Trans blot turbo cassette

Total protein lysates were extracted from whole-cell lines in phosphate-buffered saline. The Pierce BCA Protein Assay Kit (Cat#23225, Thermo Fisher) was used to determine concentration. All proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the proteins in the gel were then transferred to polyvinylidene difluoride blotting membrane (A29532146, P 0.45 GE Healthcare Life Science, Chicago, IL, USA). Trans-Blot Turbo Cassette (Bio-Rad, Hercules, CA, USA) was used for blotting. For blocking, 5% skimmed milk was used. Primary antibodies including anti-YTHDF1 (1:1000 dilution, 17479-1-AP, Proteintech), YTHDF2 (1:1000 dilution, ab170118, Abcam), PD-L1 (1:1000 dilution, A1935; ABclonal, Tokyo, Japan), and GAPDH (1:1000 dilution, Ab8245; Abcam) were incubated overnight at 4°C. Secondary antibodies for rabbit (1:20,000 dilution, NA9340; GE Healthcare Life Science) or mouse (1:20,000 dilution, NA9310; GE Healthcare Life Science) were incubated at room temperature for 1 hour. To visualize the band, we used enhanced chemiluminescence (Pierce ECL Plus Substrate, Thermo Scientific). The photo was detected by a chemi-doc (Bio-Rad). Signal intensity was quantified using Image Labo Software (Bio-Rad).