Pparγ 16643 1 ap

PPARγ (16643‐1‐AP), Tubulin (11224‐1‐AP), PD‐L1 (66248–1), ATG7 (10088‐2‐AP), LC3B (14600‐1‐AP), GST (66001‐2), Lamp1(21997‐1‐AP), and Flag (66008‐4) antibodies were obtained from Proteintech. Secondary antibodies were obtained from Jackson Immunoresearch. Cells were lysed in lysis buffer (50 mM Tris–HCl pH 7.4, 250 mM NaCl, 0.5% Triton X100, 10% glycerol, 1 mM DTT, protease inhibitor cocktail). Protein concentration in the supernatant was determined by the Pierce BCA Protein Assay Kit (Thermo). For immunoprecipitation, cell lysates were precleared with protein A/G magnetic bead (Cat: B23202; Bimake). Precleared lysates were subjected to immunoprecipitation using the indicated antibodies with protein A/G magnetic beads as described previously.⁴⁹, ⁵⁰ The samples were subjected to SDS‐PAGE and immunoblotted with indicated antibodies. Data are triplicates from three independent experiments. Blots were quantified by Image J.

RAW264.7 cells were purchased from Cell Resource Center, Peking Union Medical College (Beijing, China). Human adipose-derived stem cells (hADSCs) were donated from School of Basic Medicine Peking Union Medical College. RANKL (462-TEC-010) was were supplied by R&D Systems (Minneapolis, MN, USA). β-glycerophosphate disodium salt hydrate (G9422), l-ascorbic acid (A4544), dexamethasone (D4902), 3-isobutyl-1-methylxanthine (I5879) were bought from Sigma Aldrich Inc. (Merck KGaA, Germany). PF was bought from Nanjing Dilger Medical Technology (CAS: 23180-57-6; purity >98 %). Enzyme-linked immunosorbent assay (ELISA) kits for P1NP (QS47784), CTX-1 (QS47767), BALP (QS440290), and TRAP (QS440351) were obtained from Beijing gersion Bio-Technology Co., Ltd. HDL-C (A112-1-1) and LDL-C (A113-1-1) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). NFATc1 (66963-1-Ig), cathepsin K (11239-1-AP), C/EBPα (18311-1-AP), and PPARγ (16643-1-AP) antibodies were purchased from Proteintech Group (Hubei China). MEDAG (orb326838) antibody was purchased from biorbyt (UK). Phospho (p)-AMPKα (Thr172) (2535S) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Runx2 (ab236639) and OPG (ab73400) antibodies were purchased from Abcam (Cambridge, UK).

Cell lysates were subjected to SDS-PAGE (10% separation gel) and transferred to a polyvinylidene difluoride (PVDF) membrane. Primary antibodies against the following proteins were used: PPARγ (16643-1-AP; Proteintech), TGFBI (10188-1-AP; Proteintech), and actin (60008-1-Ig; Proteintech). Horseradish peroxidase-conjugated secondary antibodies were used (ZSGB-BIO). Signals were detected using an ECL (enhanced chemiluminescence) kit (Millipore).