Hpa006731

The following reagents were purchased from the indicated companies: MG-132 (474790; Calbiochem, Danvers, MA, USA) and Hoechst 33258 (H-3569; Molecular Probes, Eugene, OR, USA). The following antibodies were used in this study: anti-USP10 (A300-901A; Bethyl Laboratories, Montgomery, TX, USA; HPA006731; Sigma-Aldrich, St. Louis, MO, USA), anti-ubiquitin (sc-8017; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p62 (PM045; MBL, Nagoya, Japan, GP62-C; PROGEN, Heidelberg, Germany), anti-G3BP1 (611127; BD Transduction Laboratories, San Jose, CA), anti-G3BP2 (A302-040; Bethyl Laboratories), anti-PABP (ab21060; Abcam, Cambridge, GB), anti-HDAC6 (sc-11420; Santa Cruz Biotechnology), anti-FLAG (M2 Monoclonal Antibody; Sigma-Aldrich), anti-GFP (sc-9996; Santa Cruz Biotechnology), anti-lamin B1 (sc-374015; Santa Cruz Biotechnology), anti-α-synuclein (S5566; Sigma-Aldrich), anti-phosphorylated α-synuclein (015-25191; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), anti-β-actin (sc-47778; Santa Cruz Biotechnology), anti-HA (2367S; Cell Signaling, Beverly, MA, USA) and anti-α-tubulin (CP06 Oncogene Research Products, Boston, MA, USA).

The study using human samples was performed with the approval of the ethics committees of Niigata University (approval number: G2015-0686). Written informed consent for autopsy, collection of samples and subsequent use for research purposes was obtained from the next of kin of the deceased involved in this study. The autopsied brain tissues (temporal cortex) of AD patients (n = 3) and controls (n = 3) were used. Formalin-fixed, paraffin-embedded sections 4-μm-thick were immunostained using a rabbit polyclonal antibody against USP10 (HPA006731; Sigma-Aldrich; diluted 1:2,000, pretreated by heating) and a mouse monoclonal antibody against phosphorylated Tau (AT8; 90206; IGT; diluted 1:200). The primary antibody binding was detected by anti-immunoglobulin secondary antibodies conjugated with either Alexa488 or Alexa568 (respectively anti-rabbit or anti-mouse; Molecular Probes). Immunofluorescent imaging was observed by a confocal microscope (FV3000RS; Olympus, Tokyo, Japan).