Axin1

Some Cal-27 cells were transferred with miR-16 and then treated with R-spondin1; inhibition of Wnt signaling in the other cells was achieved by the addition of 5 mM tankyrase inhibitor XAV939. After incubated for 24 hours, both adherent and floating cells were collected and lysed with Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Haimen, China) supplemented with Phenylmethanesulfonyl fluoride (PMSF) (1 mM) for 30 minutes. Then, the cells were centrifuged at 12,000× g for 10 minutes, and the supernatant was collected. The protein concentration was detected by the Bio-Rad protein assay reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA). Equal amounts of the total protein were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Millipore Immobilon^®-P Transfer Membrane (EMD Millipore, Billerica, MA, USA). The antibody of β-catenin, β-actin, APC, Axin-1, and horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma Chemical (Perth, Australia). The expression levels of proteins were visualized by electro-chemiluminescence (Thermo Fisher Scientific).

For co-immunoprecipitation (co-IP), 8 μg of crosslinked β-catenin (Clone-14) or IgG (Clone MOPC-31C) antibody (Becton Dickinson, Oxford, UK) were incubated with 1 mg of either pre-cleared cytoplasmic or nuclear lysate overnight at 4°C (Online Supplementary Methods). Subsequently beads were washed five times prior to proteomic analyses or boiled for 95°C for 5 min following washes for immunoblotting. Immunoblotting was performed as previously described^{13 (link)} using antibodies to total β-Catenin (as above), phosphorylated β-catenin (Ser33/37/Thr41), β-Catenin (Clone-5), E-Cadherin (Clone-36), GSK3β (Clone-7; BD), α-tubulin (DM1A), lamin A/C (4C11; Sigma), Axin1 (C76H11), Axin2 (76G6), TCF-4 (C48H11), LEF-1 (C12A5), Survivin (71G4B7), CyclinD1 (92G2; Cell Signaling Technology), active β-catenin (8E7, Merck-Millipore, Watford, UK) and c-MYC (9E10; Santa Cruz, Heidelberg, Germany). β-Catenin and LEF-1 densitometry were performed as described in the Online Supplementary Methods.