Anti dkk1

Protein specimens were diluted in loading buffer and denatured at 95 °C. After electrophoresing on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), total protein (40 μg) was transferred onto the polyvinylidene difluoride membrane. Five percent skimmed milk was used to block the membrane at room temperature for 2 h. Primary antibodies including anti-β-catenin, anti-c-myc, anti-cyclin D1, anti-p62, anti-Beclin1, anti-DKK1, and anti-GAPDH, were purchased from Abcam (USA). The antibody against LC3I/II (#ABC929) was bought from Sigma-Aldrich (Saint-Louis, MO, USA). The membrane was incubated with the indicated antibodies overnight at 4^oC, followed by washing and incubation with corresponding secondary antibodies (Abcam) for 2 h. At length, protein samples were subjected to an enhanced chemiluminescence detection system (Bio-Rad lab, Hercules, CA, USA). This assay was performed more than three times.

RASFs were washed twice in cold PBS, and then lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology Jiangsu, China). The protein concentration of cell lysates was quantified by a BCA Kit (Beyotime Institute of Biotechnology Jiangsu, China), and 50 μg of each protein were separated by SDS-PAGE on 10% gels, and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membranes were blocked in 5% non-fat milk diluted with Tri Buffered Saline Tween-20 (TBST) at room temperature for 1 h and incubated overnight at 4 °C with primary antibody: anti-p21, anti-cyclin D1, anti-CDK4, anti-Bax (1:500; Cell Signaling Technology Inc., MA, USA); anti-DKK1, anti-PCNA, anti-MMP-2, anti-MMP-9 (1:1000; Abcam, USA). The membranes were then incubated with goat anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase secondary antibody (1:1000; Cell Signaling Technology Inc., MA, USA) for 2 h. The proteins were visualized using ECL-plus reagents (Amersham Biosciences Corp., USA). The density of the bands was measured using the Image J software (USA), and values were normalized to the densitometric values of α-tubulin (1:5000; Sigma, USA) in each sample.

Cells were harvested in cell lysis buffer (Cell Signaling Technology; Cat#: 9803) and heated for 5 min at 100 °C. Equal quantities of denatured protein samples were resolved on 10% SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Roche). After blocking with 5% non-fat dry milk in Tris-buffered saline/0.05% Tween 20 (TBST), the membrane was incubated with a specific primary antibody followed by a horseradish peroxidase-conjugated secondary antibody. Proteins were visualized using ECL reagents (Pierce). The antibodies used were as follows: anti-β-catenin, anti-DKK1, anti-NKD1 and anti-GSK3β (Abcam, Cambridge, MA, USA), and p-GSK3β (Ser9) (Cell Signaling Technology). The membranes were stripped and reprobed with an anti–α-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA) as the loading control.

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).

The cSCC cells or frozen tumour tissues were lysed by RIPA lysis buffer. Proteins were extracted, measured and analysed by western blotting. Briefly, 40 μg per lane of protein was separated by 10% (w/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane. Then, 5% (w/v) bovine serum albumin (BSA) was used to block nonspecific binding in the membrane. After washing with Tris buffered saline with Tween buffer, membranes were incubated overnight at 4°C with primary antibodies. Primary antibodies included anti‐C3 (1:800; Abcam, Cambridge, MA, USA), anti‐cyclin D1 (1:800; Abcam), anti‐cyclin E1 (1:800; Abcam), anti‐VEGF (1:800; Abcam), anti‐pro‐MMP1 (1:800; Abcam), anti‐pro‐MMP2 (1:800; Abcam), anti‐Sox‐2 (1:800; Abcam) and anti‐DKK‐1 (1:800; Abcam). Horseradish‐peroxidase‐conjugated secondary antibody was then added, and cells were incubated for 2 hours at room temperature. Signals of target protein were visualized by incubating the membranes with chemiluminescence reagents and then quantifying with the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Β‐actin was used as the loading control for normalization.

Cells were harvested and lysed for total protein in RIPA lysis buffer (Thermo Scientific, EU, Lithuania) with protease inhibitors. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were then incubated with anti-DLX3, anti-CyclinD1, anti-C-myc, anti-Tcf-7, anti-DKK1, anti-β-actin, anti-GAPDH (all from Abcam, Cambridge, MA, United States), and anti-active-β-catenin antibodies (Millipore, Burlington, MA, United States) overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000) and chemiluminescence substrate were used to detect the immunoreactive bands with autoradiography film. Protein levels were quantitatively analyzed with Image J (National Institutes of Health, Bethesda, MD, United States) with β-actin as the loading control.