Horseradish peroxidase conjugated secondary antibody

Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 4% skim milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature. Proteins were detected with primary antibodies anti-DMP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-RUNX2 (MBL, Woburn, MA, USA), anti-BMP2 (Abcam, Cambridge, UK), anti-OCN (Abcam), and anti-β-actin (Santa Cruz Biotechnology), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Enzo Life Sciences, Minneapolis, MN, USA). It was detected using an enhanced chemiluminescence detection system (GE Healthcare, Buckinghamshire, UK), according to the manufacturer's instructions. Signals were detected using a Fusion Solo X (Vilber, Paris, France). The protein expression levels were normalized to that of β-actin. All Western blot analyses were repeated three times under the same conditions.

Protein expression levels were evaluated by Western blot analysis, as previously described [28 (link)]. In brief, 5 × 10⁵ cells were incubated in 60 mm culture dishes (BD Falcon) with the indicated doses of ATO and VPA alone and in combination for 24 and 72 h. Cells were washed with PBS and then suspended in five volumes of lysis buffer (PRO-PREP^TM Protein Extraction Solution; Intron Biotechnology, Seongnam, Korea). Protein concentrations were determined using the Bradford (Bio-Rad, Hercules, CA, USA) method. Supernatant samples containing 30 µg total protein were resolved in 12.5% and 15% SDS-PAGE gels, transferred to Immobilon-P PVDF membranes (Millipore, Bedford, MA, USA) by electroblotting, and probed with anti-poly(ADP-ribose) polymerase (PARP), anti-cleaved PARP, anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9 (Cell Signaling Technology Inc., Danvers, MA, USA), and anti-GAPDH antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Enzo Life Sciences, Farmingdale, NY, USA). Blots were developed using an ECL kit (DoGen Bio Co., Seoul, Korea).

Cells were washed twice with ice-cold PBS, lysed with Triton lysis buffer containing phosphatase and protease inhibitors (1 mM PMSF, 1 mM EDTA, 0.2 mM Na₃VO₄, 0.5 mM NaF, and 1 µg/ml leupeptin) on ice for 30 min. Lysates were centrifuged at 13,000 rpm for 15 min at 4 °C, and supernatants were collected. Protein concentrations were measured with the Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). Lysates were boiled with sample buffer containing β-mercaptoethanol for 5 min, and equal amounts of protein were separated on 6–13% SDS-PAGE followed by transferred to nitrocellulose blotting membranes (GE Healthcare Life Sciences, Chicago, IL, USA). Blots were blocked with 5% skim milk in TBS containing Tween 20 (TBS-T) for 1 h and incubated with primary antibodies overnight at 4 °C. The following day, blots were hybridized with horseradish peroxidase-conjugated secondary antibodies (Enzo Life Sciences, Farmingdale, NY, USA). Between each step, the blots were washed with TBS-T three times for 10 min each. The protein bands were detected by ECL chemiluminescence kit (Biomax, Seoul, Korea).

Hep G2 cells (2 × 10⁶ cells/well in six-well plate) were harvested by centrifugation at 200× g for 3 min. Cells were then washed with Tris-buffered saline (TBS; 20 mM Tris, pH 7.5, 130 mM NaCl) containing protease inhibitor and phosphatase inhibitor cocktails and placed on ice shortly after. Subsequently, cells were lysed by the addition of RIPA buffer directly to the dish. The nuclear/cytosol fractionation kit (Bio Vision Technology Inc., New Minas, NS, Canada) was used to separate nuclear and cytoplasmic proteins according to the manufacturer’s protocol. After isolation, protein concentration of the samples was determined using a micro BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples (20 µg) were separated on a 12% reducing SDS-PAGE and were transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk and was sequentially incubated with anti-Nrf2, anti-HO-1, or anti-GAPDH antibodies at 4 °C overnight. All antibodies were purchased from Cell Signaling (Danvers, MA, USA) and were used at 1:1000 dilution. Immunoreactive bands were visualized by horseradish peroxidase-conjugated secondary antibodies (1:1000 dilution, Enzo Life Sciences, Farmingdale, NY, USA) followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Images were captured using a FluorChem E system (ProteinSimple, Santa Clara, CA, USA).

Pilocarpine hydrochloride, streptozotocin (STZ), and citric acid were procured from Sigma Chemical Company (St. Louis, MO, USA). The following proteins were used in this study: antibodies against anti-amylase (#4017, Cell Signaling Technology, Danvers, MA, USA), anti-NHE-1 (sc-28758, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-AQP-5 (sc-514022, Santa Cruz Biotechnology, CA, USA), anti-GRP78 (sc-376768, Santa Cruz Biotechnology, CA, USA), anti-CHOP (#2895, Cell Signaling Technology, Danvers, MA, USA), p-IRE1α (ab48187, Abcam, Cambridge, MA, USA), IRE-1α (#3294, Cell Signaling Technology, Danvers, MA, USA), anti-p-eIF2α (#9721, cell signaling, Danvers, MA, USA), anti-eIF2α (sc-133132, Santa Cruz Biotechnology, CA, USA), and anti-β-actin (sc-130300, Santa Cruz Biotechnology, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, USA).

THP-1 were seeded in 6-well plates (1 × 10⁶ cells/well) and differentiated into M0 as described above. Following that, M0 cells were pre-treated with ‘Capriccio’, ‘Performance’, and ‘Leonida’ (50 and 100 µg/mL) for 1 h. The medium was changed and Free Radical Initiator in PBS1X were added for 1 h. After incubation, the cells were lysed with RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate). Protein concentration was determined by a Bradford solution (Applichem, Darmstadt, Germany) using BSA as a standard. 30 µg of proteins were loaded on 12%-SDS-PAGE, transferred into nitrocellulose membranes, and immunoblotted with the SOD1 primary antibody (Enzo Life Sciences, Farmingdale, NY, USA) and with horseradish peroxidase-conjugated secondary antibody. Signals were visualized by enhanced chemiluminescence (Amersham Biosciences-GE Healthcare, New York, NY, USA). Densitometric analysis was carried out using the ImageJ software Java8.