Wnt5a

Protein concentration was determined using BCA Protein Assay Kit (Thermo, USA). For western blots, CDK3 antibody was obtained from Abcam (ab96847), p-LRP6, LRP6, Axin1, Dvl2, β-catenin, Wnt3a and Wnt5a antibodies were purchased from Cell Signaling (Wnt Signaling Antibody Sampler Kit #2915).

To detect the expression of target proteins, western blotting was performed. Total cell protein was extracted in cold RIPA lysis buffer (Keygen Biotech) after being cultured with or without Wnt5a shRNA/Ror2-siRNA/recombinant Wnt5a (R&D System)/IFN-γ (Beyotime Biotechnology)/AG490 (MCE). Protein concentration was measured using the BCA protein assay kit (Thermo Scientific). A total of 30 μg of protein was electrophoresed on SDS–PAGE and then electrotransferred to a PVDF membrane (Millipore). Nonspecific binding was blocked with PBST containing 5% nonfat dry milk, followed by incubation with primary antibody against Wnt5a (Cell Signaling Technology), β-catenin (Abcam), p16^INK4A (Abcam), JAK2 (Proteintech), p-JAK2 (Abcam), STAT3 (Proteintech), p-STAT3 (Abcam) and GAPDH (Proteintech) at 4 °C overnight. Immunoreactive bands were incubated with secondary antibody, before detecting it with ECL reagents (Keygen biotech). The gray value of each band was measured, data of which were presented as a ratio to GAPDH.

Proteins were obtained as previously described [42 (link)], and the following antibodies were used for Immunoblotting: β-catenin (Cell Signaling), active β-catenin (Millipore), Tubulin (Sigma), FLAG (M2; Sigma), Wnt2 (SantaCruz), Wnt3a (Cell Signaling), Wnt5a (Cell Signaling), pLRP6 (Cell signaling), LRP6 (Cell signaling), LRP5 (Cell signaling), Dvl2 (Cell signaling), Dvl3 (Cell signaling), Axin1 (Cell signaling) and GSK3β (BD Bioscience), EZH2 (Cell signaling), EED (Millipore), and SUZ12 (Abcam).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were done as previously described. Primary Antibodies used were CaMKII (pan) (#3362S), Dvl2 (#3216), LEF1(C12A5)(#2230), PJNK (Thr183/Tyr185) (#9251), TCF1 (C63D9) (#2203), TCF3 (D15G11) (#2883), TCF4 (C48H11) (#2569), TGFβ (56E4) (#3709), TJNK (#9252), Wnt5a (C27E8) (#2530) and αTubulin (#2144) (1 in 1000) (Cell Signal), Wnt5a (#55184-1-AP), Wnt3a (#21414-1-AP), Fzd2 (#24272-1-AP) (1 in 1000), sFRP4 (#15328-1-AP) (1 in 500) (Proteinech). GAPDH (ab9485), β-Actin(AC-15) (ab6276), β-Catenin (Total) (ab6302), Fzd8 (ab75235) (1 in 2000) (Abcam), p84 (5E10) (GT70220) 1 in 1000, (Genetex), β-Catenin(Active) (8E7) (#05–665) 1 in 500 (Millipore).

Whole-cell lysates were obtained from DPSCs cultured under control conditions or stimulated with ferutinin (10 μg/ml in αMEM) for 12, 24, and 48 h. Protein was quantified by colorimetric assay using the Bradford method (Bio-Rad, Hercules, CA). A polyacrylamide gel was cast and denatured proteins (20 µg each) were loaded and separated through the gel by electrophoresis and a protein ladder was loaded as a marker (Sigma, St. Louis, MO). The proteins were transferred from the gel to a 0.45 µm nitrocellulose membrane (Bio-Rad) at 4 °C. The membrane was blocked for 1 h at room temperature (RT) with a blocking buffer composed of 5% non-fat milk in TBS-Tween-20 (TBST) (Boston BioProducts, Ashland, MA). The membrane was washed and incubated with primary antibody against LRP6, Wnt5a, Dvl3, β-catenin, Runx2, GAPDH (Cell Signaling, Danvers, MA), osteocalcin, and osteonectin (Santa Cruz Biotechnology, Dallas, TX) (1:1000 diluted in a solution of 5% BSA in TBST) for 2 h. The membrane was washed, then incubated in secondary antibody (1:3000 in a solution of 5% milk in TBST) (Cell Signaling). The membrane was washed, placed in the cassette holder, and incubated briefly in the chemiluminescent substrate (Sigma). Films were then exposed and developed. Densitometric quantification of bands was performed using ImageJ software (NIH).

Western blot analysis was carried out as described previously [31 (link)]. Membranes were incubated with primary antibodies against WNT5A and RUNX2 (1:1000 dilution, Cell Signaling Technology); GAPDH (1:3000, Cell Signaling Technology); aggrecan, COL2A1, and MMP13 (1:1000, Abcam, Cambridge, MA, USA); and SOX9 (1:2000, EMD Millipore, Burlington, MA, USA). The blots were then incubated with appropriate secondary antibodies (1:3000 dilution, Cell Signaling Technology) at 4 °C overnight, after which they were developed with an ECL chemiluminescence Kit (EMD Millipore).