Fig. 6: USP40 deubiquitinates HSP90β, resulting in HSP90β hyperacetylation and inactivation.

a Proteomic profiling of USP40interacting protein complexes in HLMVECs. b Schematic diagram showing the role of HSP90 in EC inflammation and hyperpermeability. c HLMVECs were treated with 17-AAG prior to stimulation with LPS (0.2 μg/ml) for 6 h. ICAM1 expression was analyzed by immunoblotting. Quantification of ICAM1 expression relative to β-actin expression was performed. d HLMVECs were treated with LPS (0.2 μg/ml) for 30 min, and cell lysates were subjected to IP with an anti-USP40 antibody or an anti-HSP90β antibody, followed by immunoblot analysis of USP40 or HSP90β. e HLMVECs were transfected with the USP40-V5 plasmid for 48 h. The cells were subjected to immunofluorescence staining with anti-V5 (green) and anti-HSP90β (red) antibodies. Scale bars = 50 µm. f HLMVECs were transfected with empty vector or the Flag-USP40 plasmid for 48 h. Denatured proteins in cell lysates were subjected to IP with an anti-HSP90β antibody followed by immunoblotting with anti-ubiquitin or anti-acetylated lysine (AcK) antibodies. g HLMVECs were transfected with empty vector or the USP40-V5 plasmid for 48 h, and denatured proteins in cell lysates were subjected to IP with an anti-HSP90β antibody followed by immunoblotting with an anti-K63-linked ubiquitin antibody. h HLMVECs were cotransfected with the HSP90β-HA plasmid and empty vector or the Flag-USP40 plasmid for 48 h. HSP90β-HA was immunoprecipitated, and its ATPase activity was measured (n = 4). i HLMVECs were transfected with empty vector or the Flag-USP40 plasmid for 48 h, followed by LPS treatment for 1 h. Cell lysates were subjected to IP with an antibody against HSP90β followed by immunoblotting with the indicated antibodies. j HLMVECs were treated with TNFα (10 ng/ml) for 30 min. Cell lysates were subjected to IP with an antibody against HSP90β followed by immunoblotting with the indicated antibodies.