Ab1906

Secreted proteins were concentrated using Amicon® Ultra-15 and Ultra-0.5 Centrifugal Filters (Millipore). Cells were lysed in RIPA buffer (Tris-HCl 50 mM pH = 7.4, NaCl 50 mM, sodium deoxycholate 0.5%, ethylenediaminetetraacetic acid (EDTA) 5 mM, Triton X-100 1%, protease inhibitor 1X). For Western blots, primary antibodies were against p.MT-CO1 (1:1000, 459600, Invitrogen™), SDHA (1:5000, 459200, Invitrogen™), Actin (1:2000, A2066, Sigma), APOE (1:1000, ab1906, Abcam), FN1 (1:400, ab2413, Abcam) and OXPHOS human WB antibody cocktail (Abcam, ab110411). Primary antibodies against TIM21, MRPL45, NDUFA9, p.MT-CO1, p.MT-CO2, COX4-1 and ATP5B were raised in rabbit. These antigen-antibody complexes were detected by horseradish peroxidase (HRP)-coupled secondary antibodies and enhanced chemiluminescence on X-ray films. For blue native-polyacrylamide gel electrophoresis (BN-PAGE), mitochondria were solubilized in buffer (1% digitonin, 20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% (w/v) glycerol, and 1 mM phenylmethylsulfonyl fluoride) to a final concentration of 2 mg/ml for 30 min at 4 °C. Lysates were cleared by centrifugation (20,000×g, 15 min, 4 °C) before addition of 10X loading dye (5% Coomassie brilliant blue G-250, 500 mM 6-aminohexanoic acid, and 100 mM Bis-Tris, pH 7.0) and separated on 4–14% polyacrylamide gradient gels as described [29] (link).

Cells were harvested at the specified times and lysed with RIPA buffer (Beyotime, Shanghai, China) containing 2% PICT and 1% PMSF. The BCA protein assay kit (KeyGen Biotechnologies, Nanjing, China) was used to determine protein concentration. Then, 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was used to separate the proteins. The membrane was blocked with 5% nonfat milk for 60 min at 37 °C and then incubated overnight with specific primary antibodies (anti-APOE, mouse monoclonal antibody, 1:1000 diluted, ab1906, Abcam, Cambridge, UK; anti-β-actin, 1:1000 diluted, Santa Cruz Biotechnology) at 4 °C, followed by horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rat IgG secondary antibodies. The bands were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to β-actin.