Chemiluminescence solution

Western blotting was used to detect ARAF protein levels in tissues and cell lines. RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Nantong, China) was used to extract the total protein from tissues and cells. The protein concentration was determined using the BSA method (Beyotime Institute of Biotechnology). Briefly, 30 μg protein was loaded onto 10% SDS–PAGE gels, electrophoresed, and transferred onto to polyvinylidene fluoride membranes, which were blocked by 5% skim milk powder in TBST, incubated with primary antibodies overnight, and then with horseradish peroxidase-conjugated secondary antibodies (cat. nos. A0208 and A0216; Beyotime Institute of Biotechnology) at 1 : 10000 dilution for 2 h at room temperature. Primary antibodies against ARAF (dilution 1 : 1000; cat. no. 4432) and phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204, dilution 1 : 1000; cat. no. 4370) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against PCNA (dilution 1 : 1000; cat. no. 60097-1-Ig), cyclin D1 (dilution 1 : 1000; cat. no. 26939-1-AP), and β-actin (dilution 1 : 1000; cat. no. 20536-1-AP) were purchased from Proteintech (Rosemont, IL, USA). β-Actin was used as the endogenous control. Immunoreactive bands were visualized using a chemiluminescence solution (Beyotime Institute of Biotechnology).