Anti axin2

Total protein was isolated with the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Protein concentrations were measured using a BCA protein kit (Thermo Fisher Scientific, Rockford, IL), and proteins were separated on SDS-polyacrylamide gels and transferred onto PVDF membranes (Immobilon-P; Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat dried milk in TBST (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature and then blotted overnight with primary antibodies at 4°C. The following antibodies were used as primary antibodies: anti-cleaved caspase-3 (1:1000 dilution; Cell Signaling Technology), anti-caspase-3 (1:1000 dilution; Abcam), anti-axin2 (1:1000 dilution; Abcam), and anti-tcf7l2 (1:1000 dilution; Abcam).

Postnatal and adult mouse femurs were dissected and fixed in 4% PFA/PBS for 2 days at 4 °C. Bone tissues were decalcified in 20%EDTA for 5days. Embryonic and neonatal tissues were fixed overnight in 4%PFA/PBS, but not decalcified. Fixed skeletal tissues were dehydrated through ethanol and embedded in paraffin; 8-μm serial sections were then prepared for histological analysis49 (link). Hematoxylin and Eosin (HE) staining and immunohistochemical analyses were carried out by standard procedures using the following antibodies (Ab)50 (link): anti-Cathepsin K (1:300, cat. no. ab19027, abcam)51 (link), anti-Osterix (1:1000, cat. no. ab22552, abcam)52 (link), anti-Axin2 (1:300, cat. no. ab32197, abcam)53 (link), anti-Osteocalcin (1:1000, cat. no. M173, Takara)54 (link), anti-Alkaline Phosphatase (1:500, cat. no. M190, Takara)55 (link). Bone-tissue cells with positive immunohistochemical signals representing equal areas of control and sFRP4 KO mice were counted and their average numbers were compared.

Immunohistochemistry assays were performed on the paraffin-embedded NSCLC tissue57 (link), using the following primary antibodies: anti-AXIN2 (1:100; Abcam), anti-DKK3 (1:100; GeneTex), anti-SFRP1 (1:100; Abcam) and anti-β-catenin (1:200; Cell Signaling). The degree of immunostaining of indicated proteins was evaluated and scored by two independent observers, who scored both the proportions of tumour cells that stained positively and the intensity of the staining. The proportion of positively stained tumour cells was graded as follows: 0 (no positive tumour cells); 1 (<10% positive tumour cells); 2 (10–50% positive tumour cells); and 3 (>50% positive tumour cells). The intensity of the staining was graded as follows: 0 (no staining); 1 (weak staining=light yellow); 2 (moderate staining=yellow brown); and 3 (strong staining=brown). The staining index (SI) was calculated as the product of the staining intensity × the percentage of positive tumour cells, resulting in scores of 0, 1, 2, 3, 4, 6 and 9. Cutoff values for high and low expression of the protein of interest were chosen based on a heterogeneity measurement using the log-rank test with respect to OS. An SI score ≥4 was considered high expression, and an SI score ≤3 was considered low expression.

Western blotting analysis was performed as described previously [23 ]. Primary antibodies used were as follows: anti-β-catenin (1:1000), anti-C-MYC (1:1000), anti-AXIN2 (1:1000), anti-DKK1 (1:1000), anti-Nanog (1:1000), anti-BMI1 (1:1000), anti-BAX (1:1000), anti-BCL2 (1:1000), and anti-GAPDH (1:5000) (all from Abcam).