K48 linked polyubiquitin

The following antibodies were used for Western blots: γ-tubulin and DNP (2,4-dinitrophenylhydrazone) (Sigma); LC3 (Nanotools), phospho-p70S6 kinase (Thr389), phospho-ULK1 (Ser757), p70S6 kinase and ULK1 (Cell Signaling Technology); polyUbiquitin (Dako) and K48-linked polyUbiquitin (Cell Signaling); amyloid precursor protein (Boehringer); p62 and 8-oxodG (Abcam); Iba-1 (Wako); and GFAP, synaptophysin, goat anti-rabbit and anti-mouse IgG linked to horseradish peroxidase (DakoCytomation). The fluorogenic peptides Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVT-AMC) and 7-amino-4-methylcoumarin (AMC) were obtained from Calbiochem. Hexacosanoic acid (C26:0) and bafilomycin A1 were purchased from Sigma.

All reagents were purchased from commercial sources; LPS (InvivoGen, San Diego, CA, USA), SB203580 (Wako, Osaka, Japan), SP600125 (Wako), U0126 (Wako), 5z-7 (Santa Cruz, Dallas, TX, USA), ML120B (Sigma, St. Louis, MO, USA), TPL2 kinase inhibitor (Cayman Chemical, Ann Arbor, MI, USA), MG132 (Santa Cruz), poly(I:C) (InvivoGen), and Flagellin (InvivoGen). Antibodies against the following proteins were used in this study: p38, phospho-p38, JNK, phospho-JNK, ERK, phospho-ERK, p65, MKP1, MKP5, K48-linked polyubiquitin (Cell Signaling, Danvers, MA, USA), Myc-562 (MBL, Tokyo, Japan), FLAG-1E6 (Sigma), FLAG-M2 (Wako), HA (Roche, Basel, Switzerland), and PARP1, Myc-9E10, and β-actin (Santa Cruz). Mouse IgG, rat IgG, and rabbit IgG (Cell Signaling) were also used in this study. Anti-LINCR was produced by Cosmobio (Tokyo, Japan) using a part of the mouse-LINCR sequence (CDDQRSTARRRSTFHDGIV) as the antigen.