Anti cenpf

Cell lysates were extracted with mammalian protein extraction reagent (M-PER) lysis buffer supplemented with a protease inhibitor and phosphatase inhibitors. The protein concentrations were measured by a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher, Waltham, MA, USA). A total of 30 µg of protein was loaded onto a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and run with 1× SDS running buffer. Proteins were transferred onto a 0.45 µm polyvinylidene fluoride (PVDF) membrane, which was later blocked with 5% milk in tris-buffered saline (TBS)-0.1% Tween-20. Then, the membrane was incubated with anti-CENPF (1:1,000; Proteintech, Rosemont, IL, USA) or anti-β-actin (1:5,000; Proteintech) primary antibody overnight at 4 ℃ and secondary antibody for 1 hour. Finally, proteins were detected using enhanced chemiluminescence (ECL).

Formalin-fixed paraffin-embedded (FFPE) tissue samples were cut into 4 µm thick sections and dried at 60 ℃ for 30 minutes. After blocking endogenous peroxidase activity using blocking reagent (Dako, Santa Clara, CA, USA) for 5 minutes at room temperature, epitopes were retrieved using pH 9.0 Tris-ethylenediamine tetraacetic acid (EDTA) buffer for 40 minutes at 95 ℃ followed by incubation with anti-CENPF (1:200, Proteintech) antibody at room temperature for 30 minutes. Subsequently, the slides were incubated with peroxidase/diaminobenzidine (DAB)-10 min K5007 from a DAKO EnVision^TM Detection Kit (Dako) for 30 minutes.