Rap1a

The total protein of the tissue and cells was extracted according to the manufacturer's recommended protocol (Vazyme, Nanjing, China). The protein concentrations were determined using the BCA protein assay kit (Vazyme, Nanjing, China). Samples with equal amounts of protein (50 μg) were fractionated on 10% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes and blocked with 5% skim milk in TBST for 1.5 hat 25°C. The membranes were then incubated at 4°C overnight with 1:1000 dilutions (v/v) of the primary antibodies. This study used Rap1a, a MAPK pathway sampler kit, MMP-9 antibodies purchased from Cell Signaling Technology (Beverly, MA, USA), and caspase-3, caspase-9 and PARP1 antibodies purchased from Abcam (Cambridge, UK). After washing the membranes with TBST, incubations with 1:4000 dilutions (v/v) of the secondary antibodies were conducted for 2 hrs at 25°C. Protein expression was detected using an enhanced chemiluminescence detection system. β-actin was used as a loading control.