Anti dj 1

Cell lysates were obtained using Tris-Triton lysis buffer [10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, and 0.1% SDS] supplemented with a protease inhibitor cocktail (Roche, Schlieren, Switzerland). Lysates were subjected to Bradford assay for protein quantification and denatured by adding 2 × Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) supplemented with 5% 2-Mercaptoethanol. The primary antibodies used in this study were as follows: anti-DJ-1 (Santa Cruz Biotechnology; sc-55572, 1:100, USA), anti-FLAG (Sigma-Aldrich; F3165, 1:20000, USA), anti-Myc (Invitrogen, 46–0603, 1:5000, USA), anti-tubulin (Sigma-Aldrich; T5168, 1:5000), anti-ubiquitin (Cell Signaling Technology; 3936, 1:1000, USA), anti-TOM20 (Abcam; ab186735, 1:2000, UK), anti-HTRA2 (Abcam; ab32092, 1:500), and anti-HSP60 (Santa Cruz Biotechnology; sc-59567, 1:800) antibodies. For cycloheximide (CHX) chase experiments, CHX and MG132 were purchased from Sigma-Aldrich.

Nucleoproteins were extracted using a Nucleoprotein Extraction Kit (Sangon Biotech). Protein concentrations were determined according to the Bradford method using BCA assay reagent (Beyotime, Beijing, China). Samples (25 mg of protein) were loaded onto 8%–12% SDS-PAGE gels, and the proteins were then electrophoretically transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% BSA and then incubated overnight at 4°C with the following antibodies: anti-DJ-1 (1:400; Santa Cruz), anti-HIF-α (1:1,000; Abcam), anti-p-AKT (1:150; Cell Signaling), anti-PI3K-p110α (1:500; Cell Signaling), and anti-PCNA (1:500; Cell Signaling). After the membranes were washed, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling) at room temperature for 1 hour. The proteins were visualized using an enhanced chemiluminescence (ECL) Kit (Amersham Life Sciences, Arlington Heights, IL, USA) and exposed using a Chemiluminescence Imaging System (Fusion Solo S, Vilber, France).