Ccnd1

Proteins were extracted from cell lysis with the Radio Immunoprecipitation Assay (RIPA) buffer (Thermo Scientific, Carlsbad, California, USA) containing a protease inhibitor cocktail (Selleck, Huston, Texas, USA). BCA kit (Thermo Scientific, Carlsbad, California, USA) was used to quantify proteins and equal amounts was resolved by sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel and transferred to the PVDF membrane (Millipore, Billerica, MA). The membranes were blocked for 2h with 5% skim milk at room temperature and incubated with primary antibodies at 4°C overnight with gentle shake. The PVDF membrane was then incubated with appropriate secondary antibody for 1 h at room temperature. The bands in the membranes were then detected using the enhanced chemo luminescence substrate kit (Thermo Scientific, Carlsbad, California, USA) and scanned with Image J software. The antibodies used in our study including MMP9, CCND1, XIAP, TWIST1, IκBα, p-IκBα, p-IKKα/β, IKKα, IKKβ, N-Cadherin, E-Cadherin, Vimentin, Lamin B1 and GAPDH are purchased from Cell Signaling (CST, Danvers, MA, USA).
Subcellular fractions were prepared from ESCC cells with a Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent biotech, Eden Prairie, USA) according to manufacturer’s instructions.

Samples were lysed in RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail, phosphatase inhibitors and phenylmethylsulfonyl fluoride (PMSF). Equal amounts of protein (30–50 μg) were loaded and separated on a 10% SDS-polyacrylamide gel, electrotransferred onto PVDF membranes (Millipore, Billerica, MA, USA), and blocked in 5% bovine serum albumin in Tris-buffered saline containing Tween 20 for 1 h at room temperature. The membranes were probed with primary antibodies against p-AKT (S473), AKT, CCND1, CCNA2, CLND1, CDH1 (Cell Signaling Technology, MA, USA), HSP90 (Proteintech, IL, USA) and SULT2B1 (R&D systems, MN, USA) overnight at 4 °C. After three 10 min washes with Tris-buffered saline with Tween-20 (TBST), the membrane was incubated at room temperature for 1 h with an appropriate secondary antibody conjugated to horseradish peroxidase. HSP90 was used as the loading control. The protein signals were visualized with a Pierce ECL Western Blotting kit (Life Technologies, Thermo Fisher Scientific, USA).

Tissue and cellular proteins were extracted with radioimmunoprecipitation assay buffer (Beyotime, Jiangsu, China) mixed with phenyl-methylsulfonyl fluoride (PMSF), and quantified with BCA protein assay kit (Beyotime, Jiangsu, China). Equal amount of proteins were loaded and analyzed by 10% SDS-PAGE gels. The isolated proteins were transferred to PVDF membranes (Bio-Rad, CA, USA). After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies against MYC (1:1000), FUBP1 (1:1000), FIR (1:1000), nm23-H1(1:1000), MMP2 (1:1000), MMP9 (1:1000), CDK4 (1:1000), CCND2 (1:1000), CCND1 (1:1000), CCNE1 (1:1000), Bcl-2 (1:1000), Bax (1:1000) and cleaved Caspase-3 (1:1000) (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C. The membranes were washed by TBST, and then incubated with goat anti-rabbit secondary antibodies (Thermo Fisher Scientific, USA) for 2 h. Finally, the bands were illuminated with Pierce ECL (Thermo Fisher Scientific, USA).

Total protein was extracted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto 0.45 μm PVDF membrane (Millipore). The following primary antibodies were used in the immunoblotting assays: antibodies for Smad7 (sc-101152; Santa Cruz Biotechnology, Santa Cruz, CA, USA), vimentin (no. 3390; Cell Signaling Technology, Danvers, MA, USA), β-actin (no. 3700; Cell Signaling Technology), CCND1 (no. 2926; Cell Signaling Technology), c-myc (ab17356; Abcam, Cambridge, MA, USA), Slug (no. 9585; Cell Signaling Technology), P21 (sc-6246; Santa Cruz), P27 (no. 3688; Cell Signaling Technology) and β-catenin (no. 8480; Cell Signaling Technology).

At 72 h after transfection, aliquots of 5 × 10⁶ cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology, China), and the lysate was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). A total of 30 μg protein per lane was resolved by 10% SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, USA). The membrane was blocked with 5% non-fat milk and then incubated overnight with primary antibodies against β-actin (Sigma-Aldrich, St. Louis, Missouri, USA), CDK-4 (Cell Signaling Technology, USA), CCND1 (Cell Signaling Technology, USA), or CCND2 (Cell Signaling Technology, USA). The membrane was washed in TBST and incubated with the secondary goat anti-mouse IgG antibody (Beyotime Institute of Biotechnology, Shanghai, China). An enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) was used to detect the protein bands using a FluorChem E System (ProteinSimple, CA, USA).