Sc 517576

Anti-FLAG tag antibody (3165) was purchased from Sigma-Aldrich. Antibodies against green fluorescent protein (GFP) (sc-9996), Sox9 (sc-20095), Sox9 (sc-166505), Tankyrase-1/2 (sc-8337), Tankyrase-1/2 (sc-365897), Ubiquitin (sc-8017), Actin (sc-1615), α-Tubulin (sc-23948), and Histone H3 (sc-517576) were obtained from Santa Cruz Biotechnology. Sox9 (sc-20095) antibody was used only in Fig. 3e, n and Sox9 (sc-166505) antibody was used only in Fig. 3f. Tankyrase-1/2 (sc-365897) antibody was used only in Fig. 6a, b, 7k and Supplementary Fig. 4. Antibodies against aggrecan (AB1031), type II collagen (MAB8887), and human mitochondria (MAB1273) were purchased from Millipore, and antibodies against Myc tag (2276) and Sox9 (82630) were purchased from Cell Signaling Technology. Prior to detection of aggrecan, samples were treated with chondroitinase ABC (C3667) from Sigma-Aldrich. Antibodies against HA tag (ab9110) and MMP13 (ab51072) were purchased from Abcam. Anti-β-catenin antibody (610154) was obtained from BD Biosciences. Anti-Poly(ADP-ribose) antibody (AG-20T-0001) was purchased from AdipoGen and was used only in Fig. 3l. Anti-Poly(ADP-ribose) antibody (4335-AMC) was purchased from Trevigen. All antibodies were used according to the manufacturer’s protocol.

Proteins were extracted with RIPA lysis buffer (Thermo Fisher Scientific, Inc., Waltham, MA). Anti-p113 polyclonal antibody was prepared by immunizing rabbits with synthesized peptide corresponding to C-terminus of p113 (EQQLSAKNSTLKGRRD; ABclonal Biotechnology Co., Ltd., Wuhan, China). Western blot analysis was performed as previously described [14 (link)–16 (link)], with antibodies specific for CUX1 (ab230844), transcription factor 3 (TCF3, ab69999), ALDH3A1 (ab186726), NDUFA1 (ab249923), NDUFAF5 (ab240971), β-actin (ab179467, Abcam Inc., Cambridge, MA), CUX1 (sc-514,008), histone H3 (sc-517,576), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sc-47724), glutathione S-transferase (GST, sc-33614, Santa Cruz Biotechnology, Santa Cruz, CA), Flag-tag (14793S), ZRF1 (12844S), BRD4 (13440S), hemagglutinin (HA)-tag (3724S), or His-tag (12698S, Cell Signaling Technology Inc., Danvers, MA).

Proteins from tissues and cells were extracted with a RIPA buffer (89900, Thermo Fisher Scientific) containing a protease and phosphatase Inhibitor Cocktail (GenDEPOT, Baker, TX, USA). The protein lysates (20–40 µg) were separated by 12–15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Merck Millipore, Darmstadt, Germany). Membranes were blocked in 5% skimmed milk with a Tris-buffered saline containing 0.1% Tween 20 for 1 h and probed with primary antibodies overnight at 4 °C as follows: α-SMA (0.341 µg/mL, ab7817, Abcam), Histone 3 (1:1000, sc-517576, Santa Cruz Biotechnology) and Acetylated Histone 3 (1:1000, sc-56616, Santa Cruz Biotechnology). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000, 1706515, 1706516, Bio-Rad, Hercules, CA, USA) for 1 h. The signal was developed with ECL Western Blotting Substrates (Bio-Rad). The quantification was performed using ImageJ and normalized against β-actin. Meanwhile, acetylation of Histone 3 was normalized against Histone 3. The images of the whole membrane were presented in Figures S2–S4.