Ab131368

Western blotting was performed to measure the expression of Gli3, cleaved-caspase-3 and cleaved-PARP proteins. SiHa cells were homogenized in lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and the total protein was extracted by centrifuged at 16,000 × g at 4°C for 5 min. The proteins (20 µg/lane) were quantified according to the protocol of the BCA kit (cat. no. AR0146; Wuhan Boshide Biological Engineering Co. Ltd., Wuhan, China), separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. Membranes were subsequently incubated with primary antibodies against Gli3 (1:1,000, PA5-19822; Invitrogen, Thermo Fisher Scientific, Inc.), cleaved-caspase-3 (1:500; ab49822) cleaved-PARP (1:1,000; ab32064), cyclin B1 (1:500; ab72), cyclin D1 (1:10,000; ab134175; all Abcam, Cambridge, UK) and GAPDH (1:1,000 dilution; MA5-15738; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. GAPDH was the internal control. Membranes were then incubated with horseradish peroxidase-conjugated anti-mouse (1:1,000; ab131368) and anti-rabbit (1:1,000; ab191866; Abcam) antibodies at room temperature for 2 h. Finally, membranes were incubated with BeyoECL Plus (Beyotime Institute of Biotechnology) and the detected using Chemi Doc™ XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The total protein was abstracted by adding the corresponding pyrolysis solution (4°C for 30 minutes) (28–9425–44, ReadyPrep; GE Healthcare Life Sciences). After centrifugation at 876×g for 10 minutes, the supernatant was collected. Protein concentration was determined using bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins were processed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and later transferred onto nitrocellulose membrane as previously described [11 (link)]. After blocking with 5% nonfat milk (with Tween-20) at room temperature for 2 hours, the membrane were incubated with the primary antibodies against GAPDH (1:1000, TA-08, ZSbio, China), VEGF (1:1000, bs-1313R, Bioss), Sirt3 (1:1000, AF5135, Affinity), and LC3 (1:1000, ab48394, Abcam) overnight at 4°C. The secondary antibody (1:10 000, ab131368, Abcam, USA) was incubated with the membrane for 2 hours at room temperature. The expression of target proteins was normalized to GAPDH. At least 6 repeats were included in the western blotting.

Cell lysates were collected by 2% SDS lysis buffer and aliquots were used as input. Non-SDS lysis buffer was used to dilute to the rest of lysates. A total of 2 μg of anti-p53 (sc-126, Santa Cruz Biotechnology), anti-Ub (sc-8017, Santa Cruz Biotechnology) or normal IgG (sc-2025, Santa Cruz Biotechnology) was incubated with lysates. Dynabeads (ThermoFisher Scientific) were used for immunoprecipitation according to the manufacturer’s protocol. Immunoblotting was performed as above and anti-mouse IgG VeriBlot for IP secondary antibody (HRP) (ab131368, Abcam, Cambridge, UK) was used.

Total protein obtained from indicated HBMSCs was extracted using RIPA buffer. Subsequent to separation on SDS-PAGE, proteins were transferred to polyvinylidene fluoride membranes and then blocked in 5% skim milk. The membranes were subsequently incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies (ab131368, Abcam, Cambridge, MA, UK) for 1 h. After washing with Tris-buffered saline with Tween, the proteins were measured via enhanced chemiluminescence detection system. Primary antibodies used in this experiment were as follows: Anti-ETS1 (ab220361, Abcam), Anti-H3K9me2 (720092, Invitrogen), Anti-β-actin (K200058M, Solarbio, Beijing, China), Anti-KDM3A (ab243641, Abcam), Anti-β-catenin (ab32572, Abcam), Anti-RUNX2 (ab236639, Abcam), Anti-ALP (ab229126, Abcam), Anti-OSX (ab209484, Abcam), Anti-USP2 (ab66556, Abcam), Anti-GAPDH (ab8245, Abcam) and Anti-OCN (ab93876, Abcam).