Anti n cadherin antibody

PANC-1 and SW1990 were washed twice with PBS and lysed for 10 min in RIPA buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% NP40, 10% glycerol and 20 μM EDTA) that was supplemented with protease and phosphatase inhibitors. Cell debris was removed by centrifugation at 10,000
g for 10 min at 4°C. Then, 20 μg of whole cell lysates were denatured in SDS loading buffer and subjected to electrophoresis in a denaturing 10% SDS-polyacrylamide gel. The samples were then transferred to a membrane for subsequent blotting with specific antibodies, including anti-RAP2 antibody (Sigma-Aldrich, St Louis, USA), anti-N-cadherin antibody (Cell Signaling Technology, Beverly, USA), anti-Vimentin antibody (Cell Signaling Technology), anti-E-cadherin antibody (Cell Signaling Technology), anti-c-Myc and anti-β-actin antibodies (Proteintech, Rosemont, USA), followed by incubation with the corresponding horseradish peroxidase (HRP)-linked anti-mouse or rabbit IgG secondary antibodies (Cell Signaling Technology). SuperSignal West Pico PLUS (Invitrogen) was utilized as HRP substrate to visualize the protein bands. Target proteins were detected using the Fujifilm LAS-3000 imaging system.

Lung specimens fixed in 10% buffered formalin and were embedded in paraffin blocks. Paraffin-embedded lung sections were heat-fixed, deparaffinized, rehydrated, antigen retrieval, blocked with 3% goat serum and incubated with anti-α-SMA antibody (1:200, Cell Signaling Technology, MA) or anti-COL1A1 antibody (1:200, Cell Signaling Technology, MA) or anti-E-cadherin antibody (1:150, Cell Signaling Technology, MA) or anti-N-cadherin antibody (1:150, Cell Signaling Technology, MA) overnight at 4 °C, then the slides were detected using Real Envision Detection kit (GeneTech, Shanghai, China) according to the manufacturer’s instructions. Observe the sections with a microscope and take pictures. Image J software calculated the ratio of positive expression area to the total field of immunohistochemical staining of α-SMA, COL1A1, E-cadherin and N-cadherin.

Western blot analysis was performed using standard procedures. The following primary antibodies were used in the experiments: anti-FOXM1 antibody (Santa Cruz), anti-pAKT antibody (Peprotech, USA), anti-AKT antibody (Cell Signaling Technology, Beverly, MA, USA), anti-pGSK3β antibody (Proteintech) at 1:1000, anti-Snai1 antibody (Cell Signaling Technology), anti-pIGF1R antibody (Abcam, Cambridge, UK), anti-IGF1R antibody (Abcam, Cambridge, UK), anti-E-cadherin antibody (BD Biosciences, USA), anti-Vimentin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Cell Signaling Technology), anti-pERK1/2 antibody (Abcam), anti-ERK1/2 antibody (Abcam), anti-HIF1α antibody (Novus Biologicals, USA), anti-ETS1 antibody (Abcam) and anti-β-actin antibody (Sigma, St. Louis, MO,USA).

Western blot was performed as previously described.²⁴ Total protein was extracted from cells in RIPA lysis buffer (Beyotime, #P0013B) and quantified using a Bradford assay. In total, 30 μg of protein was separated using 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, #ISEQ0010). The membrane was blocked in a 5% powdered milk solution and incubated in primary antibody overnight at 4℃. After washing, the membrane was incubated with a horseradish peroxidase‐conjugated secondary antibody (dilution 1:4000) at 37℃ for 1 h. Protein bands were visualized using Western Bright ECL (Millipore, #WBKLS0500) and detected using ImageQuant LAS4000‐mini (General Electric, USA). Relative protein levels were calculated based on a β‐Actin loading control. The antibodies used for Western blot were listed below: anti‐BK channel antibody (Alomone Labs, APC‐151, dilution 1:500), anti‐HIF1α antibody (Cell Signaling Technology, #36169, dilution 1:1000), anti‐N‐cadherin antibody (Cell Signaling Technology, #13116, dilution 1:1000), anti‐E‐cadherin antibody (Cell Signaling Technology, #3195, dilution 1:1000), anti‐Vimentin antibody (Cell Signaling Technology, #5741, dilution 1:1000) and Anti‐β‐Actin Mouse Monoclonal Antibody (TransGen Biotec, #HC201‐01, 1:4000).

Human ESCC TE1 cells were grown in the RPMI-1640 medium (Gibco), human ESCC KYSE180 cells were grown in the DMEM medium (Gibco), with supplementation of 10% (v/v) fetal bovine serum (Gibco) and 100 units/ml streptomycin and penicillin (Millipore) in a humidified chamber at 37 °C. For the establishment of stable circGSK3β-depleted cell line, TE1 cells were cultured for 24 h to ~ 80% confluence before transfected with the circGSK3β-shRNA constructs in pLKD vector. The pLKD empty vector was used as a negative control. Twenty four hours post-transfection, the cells were selected with 2 mg/ml puromycin for 2 weeks. The efficiency of the depletion was determined by real-time RT-PCR. The anti-GSK3β antibody, anti-GAPDH antibody, anti-E-cadherin antibody, anti-N-cadherin antibody, anti-Vimentin antibody, anti-Claudin antibody, anti-β-Actin antibody, and anti-β-catenin antibody were from Cell Signaling.

Western blotting was performed as described previously^{19 (link)}. The primary antibody information is as follows: anti-ESRP1 antibody (Abcam, ab107278, 1:200), anti-STAT1 antibody (Abcam, ab92056, 1:500), anti-STAT2 antibody (Abcam, ab32367, 1:5000), anti-ISG15 antibody (Santa Cruz Biotechnology, sc-166755, 1:500), anti-CREB antibody (Abcam, ab32515, 1:1000), anti-E-cadherin antibody (Cell Signaling Technology, #14472, 1:500), anti-N-cadherin antibody (Cell Signaling Technology, #13116, 1:500), anti-Vimentin antibody (Abcam, ab8069, 1:500) and anti-GAPDH antibody (Cell Signaling Technology, #3700s, 1:5000).