Chemidoc xrs plus apparatus

J774.1 cells were cultured in 48-well culture plates at 5.0 × 10⁵ cells/well in 10% FBS-RPMI 1640 medium at 37 °C overnight. After that, the cells were treated with 2.5 mg/mL of OSA-PG or 200 ng/mL of LPS in 10% FBS-RPMI 1640 medium for 30 min at 37 °C. Total cell lysate was prepared as previously described [45 (link)]. Cytosolic and nuclear proteins were each isolated using a CelLytic NuCLEAR Extraction kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Protein concentration was quantified using a DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Heat-denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Hybond-P; GE Healthcare, Buckinghamshire, UK). Immunoblotting with various antibodies was performed as previously described [23 (link)]. Bands were analyzed using a ChemiDoc XRS Plus apparatus (Bio-Rad Laboratories), and the chemiluminescent intensity was quantified using the Quantity One software (Bio-Rad Laboratories).

RBL-2H3 cells were seeded into 35 mm dishes (Corning) at 4.0 × 10⁵ cells/well and sensitized with anti-DNP IgE (50 ng/mL) in DMEM containing 10% FBS at 37 °C overnight. After washing, the cells were treated with 17, 34, 67, or 135 µM DHEA or with 0.1% (v/v) ethanol (vehicle) for 30 min at 37 °C. The cells were then stimulated with DNP-HSA (50 ng/mL) and further incubated for 5 min. After removing the added reagents, cells were immediately lysed and immunoblotting was performed as previously reported^{39 (link)}. Blots were developed by ImmunoStar LD (Fujifilm Wako Pure Chemical). Bands were visualized using a ChemiDoc XRS Plus apparatus (Bio-Rad Laboratories, Hercules, CA, USA), and the chemiluminescent intensity was quantified using the Quantity One software (Bio-Rad Laboratories).

RAW 264.7 cells were seeded into a 6-well culture plate at 1.0 × 10⁶ cells/well and precultured at 37 °C for 16 h. After removing the medium, the cells were cultured in the medium containing 1.25 mg/mL of CLE at 37 °C for 24 h. After removing the medium, the cells were stimulated with 1.0 µg/mL of LPS in the medium for 15 min at 37 °C. Cytosolic and nuclear proteins were then prepared using a CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Immunoblot analysis was conducted as previously described [41 (link)]. Bands were visualized using a ChemiDoc XRS Plus apparatus (Bio-Rad Laboratories). The chemiluminescent intensity was quantified using the Quantity One software (Bio-Rad Laboratories).