Bicinchoninic acid bca reagent

Huh7 and HepG2 cells were acquired from the American Type Culture Collection. The cells were maintained in DMEM containing 10% fetal bovine serum and 1% streptomycin/penicillin (all from Thermo Fisher Scientific, Inc.) at 37°C in a humidified 5% CO₂ incubator. Prednisolone, MTT and doxorubicin were obtained from Sigma-Aldrich (Merck KGaA). The Muse^® Annexin V Dead cell kit and cell cycle arrest package for flow cytometry analysis, and the PVDF membranes were purchased from EMD Millipore. For nick-end labeling, the DeadEnd™ Fluorometric TUNEL System was purchased from Promega Corporation. The human apoptosis proteome array kit was obtained from Bio-Rad Laboratories, Inc. The ECL detection reagent was bought from GE Healthcare. RIPA buffer was procured from Rockland Immunochemicals, Inc. Bicinchoninic acid (BCA) reagent was obtained from Thermo Fisher Scientific, Inc. The cytochrome c release kit (cat. no. ab65311) was from Abcam. Anti-GAPDH, anti-cleaved caspase 3, anti-cleaved caspase 9, anti-caspase 9, anti-cleaved poly-ADP ribose polymerase (PARP), anti-PARP, anti-caspase 3, anti-Bcl-2, anti-Bax, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse (cat. nos. 2118, 9664, 9505, 9502, 9541, 9532, 9662, 2870, 5023, 7074 and 7076, respectively) were obtained from Cell Signaling Technology, Inc.

The YFP-Parkin-C431S mutant was generated using PCR-based site-directed mutagenesis (forward primer: 5′-GTGGAAAAAAATGGAGGCAGCATGCACATGAAGTGTC-3′; reverse primer: 5′-GACACTTCATGTGCATGCTGCCTCCATTTTTTTCCAC). HEK239T cells were transiently transfected with YFP-Parkin or YFP-Parkin-C431S or YFP-Parkin-SA3/C431S by using Lipofectamine 2000 (Invitrogen) according to the manufacture’s protocol. Twenty-four hours after transfection, the indicated drug treatments were given for 2 hours with a media change. Cells were lysed on ice for 30 min in 50 mM tris (pH 7.5), 150 mM NaCl, 0.5% NP-40, and 10% glycerol with complete protease inhibitors (Roche). Lysates were clarified for 10 min at 16,000g centrifugation at 4°C, and the supernatant protein was quantified by the bicinchoninic acid (BCA) reagent (Thermo Fisher Scientific). To remove oxyester-linked Parkin, a portion of each lysate was treated with 100 mM NaOH at 37°C for 1 hour. Lysates were mixed with SDS sample buffer and boiled for 10 min before SDS-PAGE analysis. For optimal cellular signaling detection, duplicated plates were lysed in the CST lysis (Cell Signaling Technology) buffer with phosphatase and protease inhibitors.