Plrp6

Cells were lysed in RIPA buffer, proteins were separated on a 10% SDS-polyacrylamide gel by electrophoresis and transferred onto Nitran membrane (Sigma-Aldrich, USA). These were blocked with 5% non-fat dry milk and incubated with primary antibody (ZNF471 (1:3000; Sigma, USA), p-AKT (Ser473), total AKT, anti-CTNNB1, non-phospho-CTNNB1, CDH1, p-ELK-1(Ser383), total ELK-1, SNAI2, CCND1, c-MYC, DVL2, WNT3A, p-LRP6, total LRP6, β-actin (1:3000, Cell Signaling Technologies, USA), RAF1, (1:3000, Santa Cruz Technologies, USA) and SNAII, CDH2, VIM (1:3000, Cloud Clone, USA), Ki67, and Caspase 3 (1:3000, Novus Biological, USA)) overnight at 4°C and subsequently by secondary antibodies either anti-mouse IgG-HRP or anti-rabbit IgG-HRP (1:5000, Cell Signaling, USA). Proteins were visualized by SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, USA), and imaging was performed with Image Quant LAS 4000 (GE Healthcare, USA).

Protein concentration was determined using BCA Protein Assay Kit (Thermo, USA). For western blots, CDK3 antibody was obtained from Abcam (ab96847), p-LRP6, LRP6, Axin1, Dvl2, β-catenin, Wnt3a and Wnt5a antibodies were purchased from Cell Signaling (Wnt Signaling Antibody Sampler Kit #2915).

Proteins were obtained as previously described [42 (link)], and the following antibodies were used for Immunoblotting: β-catenin (Cell Signaling), active β-catenin (Millipore), Tubulin (Sigma), FLAG (M2; Sigma), Wnt2 (SantaCruz), Wnt3a (Cell Signaling), Wnt5a (Cell Signaling), pLRP6 (Cell signaling), LRP6 (Cell signaling), LRP5 (Cell signaling), Dvl2 (Cell signaling), Dvl3 (Cell signaling), Axin1 (Cell signaling) and GSK3β (BD Bioscience), EZH2 (Cell signaling), EED (Millipore), and SUZ12 (Abcam).

Paclitaxel (PTX) was purchased from Bristol-Myers Squibb, dissolved in Ethanol to make 7 μM stock concentration. JNK-IN-8 (Calbiochem, UK), with a working concentration of 10 µM was prepared in DMSO. MDR1/ABCB1, E-cadherin, N-cadherin, Vimentin, Snail, TCF8/ZEB1, β-catenin, Axin-1, Claudin, PI3K p110γ, p-PDK-1, Akt, p-Akt, p-c-Raf, Cyclin D1, p-GSK3β, p-p38 MAPK, p38 MAPK, p-p44/42, c-jun, p- SAPK/JNK, SAPK/JNK, p-AMPK, Wnt3a, Wnt5a/b, p-LRP6, LRP6, Dvl2, Dvl3, β-actin, GAPDH primary, and secondary antibodies were used at 1:1000 dilutions and were from Cell Signaling Technology (CST, Beverly, MA, USA).