Hoxa1

Total cellular proteins were isolated using RIPA buffer (Thermo Scientific) and quantified with a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein lysate was then separated by 10% SDS‐PAGE, followed by transfer onto PVDF membrane (Invitrogen). After blocking with 5% skim milk in TBST at room temperature for 2 hours, the membrane was incubated at 4°C overnight with specific primary antibodies: HOXA1 (1:1000, Abcam), cyclin D1 (1:1000, Abcam), c‐Myc (1:1000, Abcam), β‐catenin(1:1000, Abcam), p‐GSK3β (1:1000, Abcam), E‐cadherin (1:2000, Abcam), N‐cadherin(1:2000, Abcam), Vimentin (1:1000, Abcam) and GAPDH (1:1000, Abcam). After washing with TBST, the membrane was incubated with HRP‐labeled secondary antibody (1:3000, Abcam) at room temperature for 2 hours. GAPDH was used as an internal control. Finally, the bands for proteins were visualized using ECL western blot detection reagents (Beyotime).

Total protein was extracted from tissues and cells using radioimmunoprecipitation assay buffer (RIPA buffer, Beyotime, China) with protease inhibitors (Beyotime). Protein concentration was then measured using a BCA Protein Assay Kit (Beyotime). Total protein was separated by 10% SDS-PAGE (Beyotime) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA) by wet transfer. After blocking for 2 hrs with TBST solution containing 5% skimmed milk, the membranes were incubated with specific primary antibodies overnight at 4°C. The following morning, the membranes were incubated with a corresponding secondary antibody for 2 hrs at room temperature. Finally, the intensity of the protein bands was measured with C300 imaging system (Azure Biosystem, USA) by enhanced chemiluminescence (ECL, Thermo Scientific, USA). The relative-integrated density was analyzed using ImageJ software based on GAPDH as an internal control. The primary antibodies included HOXA1 (1:1000, Abcam, Eugene, USA), E-cadherin (1:1000, Cell Signaling Technology, CA, USA), N-cadherin (1:1000, Cell Signaling Technology), CyclinD1 (1:1000, Cell Signaling Technology) and GAPDH (1:10,000, Proteintech, Chicago, IL, USA). We also used an HRP-conjugated secondary antibody (1:2000, ZSGB-Bio Origene, Beijing, China). All assays were performed in triplicate.