Dnaja3

Reagents: 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid (CDDO) (Cayman Chemical) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich), cycloheximide (Sigma-Aldrich), oligomycin (Sigma-Aldrich), MKT-077 (Sigma-Aldrich). Antibodies for Western blot analysis: LONP1 (Proteintech, 1:3000), OXA1L (Proteintech 1:10000), mtHSP70 (Proteintech, 1:10000), GRPEL1 (Proteintech, 1:5000), TIM44 (Proteintech, 1:5000), CLPX (Proteintech, 1:3000), VDAC1 (Abcam, 1:3000), NDUFA9 (Abcam, 1:3000), DNAJA3 (Santa Cruz BioTech, 1:1000), HSP60 (Santa Cruz BioTech, 1:3000), TOM20 (Santa Cruz BioTech, 1:3000), anti-FLAG M2 (Sigma-Aldrich, 1:10000), anti-HA.11 (Covance, 1:5000).

Whole proteins were extracted and resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (PVDF) (Millipore). The membrane was blocked in 5% nonfat milk at room temperature for 1 h, followed by incubation with primary antibodies at 4 °C overnight. Primary antibodies were used to detect tau (1:1000, GeneTex, Irvine, CA, USA), HSPA8 (1:1000, Novus Biologicals, Centennial, CO, USA), HSPA1A (1:1000, Santa Cruz, Dallas, TX, USA), BiP (1:1000, Abcam), HSPA9 (1:1000, Thermo Fisher Scientific), DNAJB6 (1:1000, Abcam), β-actin (1:1000, Proteintech, Rosemont, IL, USA), V5 (1:1000, Thermo Fisher Scientific), α-synuclein (1:1000, GeneTex), and DNAJA3 (1:500, Santa Cruz, Dallas, TX, USA). Signals were developed using Luminata™ Crescendo Western HRP Substrate (Millipore). The image was quantified by ImageJ software.