Ab245117

Total protein was extracted from AGS cells using RIPA lysis buffer and quantified by a Pierce BCA Protein assay kit (Thermo Fisher Scientific). After denaturing, proteins were separated via 12% SDS-PAGE and the separated proteins were subsequently transferred onto PVDF membranes, which were blocked with 5% fat-free milk for 2 h at room temperature. After washed, membranes were incubated with primary antibodies at 4°C overnight and subsequently incubated with an IgG-HRP-conjugated goat anti-rabbit secondary antibody for 1 h at room temperature. Protein bands were visualized using ECL reagent (Millipore). Densitometric analysis were performed using Image J v1.46 software. Information about antibodies are as follows: E-cadherin (ab40772; 1:10,000), N-cadherin (ab245117; 1:1,000), Vimentin (ab92547; 1,000), snail family transcriptional repressor 1 (Snail; ab216347; 1,000), ST8SIA1 (ab253021; 1:300), SPI1 (ab227835; 1:1,000), secondary antibody (ab6721; 1:5,000; all Abcam).

The total protein from each group was diluted to the same concentration. Proteins were denatured, electrophoresed, and then transferred to polyvinylidene fluoride membranes (1620177; Bio-Rad, Hercules, CA, USA). Blots were blocked with 5% BSA and washed. The membrane was then incubated with the following antibodies overnight. The antibodies were anti-YAP1 (1:1000, ab205270; Abcam), anti-neurogenic locus notch homolog protein 1 (Notch 1) (1:1000, ab52627; Abcam), anti-jagged canonical notch ligand 1 (Jagged 1) (1:1000, ab7771, Abcam), anti-GAPDH (1:10000, AF7021; Affinity, Shanghai, China), anti-Ki67 (1:1000, ab16667, Abcam), anti-PCNA (1:2000, ab92552; Abcam, UK), anti-Bcl-2-associated X protein (Bax) (1:5000, ab32503; Abcam), anti-Bcl-2 (1:5000, ab32124, Abcam), anti-N-cadherin (1:5000, ab245117; Abcam), anti-Vimentin (1:1000; Abcam), and anti-E-cadherin (1:2000, ab40772; Abcam) antibodies. After washing, the membranes were incubated with anti-rabbit (7074, CST, USA) or anti-mouse (7076, CST) IgG HRP-linked antibodies. Proteins were visualized using a chemiluminescence imaging analysis system (610020-9Q; Qinxiang, China) and ImageJ software (NIH, Bethesda, MD, USA).

Proteins were extracted from MG63 or Saos-2 cells using lysis buffer (Solarbio), and the process of transferring and incubating were performed on the basis of the manufacturer’s protocols. Protein concentration was quantified using a BCA kit (Takara Bio, Inc.). The proteins were separated via SDS-PAGE on 8% gel and then electroblotted onto a PVDF membrane. Following blocking with 5% blocking reagent at room temperature for 1 h, the membranes were incubated with the primary antibodies in 5% BSA overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies at room temperature for 1 h. Proteins were visualized using ECL reagent. The primary antibodies use were the following: anti-BYSL (1:1,000, NBP1-89501; Novus Biologicals), anti-Bcl-2 (1:1,000, ab196495; Abcam, Cambridge, United Kingdom), anti-Histone H3 (1:1,000, ab18521; Abcam), anti-Bax (1:2000, ab32503; Abcam), anti-E-cadherin (1:5,000, 20874-1-AP; Proteintech, Rosemont, IL, United States), anti-N-cadherin (1:1,000, ab245117; Abcam), anti-Nrf2 (1:1,000, ab137550; Abcam), anti-β-actin (1:1,000, AC026, Abclonal), and ani-vimentin (1:2,000, 10366-1-AP; Proteintech).