Bs 4590r

Total protein from adipocytes was extracted using a commercial protein extraction kit containing lysate buffers, phosphatase inhibitors, and protease inhibitors (C510003; Sangon Biotech Co. Ltd.) according to the manufacturer's instructions. Nuclear and cytoplasmic protein were extracted from adipocytes using a commercial kit containing nuclear and cytoplasmic extraction buffers (P1200; Applygen Technologies Inc.) according to the manufacturer's instructions. Protein concentrations were quantified using the bicinchoninic acid Protein Assay Kit (P1511, Applygen Technologies Inc.). Twenty micrograms of protein from each sample was separated by 12 or 15% SDS-PAGE and electrophoretically transferred onto 0.45-μm polyvinylidene fluoride membranes blocked in Tris-buffered saline solution with 0.01% Tween-20 (TBS-T) containing 3% BSA for 4 h at room temperature. Subsequently, blocked membranes were incubated overnight at 4°C with primary antibodies against PPARγ (bs-4590R, Bioss Biotechnology Co. Ltd.; 1:1,000), histone H3 (4499, Cell Signaling Technology Inc.; 1:1,000), β-actin (ab8226, Abcam; 1:2,000), β-tubulin (10094-1-AP, Proteintech; 1:1,000). After being washed 3 times with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Boster Biological Technology Co. Ltd.) for 45 min at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence reagent (WBKLS0500, Millipore) via a protein imager (ProteinSimple). Last, all bands were quantified by calculating the integrated optical density from the area and optical density of each protein band using Image-Pro Plus 6.0 (Media Cybernetics Inc.) according to the manufacturer's instructions. Background subtraction was performed during quantification. Total protein abundance of PPARγ was standardized by β-actin, nuclear protein abundance of PPARγ was standardized by histone H3, and cytoplasmic protein abundance of PPARγ was standardized by β-tubulin. For the analysis of adiponectin multimers in the supernatant of adipocytes, 10-μL supernatant samples collected after centrifugation at 350 × g for 10 min at room temperature were subjected to 2 to 15% SDS-PAGE under nonreducing and non-heat-denaturing conditions as previously described (Waki et al., 2003) (link). Subsequently, Western blot analysis was performed as described above by using anti-C-terminal globular domain antibody against adiponectin (ab181699, Abcam; 1:1,000). Protein abundance of HMW adiponectin in supernatant was standardized by intracellular β-actin.