Anti dkk3

Western blot analysis was performed according to a previously described standard method51 (link) using anti-AXIN2 (1:500; Abcam, Cambridge, MA), anti-DKK3 (1:500; GeneTex, San Antonio, TX), anti-SFRP1 (1:500; Abcam) or anti-β-catenin (1:1,000; Cell Signaling, Danvers, MA) antibodies. Blotted membranes were stripped and re-blotted with anti-GAPDH rabbit monoclonal antibody (1:2000; Sigma, St. Louis, MO) as a loading control. Original data of immunoblotting are presented in Supplementary Fig. 8.

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.