Anti c ebpα

Western blotting (WB) was used to detect protein quantity. In brief, the sample was lysed in RIPA lysis solution to extract total protein and denatured. Next, the sample proteins were separated via SDS-PAGE gel electrophoresis under conditions involving a constant voltage of 100 V. In this study, the proteins were transferred from SDS-PAGE gel to PVDF membranes using a semi-dry transfer method. The membranes were then blocked for 4 h, and incubated with primary antibodies Anti-CEBPα (Abcam, 1:1000) Anti-PPARγ (Abcam, 1:1000) and FABP3 (Cell Signaling, 1:1500) overnight at 4 °C. Next, the membranes were washed thrice with TBST (Tris-buffered saline) and incubated with secondary antibody, rabbit anti-goat IgG antibody (Novogene, 1:5000) at room temperature for 1.5 h. Finally, a BeyoECL plus kit (Beyotime) was used to detect the protein signal. β-tubulin (Cell Signaling, 1:2000) was used as a reference protein and blots were analysed using IPWIN software.

Immunoblot analyses were performed as previously described [29 (link)]. For ABR protein detection, a mouse monoclonal antibody anti-ABR (Abcam), and for C/EBPα protein detection, a rabbit monoclonal antibody anti-C/EBPα (Abcam) was used. Polyclonal rabbit anti-GAPDH (sc-25778; Santa Cruz Biotechnology) and b-tubulin (sc-9104) antibodies were used for normalization. The immunoreactivity was determined using an enhanced chemiluminescence method (Amersham Biosciences) according to the manufacturer’s instructions. The band intensities were quantified using ImageJ software (National Institute of Health, Bethesda, MD).

ChIP was performed as previously described56 (link). When cellular confluence reached 80%, hADSCs were cross-linked with 1% formaldehyde (Sigma) at 37 °C for 15 min. The cells were then lysed, and DNA-protein complexes were immunoprecipitated. Next, the formaldehyde-cross-linked DNA was reverse cross-linked using a ChIP Assay Kit (Millipore) according to the manufacturer’s protocol. DNA-chromatin complexes were immunoprecipitated with anti-C/EBPα (1:300, Abcam) or mouse IgG (Millipore) as an internal control. The primers used for analysing the precipitated DNA are listed in Table 2.

Whole-cell lysates were prepared from cells collected in lysis buffer (50 mM Tris pH7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 1.0% SDS, 2 mM NaF, 2 mM Na₃VO₄, and protease inhibitors (Roche) followed by sonication and centrifugation at 13,000g for 10 minutes. Tumor lysates were prepared by homogenizing tumor tissue in lysis buffer and centrifuging at 13,000g for 10 minutes at 4°C. Proteins were quantified using a BCA assay kit, separated by electrophoresis on SDS PAGE gels, and transferred to PVDF membranes. After one hour in blocking buffer (SuperBlock), membranes were incubated overnight with primary antibodies. The following antibodies were used: anti-p-Akt (Cell Signaling), anti-Akt (Cell Signaling), anti-rpS6 (Abcam), anti-p-rpS6 (Abcam), anti-mTOR (Sigma-Aldrich), anti-p-mTOR (Sigma-Aldrich), anti-ERK (Abcam), anti-p-ERK (Abcam), anti-LC3B-II (Abcam), anti-Atg7 (Santa Cruz Biotechnology), anti-Atg12 (Santa Cruz Biotechnology), anti-Beclin-1 (Abcam), anti-C/EBPα (Abcam), anti-PPARγ (Abcam), anti-FABP4 (Abcam), anti-FAS (Abcam), anti-Tubulin (Abcam), and anti-β-Actin (Santa Cruz Biotechnology). The anti-Tubulin and anti-β-Actin were used as controls. Specific protein bands were imaged using secondary antibody conjugated with horseradish peroxidase and chemiluminescent ECL reagents. Image J was used for quantification.

Western blot analyses were performed using a standard protocol as previously described54 (link). Confluent hADSCs were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, China) supplemented with 1 nM of PMSF (Invitrogen), after which the collected protein contents were measured using a BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA). Proteins were separated by 10% SDS-PAGE electrophoresis and electro-blotted onto PVDF membranes (Millipore). The membranes were then incubated with optimal concentrations of the following primary antibodies: anti-Runx2 (1:1500, Abcam, Cambridge, MA, USA), anti-Ocn (1:1000, Abcam), anti-BSP (1:1000, Abcam), anti-GSK3β (1:1000, Abcam), anti-β-catenin (1:2000, Abcam), anti-C/EBPα (1:1000, Abcam) and anti-β-actin (1:3000, Abcam). Immunoreactive bands were detected using anti-rabbit (1:5000) or anti-mouse (1:5000) fluorescein-conjugated secondary antibodies (Abcam) and visualized by Odyssey V3.0 image scanning. All of the procedures were performed three times.

Protein extracts were run on 10–15% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. After blocking with 5% skim milk, the membranes were probed with primary antibodies, afterwards incubated with horseradish peroxidase-conjugated secondary antibodies. Finally, the bands were visualized using a chemiluminescence reagent (Proteintech, Wuhan, China). The primary antibodies included antibodies from Cell Signaling Technology (Danvers, MA, USA): anti-Runx2 (#12,556), anti-phospho-LRP6 (Ser1490) (#2568), anti-non-phospho-β-catenin (#8814), anti-C/EBPα (#8178), and anti-PPARγ (#2443); antibodies from Abcam (Cambridge, MA, USA): anti-NDRG1 (ab124689), anti-osterix (ab94744), anti-LRP6 (ab134146), anti-transcription factor 7 like 2 (TCF7L2) (ab76151), and anti-alkaline phosphatase (ALP) (ab108337); antibodies from Proteintech (Wuhan, China): anti-fatty acid binding protein 4 (FABP4) (12802-1-AP), anti-osteopontin (25715-1-AP), anti-β-catenin (51067-2-AP), and anti-β-actin (66009-1-Ig).