Ab231956

After washing with PBS, the cells were lysed with prechilled lysis buffer. The collected cell lysate was centrifuged at 14000 × g for 15 min at 4°C, and the protein content was measured with 5 × sample buffer (BSA; Thermo Fisher Scientific, Inc., CA, USA). Then, these protein samples were tested by WB. The 4%-20% precast gel was used to transfer the protein to the nitrocellulose membrane, and the 5% skimmed milk was used to seal the membrane at ambient temperature (Bio-Rad Laboratories) 1 h. Subsequently, specific antibodies (diluted 1 : 10 00), including anti-FGF12 (ab231956, Abcam), anti-E-cadherin (ab231303, Abcam), anti-Vimentin (ab8978, Abcam), anti-p-PI3K(#17366, Cell Signaling Technology), anti-PI3K(#4249, Cell Signaling Technology), anti-p-AKT(S473)(#4060, Cell Signaling Technology), anti-N-cadherin(ab18203, Abcam), anti-p-AKT(T308)(#13038, Cell Signaling Technology), anti-AKT(#4685, Cell Signaling Technology), and anti-β-actin (#4970, Cell Signaling Technology), were used to block membranes. Subsequently, the secondary antibody was used to incubate the membranes (Santa Cruz) at ambient temperature for 1 h and visualized them using ECL solution (Bio-Rad Laboratories). Finally, a chemiluminescence imaging system (Mini HD9; UVitec, Cambridge, UK) was used to take images.

The CRC and adjacent tissues were fixed with formalin. The paraffin-embedded tumor tissues and adjacent sections were stained with anti-FGF12 antibody (1 : 100, ab231956, Abcam), anti-Ki67 antibody (1 : 200, ab16667, Abcam), anti-Vimentin antibody (1 : 100, ab8978, Abcam), anti-E-cadherin antibody (1 : 200, ab231303, Abcam), anti-N-cadherin antibody (1 : 200, ab18203, Abcam), anti-Nanog antibody (1 : 100, ab109250, Abcam), anti-OCT4 antibody (1 : 1000, ab181557, Abcam), and anti-cleaved-caspase 3 antibody (1 : 200, #9664, Cell Signaling Technology), respectively. Stained cells are counted in each field with at least 400 cells. The 5 highest field-measured average positive cells were used to conduct subsequent data analysis. Expression of related proteins was evaluated via staining intensity and density.