Protein assay reagent kit

For total cell lysis, the cells were washed twice with ice‐cold PBS and dissolved in Nonidet P‐40 (NP‐40) buffer containing 50 mmol/L Tris‐HCl (pH 7.5), 150 mmol/L NaCl, 0.5% NP‐40, and protease and phosphatase inhibitors (Nacalai Tesque). The protein concentration of each lysate was determined using a protein assay reagent kit (BioRad, Hercules, CA, USA). After sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked for 20 min in blocking buffer (Nacalai Tesque) and probed overnight with primary antibodies for UGT2B (H‐300; detection of UGT2B family members and UGT2A1), KLK3, AR (all from Santa‐Cruz, Biotechnology, Santa Cruz, CA, USA), ACSL3, AKR1C3, FLAG and β‐actin (all from Sigma). After extensive washing, bound antibodies were visualized using HRP‐conjugated secondary antibodies and the signal was enhanced by chemiluminescence (ECL; GE Healthcare, Tokyo, Japan).

Following the removal of 22 high-abundance proteins, including albumin and IgG, using ProteoMiner low abundance protein enrichment kits (Bio-Rad Laboratories, Inc., Hercules, CA, USA), protein quantification was conducted using a Protein Assay reagent kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) based on Bradford methods, according to manufacturer's protocol. iTRAQ labeling was performed according to the manufacturer's protocol (Applied Biosystems Life Technologies, Foster City, CA, USA). Briefly, 100 µg CSF proteins from the ALS and NC groups were precipitated with cold acetone (ratio of acetone:sample, 5:1) for 1 h at −20°C and resuspended in 20 µl dissolution buffer, respectively. Following centrifugation at 2,000 × g for 15 min and disposal of the supernatant, the precipitant was dissolved into 20 ul iTRAQ solution and 1 ul 1% sodium dodecyl sulfate (SDS). Subsequently, 1 ul cysteine sealing reagent was added for 10 min at room temperature. Proteins were trypsinized (Sigma-Aldrich, St. Louis, MO, USA) at 37°C overnight (ratio of enzyme:protein, 1:20). Peptides were labeled with iTRAQ regents for 1 h at room temperature. iTRAQ regents 113 and 118 were used to label the peptides from the NC and ALS groups, respectively. Following this, samples were mixed, desalted with Sep-Pak Vac C18 cartridges (Waters Corporation, Milford, MA, USA) and dried in a vacuum concentrator.

Cells were sequentially treated with esculetin and H₂O₂ as described above. Cells were harvested and lysed using a lysis buffer (120 mM NaCl, 40 mM Tris [pH 8], and 0.1% NP 40) on ice, and the protein concentration was detected using the Protein Assay Reagent Kit (Bio-Rad, Hercules, CA, USA). Aliquots of the protein solutions were electrophoresed on a 10% SDS PAGE and transferred onto nitrocellulose membranes. Subsequently, the membranes were shaken with primary and secondary antibodies (Invitrogen, Carlsbad, CA, USA). Protein bands were examined using an Enhanced Chemiluminescence Western Blotting Detection Kit (Amersham, Little Chalfont, UK). The primary MMP-1 (CSB-PA07009A0Rb) antibody was purchased from Cusabio Technology (Houston, TX, USA). Primary antibodies against SAPK/ERK kinase (SEK)1 (#9152), phospho-SEK1 (#9156), MAPK kinase (MEK)1 (#9124), phospho-MEK1 (#98195), c-Jun N-terminal kinase (JNK1/2) (#9252), phospho-JNK1/2 (#9251), c-Fos (#2250), and phospho-c-Jun (#9261) were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary extracellular signal-regulated kinase (ERK) 2 (sc-1647) and phospho-ERK1/2 (sc-7383) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary Actin (A2066) antibody was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) [17 (link)].