Non phospho β catenin

Protein was extracted from the cells using RIPA or IP buffer containing cocktails, resolved by SDS–polyacrylamide gels and then transferred to PVDF membranes. Primary antibodies against DKK4 (Abcepta, Cat# AP11649b, RRID: AB_10819953), β-catenin (Santa Cruz, Cat# sc-7963, RRID: AB_626807), nonphospho-β-catenin (Cell Signaling Technology, Cat# 4270, RRID: AB_1903918), phospho-β-catenin (Cell Signaling Technology, Cat# 9561, RRID: AB_331729), Met (Cell Signaling Technology, Cat# 8198, RRID: AB_10858224), Cyclin D1 (Cell Signaling Technology, Cat# 2978, RRID: AB_2259616), ubiquitin (Santa Cruz, Cat# sc-8017, RRID: AB_628423), LRP6 (Santa Cruz, Cat# sc-25317, RRID: AB_627894), and β-actin (Proteintech, Cat# 66009-1-lg, RRID: AB_2687938) were used. Peroxidase-conjugated secondary antibody (ZSGB-BIO, China) was used, and the antigen-antibody reaction was visualized by an automatic digital chemiluminescence imaging system (4600SF, Tanon, China). Band intensities were measured with ImageJ software. Statistical analysis was carried out with three technical replicates using each protein sample.

For western blotting, equal amounts of extracted protein from the cells were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (PVDF). The membranes were blocked with 5% degreased milk in Tris-buffered saline and 0.1% Tween-20 (TBST) and incubated overnight with the appropriate primary antibody against PARP, caspase-3, Snail, n-cadherin, zo-1, non-phospho-β-catenin (Cell Signaling Technology, MA, USA), vimentin, E-cadherin or β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The following day, membranes were incubated with appropriate secondary antibodies conjugated with HRP. The signals on the blots were detected using an enhanced chemiluminescence (ECL) solution.

Cells were lysed in RIPA cell lysis buffer (Beyotime, Shanghai, China) in the presence of protease inhibitor cocktail (Biotool, Huston, USA) and PMSF (Beyotime). Protein concentration was quantified by BCA protein assay kit (Beyotime). 50μg of the extracts were separated on 10% SDS-PAGE and transferred to PVDF membrane. Membrane was then blocked with 5% non-fat milk and incubated overnight with the following primary antibodies, respectively, which are Keratin10 (Abcam, Cambridge, USA), Involucrin (Proteintech, Wuhan, China), OCT4 (Proteintech), SOX2 (Proteintech), GSK-3β (Abcam), β-catenin (Proteintech), non-phospho β-catenin (Cell Signaling, Boston, USA) and LEF1 (Proteintech) followed by incubation with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h. Hybridization signal was detected by enhanced chemi-luminescence (ECL) (ThermoFisher, MA,USA) according to the manufacturer's instructions. GAPDH (ZSGB-BIO, Guangdong, China) was used as reference protein and determined following the same procedure as above.

The cells were lysed at 4°C in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 5 mM EDTA (pH 8.0), and 1 mM EGTA supplemented with protease and phosphatase inhibitors. After 20 min of lysis on ice, the cell debris was removed via microcentrifugation, followed by rapid freezing of the supernatants. The protein concentration was determined using the Bradford method. In our experiments, equivalent loads of 25–50 g of protein were electrophoresed using a SDS-polyacrylamide gel and then electrophoretically transferred from the gel to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated in specific primary antibodies (NIFK: Abcam, ab13880, 1:1000; Ki-67: Dako, Code M7240, 1:500; CK1α: Abcam, ab88079, 1:1000; non-phospho-β-catenin: Cell Signaling, #8814, 1:1000; phospho-β-catenin (Ser45): Cell Signaling, #9564, 1:1000; RUNX1: Cell Signaling, #4336, 1:1000) overnight at 4°C and subsequently incubated in a corresponding horseradish peroxidase-conjugated secondary antibody for 1 hr. The membranes were visualized using the ECL-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA).

N39 cells were seeded on poly-l-lysine–coated coverslips. After treatment with Wnt3a for 24 h, cells were washed with PBS, fixed with 4 % paraformaldehyde and permeabilized in 0.2 % Triton X-100, 0.1 M glycine. The cells were then washed with PBS, blocked with BSA containing antibody diluent (Invitrogen) at room temperature (RT) and incubated with antibodies against proinsulin (1:50; R&D Systems), non-phospho β-catenin (1:500; Cell Signaling), and NeuroD1 (1:200; Santa Cruz) at 4 °C. Coverslips were incubated with secondary antibodies for 2 h at RT, and Hoechst 33342 (Invitrogen) was used for nuclear staining for 10 min at RT. ProLong Diamond Antifade Mountant (Invitrogen) was used for mounting coverslips. Each slide was analyzed with an LSM700 confocal microscope (Carl Zeiss). Images were analyzed with ZEN2012 software (Carl Zeiss).