Ripa lysis buffer

Cells or tissues were harvested in RIPA lysis buffer (Yeasen, China) and the concentrations were analysed using the Pierce BCA Protein Assay kit (Thermo Scientific, USA). The cell lysate was electrophoresed by sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred to a polyvinylidene fluoride membrane, blocked with non‐fat milk for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C: rabbit CD147 antibody (1:1000; CST), rabbit IKKα antibody (1:1000; CST), rabbit Phospho‐IKKα antibody (Ser176/180) (1:1000; CST), mouse IκBα antibody (1:1000; CST), rabbit Phospho‐IκBα antibody (Ser32) (1:1000; CST), rabbit NF‐kappa B p65 antibody (1:1000; CST), rabbit Phospho‐NF‐kappa B p65 antibody (Ser536) (1:1000; CST), rabbit alpha‐Tubulin antibody (1:1000; Proteintech, USA). Then the membrane was washed three times, incubated with rabbit or mouse HRP‐linked secondary antibodies (1:10000; CST) and visualized with ECL Ultra (New Cell and Molecular Biotech, Suzhou, China).

Western blot was performed using standard procedures. Total proteins were extracted from GBC cells using RIPA lysis buffer (Yeasen), and were quantified unsing a Micro BCA Protein Assay Kit (Thermo‐Fisher Scientific). 20 µg of total protein was electrophoresed through 10% SDS polyacrylamide gel and were then transferred to PVDF membranes (Millipore). The membranes were blocked in 5% skim milk for 1 h at room temperature and then incubated with primary antibodies at 4 °C overnight. Secondary antibodies were labeled with HRP, and the signals were detected using ECL Kit (Millipore) by the ChemiDoc XRS+ imaging System (Bio‐Rad). A β‐actin antibody was used as a control for whole‐cell lysates. The relative protein expression was calculated by Image J software and normalized to the expression of β‐actin. The antibody against DNMT3A (#32 578, dilution 1:1000) and p‐YAP (#13 008, dilution 1:1000) were purchased from Cell Signaling Tech, β‐actin (A1978, dilution 1:10 000), Flag (SAB1306078, dilution 1:10 000), and HA (H6908, dilution 1:10 000) were purchased from Sigma–Aldrich, CDH1 (ab40772, dilution 1:5000) and N‐cadherin (ab76011, dilution 1:5000) were purchased from Abcam, YAP (13584‐1‐AP, dilution 1:2000) and TAZ (23306‐1‐AP, dilution 1:1000) were purchased from Proteintech.

Dorsal CA3 tissues (1 mg) from P12 control and shRNA-virus treated mice were homogenized in RIPA lysis buffer (Yeasen, Shanghai, China) containing cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche Applied Science, Basel, Switzerland) and disrupted by ultra-sonication. Protein samples were separated on 10% tris-glycine gels before being transferred onto PVDF (0.45 μm, Merk Millipore, Darmstadt, Germany) and the PVDF was then blocked in 5% nonfat milk. Immunoblotting was conducted with HRP-conjugated secondary antibodies and ECL Western Blotting Substrate (BioSharp, Beijing). The following primary antibodies were used in this study: rabbit anti-Oxtr (Abclonal, Wuhan, China) and rabbit anti-β-tubulin (Yeasen, Shanghai, China). Loading controls were run on the same gel. Signals were acquired using Amersham Imager 600 (GE Life Science, Boston, USA), and images were analyzed using Fiji ImageJ software.

For Co‐IP analysis, whole cells were lysed in Lysis Buffer for WB/IP assays (Yeasen) containing a protease inhibitor cocktail, followed by incubation with specific antibodies and protein A/G beads (MedChemExpress) overnight. The beads were washed with 1%‐Triton buffer and eluted with 1× SDS Loading Buffer.
For Western Blot analysis, cells were lysed in RIPA Lysis Buffer (Yeasen) with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail. Protein concentrations were determined by the BCA Protein Assay Kit (Beyotime). Equal amounts of proteins were loaded and separated on an SDS‐PAGE gel, transferred onto a PVDF membrane (Millipore), and combined with appropriate antibodies for chemiluminescence system (New Cell & Molecular Biotech Co., Ltd.) detection.
Four percent of paraformaldehyde was used to fix tissues or cells, and after 15 min, samples were permeabilized with 0.5% Triton X‐100 for 20 min and then blocked with 4% goat serum for 30 min. After that, samples were incubated with appropriate primary antibodies (1:100) at 4°C overnight and proceeded to secondary antibody (1:2000) incubation. Cells and tissues were photographed under an FV3000 fluorescent microscope (Olympus).

The total proteins of tissues and OS cells were extracted by RIPA lysis buffer (YEASEN, China); then, the BCA protein detection kit (Thermo Fisher Scientific, USA) was adopted for measuring protein concentration. Samples were boiled, denatured, and separated using electrophoresis. Proteins were then transferred onto PVDF membranes. Rapid blocking solution was used to block membranes for a 30 min period. Primary antibodies were then used to incubate membranes at 4°C overnight. Afterwards, membranes were further incubated with secondary antibody for an additional 1 h at room temperature (RT). The membranes were then exposed to ECL reagent (Millipore, USA) with the Tanon 4200 automatic chemiluminescence imaging analysis system. Antibody information is shown in Additional file 2: Table S2.

HNSCC cells or tissues were harvested in RIPA lysis buffer (Yeasen, China) and whole-cell lysate was electrophoresed using sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with non-fat milk for 1 h at room temperature, and probed with primary antibodies: a rabbit polyclonal PTK7 antibody (Proteintech, 17799-1-AP, USA; 1:1000), a rabbit monoclonal p-Lrp6 antibody (CST, #2568, USA; 1:1000), a rabbit monoclonal Dvl2 antibody (CST, #3224, USA; 1:1000), a rabbit monoclonal Naked2 antibody (CST, #2073, USA; 1:1000), a rabbit monoclonal GSK3β antibody (Proteintech, 22104-1-AP, USA; 1:1000), a rabbit polyclonal p-GSK3β antibody (Signalway Antibody, #11002, USA; 1:1000), a rabbit polyclonal β-Catenin antibody (Proteintech, 51067-2-AP, USA; 1:1000), a rabbit polyclonal E-cadherin antibody (Proteintech, 20874-1-AP, USA; 1:1000), a rabbit polyclonal Vimentin antibody (Proteintech, 10366-1-AP, USA; 1:1000), a rabbit polyclonal alpha-Tubulin Polyclonal antibody (Proteintech, 11224-1-AP, USA; 1:1000) overnight at 4 °C and then with rabbit or mouse HRP-linked secondary antibodies (CST, USA; 1:10000) and visualized with ECLUltra (New Cell and Molecular Biotech, Suzhou, China).