Anti sfrp1

For western blots, total cellular protein was extracted from cells and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Nuclear extracts were prepared using the Nuclear Extraction Kit (ActiveMotif), according to the manufacturer's instructions. The following antibodies were used: anti-β-catenin (Millipore, Billerica, MA), anti-LZTFL1 (Sigma, St. Louis, MO), anti-SFRP1 (Abcam), anti-DKK2 (GeneTex, San Antonio, TX), anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GAPDH rabbit monoclonal antibody (Sigma, St. Louis, MO).

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.

Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F-12) Media was obtained from Hyclone (Utah, USA). Recombinant human and mouse IL-1β were obtained from R&D Systems (Minneapolis, MN, USA). Penicillin, streptomycin and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY, USA). EPZ005687 was obtained from MedChemExpress _(1396772-26-1). The following antibodies were used in this study: anti-EZH2 from Abcam (Cell Signaling Technology); anti-SOX9 and anti-β-catenin from Cell Signaling Technology (Danvers, MA, USA); anti-H3K27me3 (Millipore, CA, US); normal rabbit IgG (Santa Cruz, Heidelberg, Germany); anti-SFRP-1 (Abcam, Cambridge, UK); Alexa-Fluor-488- and Alexa-Fluor-545- conjugated secondary antibodies from Molecular Probes (Eugene, OR, USA); and goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680 secondary antibodies from LI-COR Biosciences (Lincoln, NE, USA).

Western blot analysis was performed using anti-SFRP1 (1:2000, Abcam, Cambridge, MA, USA); anti-AXIN2 (1:1000, Abcam, Cambridge, MA, USA), anti-ICAT (1:1000, Abcam, Cambridge, MA, USA); anti-β-catenin (1:1000, Abcam, Cambridge, MA, USA). To control sample loading, the blotting membranes were stripped and re-probed with an anti–α-tubulin or anti-p84 antibody (1:1000, Sigma, Saint Louis, MO, USA). Nuclear extracts were prepared using the Nuclear Extraction Kit (Active Motif), according to the manufacturer's instructions. Briefly, cells were collected in the PBS/phosphatase inhibitors solution and lysed in the Lysis Buffer, and then centrifuged for 5 minutes. Suspending nuclear pellet in 50 ul Complete Lysis Buffer, then incubating for 30 minutes on ice. Vortex the mixture for 30 seconds followed by centrifuge for 10 minutes at 14,000 g at 4°C, the collected nuclear extraction were store at −80°C for further examination. The expression levels of SFRP1, AXIN2, ICAT and β-catenin were determined by densitometry. The ratio of first sample (SFRP1/α-tubulin, AXIN2/α-tubulin, ICAT/α-tubulin and β-catenin /α-tubulin) was considered as 1.0.

Cells were lysed in RIPA buffer (25 μM Tris-HCl (pH 7.6), 150 μM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1% proteinase inhibitors (Cat. No: P2850, Sigma) for total protein preparation. Nuclear protein extracts were obtained with Nuclear Extraction Kit (Active Motif) according to the manufacturer's instructions. Briefly, 40 μg protein samples were analyzed by 10% SDS-PAGE and the gels were transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% non-fat dried milk in TBST (10 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20) for 1 h at room temperature, and incubated overnight at 4°C with specific primary antibodies. Membranes were washed three times with TBST buffer, then incubated for 1 h with 1:2000 secondary antibodies, and developed with enhanced chemiluminescence (ECL, Millipore, USA). The primary antibodies included anti-NKD1 (1:1000, Abcam, USA), anti-SFRP1 (1:1000, Abcam, USA), anti-GSK3β antibody (1:1000, Abcam, USA), anti-TLE3 antibody (1:1000, Abcam, USA) and anti-β-catenin (1:1000, Abcam, USA) antibody. Anti-GDPAH (1:5000, Beyotime, China) and anti-p84 (1:1000, Abcam, USA) antibodies were used as the loading control.