Focus subcell kit

MEF cells were seeded in 100-mm dishes (8 × 10⁵ cells/dish) and incubated at 37°C in 5% CO₂. The next day, cells were treated with LG2055 (100 μg/mL) for 24 h. After treatment, the cells were washed with PBS, collected by trypsinization, and fractionated into cytosolic and nuclear fractions using the Focus SubCell kit (G Biosciences, St. Louis, MO). The isolated fractions were analyzed by SDS-PAGE and western blotting.

Ten wide-type mice from the vehicle and 0.7 g/kg/d ethanol groups were euthanized and exsanguinated at the end of 8 weeks of feeding. The brains were removed and cut into six 1.75 mm-thick coronal sections. Under the microscope, the cortical tissues were collected from the parietal and temporal lobes. The nuclear fraction from the cortical tissues was isolated using FOCUS SubCell Kit (G-Biosciences, St. Louis, MO, USA) following the manufacturer’s instructions. The protein concentration was measured using a BCA assay (Thermo Scientific, Plainville, MA, USA). Nuclear PPARγ protein expression was measured by Western blot analysis as described below. Nuclear PPARγ DNA-binding activity was determined by a PPARγ transcription factor assay kit (Cayman, Ann Arbor, MI, USA) following the manufacturer’s protocol. In brief, 120 µg nuclear proteins were incubated with a biotin-labeled DNA probe containing a PPAR-specific double-stranded consensus sequence and a single-stranded capture region. The samples were digested with a double-stranded DNA-specific nuclease and subsequently transferred to a 96-well plate coated with single-stranded DNA complementary to the capture region. A chemiluminescent alkaline phosphatase substrate was added, and the output signal was measured using FLUOstar Omega microplate reader (BMG LABTECH, Cary, NC, USA).

The brains were collected and processed as described previously^{17 (link)}. Eighteen mice (n = 6 for each group) were euthanized and exsanguinated. Cortical tissues were isolated from peri-infarct area and contralateral corresponding area. The mitochondria, cytosolic and nuclear fractions from the cortical tissues were isolated using FOCUS SubCell Kit (G-Biosciences) following the manufacturer’s instructions. The protein concentration was measured using a BCA assay (Thermo Scientific). The rest eighteen mice (n = 6 for each group) were anesthetized and perfused transcardially with 1X phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M PBS. The brains were removed, fixed overnight in 4% paraformaldehyde in 0.1 M PBS, dehydrated in a graded series of sugar solutions over the course of 72 hours, then embedded in O.C.T. compound (Fisher Scientific) and quick frozen for 5 minutes in liquid nitrogen. The frozen brains were then cut into 14 μm coronal sections and placed on frost-free slides.

Mitochondrial- and cytosolic-enriched fractions were isolated from fresh spinal cord tissue using the Focus SubCell kit (G-Biosciences, St. Louis, MO) according to the manufacturer’s protocol. In brief, 100 mg of fresh spinal cord tissue was homogenized and centrifuged at 700 × g for 10 min to pellet nuclei. The supernatant was removed and centrifuged at 12,000 × g for 15 min to pellet mitochondria. The supernatant was removed from the mitochondrial pellet for a cytosolic fraction. The protein content of all samples was assessed by the Bradford method (Bio-Rad Protein Assay, Hercules, CA) and normalized prior to adding sample buffer.