Enhanced chemiluminescence reaction solution

Total protein from tissues and cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer containing phenylmethylsulfonyl fluoride (PMSF), loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and transferred electrophoretically to polyvinylidene fluoride membrane (1620177, Bio-Rad, Hercules, CA, USA). Following blockage utilizing 5% skimmed milk or 5% bovine serum albumin (BSA) for 1 h at ambient temperature, the membrane was probed with diluted primary antibodies to β-actin (4970, CST, MA, USA), GSK3β (ab32391, Abcam), FTO (ab94482, Abcam), phosphoserine (ab9332, Abcam), KLF5 (ab137676, Abcam), and c-Myc (18583, CST, MA, USA) overnight at 4°C. The membrane was reprobed with the secondary goat anti-rabbit (ab6721, Abcam) or anti-mouse (ab6789, Abcam) immunoglobulin G (IgG) antibody labeled by horseradish peroxidase (HRP) for 1 h at ambient temperature. The membrane was immersed in enhanced chemiluminescence reaction solution (1705062, Bio-Rad, Hercules, USA) at ambient temperature for 1 min and imaged on the Image Quant LAS 4000C gel imager (GE, NY, USA). The relative gray-scale ratio of the target protein to β-actin was calculated.

Protein was isolated from tissues and cells using Cell lysis buffers (Takara, Dalian, China) following the suggestion of the manufacturer. Subsequently, the protein was separated by 10% SDS-PAGE gel, transferred with PVDF membranes, and blocked with 5% non-fat milk. After that, the membranes were incubated with rabbit anti-MMP9 antibody (1:1000; ab38898, Abcam, Cambridge, MA, USA), rabbit anti-TIMP1 antibody (1:1000; ab216432, Abcam, Cambridge, MA, USA), rabbit anti-CEACAM1 antibody (1:1000; #14,771, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-GAPDH antibody (1:2000; ab8245, Abcam, Cambridge, MA, USA) overnight at 4°C. The membranes were further incubated with HRP-labeled goat anti-rabbit IgG (1:2000; ab7090, Abcam, Cambridge, MA, USA) at room temperature for 1 h. Finally, bands were visualized using an enhanced chemiluminescence reaction solution (Bio-Rad, Hercules, CA, USA). GAPDH is used as an internal reference gene to normalize protein levels.