig. 6.

USP13 promotes HMGB1 secretion. A HEK293T cells were transfected with Flag-USP13 plasmid for 48 h. Culture supernatants were harvested and immunoblotted with anti-HMGB1 antibody. B HEK293T cells were overexpressed with USP13 wildtype (WT) or USP13C345A/M664/739E (Mut) plasmid for 48 h and treated with 50 μM H₂O₂ for the last 18 h. N = 3. C HEK293T cells were transfected with Flag-USP13 plasmid for 48 h and then treated with 10 μM Spautin-1 for the last 18 h. D HEK293T cells were pre-treated with 0.1, 1, 5 μM Spautin-1 for 1 h, and 50 μM H₂O₂ was treated for 18 h. E HEK293T cells were transfected with shRNA USP13 plasmid for 48 h and treated with 50 μM H₂O₂ for the last 18 h. F RAW 264.7 cells were pre-treated with 10 μM Spautin-1 for 1 h and then with 100 ng/mL LPS for 18 h. G Stably expressed HEK293/hTLR4-MD2-CD14 cells were transfected with shRNA USP13 plasmid for 48 h and treated with 100 ng/mL LPS for the last 18 h. H HEK293T cells were transfected with Flag-USP13 plasmid for 48 h and then treated with 80 nM Wortmannin for the last 24 h. All data represent one of two (A, G, and H) or three (B–D) similar independent experiments