Ab219412

The RIPA lysis buffer containing protease inhibitors (Beyotime Biotechnology, Shanghai, China) was utilized to extract proteins from mandible tissues and cells, and the BCA kit (San Jose, USA) was employed to examine the content of isolated proteins. After SDS-polyacrylamide gel electrophoresis, the proteins were transferred to cellulose nitrate membranes, which were then blocked with 5% skim milk for one hour and incubated with the antibodies of BMP2 (1:1000, ab214821, Abcam, MA, USA), Runx2 (1:1000, ab236639), OPN (1:1000, ab8448), OCN (1:1000, ab133612), CEBPA (1:1000, ab40761), CEBPB (1:1000, ab32358), CEBPD (1:1000, ab245414), FABP4 (1:1000, ab92501), PPARG (1:1000, ab178860), ALP (1:1000, ab229126), APN (1:1000, ab181281), Wnt (1:1000, ab219412), β-catenin (1:1000, ab32572), and β-actin (1:1000, ab8227) overnight at 4° C. After washing, the membranes were incubated with peroxidase-bound secondary antibodies for one hour at room temperature. Finally, the bands were developed with an ECL kit (Amersham Pharmacia Biotech, Little Chalfont, UK).

Tumor tissues from ovarian cancer patients were fixed by formalin and embedded with paraffin. Sections from paraffin-embedded specimens were dewaxed, dehydrated, and subjected to antigen retrieval. After blocking endogenous peroxidases, the sections were incubated with primary antibody overnight at 4°C. The antibodies were as follows: KLF4 (#ab215036, rabbit monoclonal, Abcam, 1 : 2000), PAX6 (#ab195045, rabbit monoclonal, Abcam, 1 : 500), SMAD3 (#ab40854, rabbit monoclonal, Abcam, 1 : 500), and WNT3A (#ab219412, rabbit monoclonal, Abcam, 1 : 500). Detailed information of the antibodies is listed in Supplementary Table S4. Wash with 1x phosphate-buffered saline (1xPBS), followed by incubation in horseradish peroxidase- (HRP-) conjugated secondary antibody at 37°C for 1 hour and detection using diaminobenzidine (DAB). At least 10 random images from stained sections were captured by a light microscope. The intensity of DAB staining and the percentage of DAB positive cells were analyzed using IHC Profiler in ImageJ [35 (link)]. Quantification of KLF4, PAX6, SAMD3, and WNT3A for each sample was determined by H-score ([1 × (%of weak staining) + 2 × (%of moderate staining) + 3 × (%of strong staining)]) [36 (link)].

Antibodies against ITCH (#ab220637), β-catenin (#ab16051), Wnt3a (#ab219412), β-actin (#ab179467), phospho-Dvl (#ab124933) and Dvl (#ab233003) were purchased from Abcam (Cambridge, UK). HRP-conjugated goat anti-rabbit IgG (#sc-2004) was purchased from Santa Cruz Biotechnology. Gastric cancer cells and gastric cancer tissues were lysed using RIPA lysis buffer (Beyotime, China) following the manufacturer’s protocols. Subsequently, the proteins in samples were separated via 10% SDS polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. The membranes containing total proteins were blocked in 5% non-fat milk in TBST for 1 h and then incubated with primary antibodies overnight. Then, we washed the membranes with TBST 3 times and incubated them with HRP-conjugated secondary antibodies for 1 h. Finally, the protein bands were detected using enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA).

The spinal cord tissues were minced firstly, and homogenized using lysis buffer. After centrifugation (30 min at 4° C), the supernatant samples (30 μg) were subjected to SDS-PAGE, and transferred to a PVDF membrane (Sigma, USA). After blocking with TBST containing 10% non-fat milk for 1 h, the membrane was incubated with primary antibodies at 4° C for 12 h. The primary antibodies information was listed as follows. Wnt3a (1:1000, ab219412, Abcam, UK), Wnt2b (1:1000, ab178418, Abcam, UK), Wnt7a (1:1000, ab274321, Wnt5a (1:1000, ab179824, Abcam, UK), Abcam, UK), phospho-GSK-3β (1:1000, ab75814, Abcam, UK), β-catenin (1:1000, ab32572, Abcam, UK). The membrane was then incubated with secondary antibodies (Goat polyclonal Secondary Antibody to Rabbit IgG, 1:2000, ab150077, Abcam, UK) at 4° C for 2 h. Then, proteins were detected using an enhanced chemiluminescence detection kit (Sigma, USA), and protein bands was analyzed with ImageJ software.
To measure the protein expression of β-catenin in the nuclear, Nuclear Complex Co-IP Kit (Active Motif, Carlsbad, CA, USA) was used to exact nuclear firstly according to the instruction.

The drilled tooth was prepared with RIPA cell lysis buffer, the supernatants were collected, and the total protein by the BCA kit (Thermo Scientific, Rockford, IL, United States of America) was measured. It was added 20 μg total protein per line to separate by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Then, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. PVDF membranes were blocked in TBS-T at room temperature for 2 h and washed three times. Next, they were incubated with primary antibodies at 4 °C overnight.
Primary antibodies included that BMP-2 (1:1,000, ab225898, Abcam), osteocalcin (1:2,000, ab76956, Abcam), osterix (1:1,000, ab209484, Abcam), and Runx2 (1:500, ab76956, Abcam), Wnt3a (1:1,000, ab219412, Abcam), p-GSK3β (1:1,000, #5558, CST), GSK3β (1:1,000, #12456, CST), p-β-catenin (1:1,000, #2009, CST), β-catenin (1:1,000, #8480, CST) and GAPDH (1:5,000; #5174, CST). Then, membranes were incubated with goat anti-mouse IgG (H+L) HRP (1:5,000, ab6789, Abcam) and goat anti-rabbit IgG (H+L) HRP (1:5,000, s0001, Affinity) secondary antibodies for 2 h at room temperature and washed three times. PVDF membranes were detected with ECL, and the protein bands were quantified by Scion Image 4.0 software (Scion Corporation, Frederick, MD, United States of America).

Total protein was isolated from brain samples and bEnd.3 cells was lysed with RIPA lysis buffer. The protein concentration was determined by the BCA kit. Next, the same amounts of proteins from each sample were electrophoresed on a 12% SDS-PAGE gels and then transferred to the PVDF membrane. The membranes were blocked with 5% nonfat skim milk for 2 h at room temperature and probed overnight at 4 °C with the primary antibody. HRP-labeled secondary antibody were used for detection on ChemiDoc XRS system (Bio-rad). The membranes were then incubated with HRP-conjugated secondary anti-rabbit antibody (1:2000, ab288151, Abcam) or mouse antibody (1:5000, ab97040, Abcam) for 1 h at room temperature, and then visualized with enhanced chemiluminescence (ECL) reagent on ChemiDoc XRS system (Bio-rad). β-actin was cited as the internal reference. Primary antibodies were as follows: occluding (1:1000, ab216327, Abcam), claudin-5(1:500, 35–2500, Invitrogen), ZO-1(1:1000, ab276131, Abcam), ARHGAP25 (1:1000, ab192020, Abcam), β-catenin (1:1000, 9562, Cell signaling), wnt3a (1:1000, ab219412, Abcam), β-actin (1:2000, ab210083, Abcam).