Anti cyclin e2

A-498 cells transfected with miRNAs were seeded on 6-well plates, and collected and centrifuged at 700 x g for 1 min. A-498 cells were washed once in PBS and lysed in RIPA lysis and extraction buffer. The protein concentration was determined using a BCA protein assay kit. Equal sample volumes were loaded onto 8-10% polyacrylamide-SDS gel and were then transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was incubated with blocking buffer and then incubated overnight at 4˚C with anti-p53, anti-CDKN1A p21, anti-cyclin E2, anti-Bax and anti-β-actin (1:4,000; Santa Cruz Biotechnology, Santa Cruz, CA, uSA) followed by incubation with the corresponding secondary antibodies.