Ab41928

At least 3 × 10⁶ monocytes were lysed in radio immunoprecipitation assay (RIPA) buffer containing a proteinase and phosphatase inhibitor (Beyotime) for 30 min at 4°C. Lysates were centrifuged for 15 min at 14,000g at 4°C and the supernatant was discarded. Protein concentration in cell lysates were measured via the Bradford assay (HyClone-Pierce, USA). Proteins were separated by electrophoresis using 12% vertical dodecyl sulfate-polyacrylamide gel and transferred onto nitrocellulose membranes (Millipore, USA). The PVDF membranes was immersed in 5% skim milk for 1 h at room temperature and then immunoblotted with primary antibodies, icluding rabbit anti-human P50 (ab7971, 1:5,000, Abcam, USA) or mouse anti-human PPAR-γ antibody (ab41928, 1:1,000, Abcam, USA) for 12–16 h at 4°C, followed by incubation with the secondary Goat anti-mouse or anti-rabbit IgG antibody (H&L) (GenScript, USA). The band intensity was measured by an ECL Western blot detection kit (Thermo Scientific, USA). The expression level of PPAR-γ and P50 was quantified by densitometry with normalization to GAPDH.

ChIP analysis was conducted with a ChIP Assay Kit (Millipore) following the manufacturer’s instructions. Sorted monocytes were cross-linked in 1% formaldehyde for 10 min at room temperature, and then, we added glycine to stop the reaction. The fixed cells were washed with 20 ml ice-cold phosphate-buffered saline twice and then lysed. The cell lysates were centrifuged and resuspended before being sonicated to generate 500- to 1,000-bp fragments. After that, the indicated antibodies were added and incubated with the sheared DNA. The immune compounds were precipitated with protein A agarose beads, and then washed and eluted. The precipitated DNA was purified, and then, the target DNA was amplified by qPCR. The primers used are presented in Supplementary Table S3. Anti-PPAR-γ (Abcam, ab41928) and anti-SIRT1 antibodies (Abcam, ab12193) were of ChIP grade and were purchased from Abcam. An anti-histone H3ac (pan-acetyl) antibody (pAb) (cat. no. 39139) and an anti-histone H4ac (pan-acetyl) antibody (pAb) (cat. no. 39243) were purchased from Active Motif.

The left distal femur was embedded in olefins for subsequent histological and immunohistochemical analysis after completing the microstructure of bone investigation. Hematoxylin and eosin staining was used to examine the morphology of trabecular bone tissue. Additionally, osteocalcin staining (OCN) (ab41928; Abcam Biotechnology, MA, USA; the dilution ratio is 1:100) was utilized to assess the activities of osteoblast, and TRAP staining (Sigma-Aldrich, St. Louis, MO, USA) was used to assess the osteoclast activity. In semiquantitative immunohistochemistry analysis, we captured four distinct images at 400 × magnification using Image Pro Plus software (Media Cybernetics, MD, USA) for each slide. The number of positive cells was quantified by counting them on each bone surface (BS), and staining intensity was evaluated using the integrated optical density (IOD/area, mean density) of each positive cell area.

Adipose tissue of Nob3.14 mice were homogenized in TES buffer (20 mM TrisHCl, 1 mM EDTA, 8.7% sucrose, pH 7.4, supplemented with protease inhibitor cocktail). Proteins were separated by SDS-PAGE, transferred to a PVDF membrane (Immobilon-P Membrane, Merck Milipore, Darmstadt, Germany) and targeted proteins were detected by ECL Prime Detection Reagent (GE Healthcare Europe GmbH, Freiburg, Germany) using the FUSION-SL4 advanced chemiluminescence system (Peqlab Biotechnologie GmbH, Erlangen, Germany). Primary antibodies against PPARγ (ab41928, Abcam, Cambridge, UK), pHSL (#4139S, Cell Signaling, Beverly, MA, USA), tHSL (#4107S, Cell Signaling), β- ACTIN (A3854, Sigma-Aldrich, St. Louis, USA), and appropriate horseradish peroxidase-labeled secondary antibodies (Dianova, Hamburg, Germany) were applied.