Anti cyclin e he12

Protein aliquots (25 μg) were separated by SDS‐PAGE (Bio‐Rad, Hercules, CA, USA) and transferred to PVDF membranes (Bio‐Rad). The membranes were washed three times and then incubated with Blocking One solution (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies against anti‐MET (25H2), anti‐phospho‐MET (pMET) (Tyr1234/1235), anti‐protein kinase B (AKT), anti‐phospho‐AKT (Ser473), anti‐cleaved poly(ADP‐ribose) polymerase (PARP) (Asp214), anti‐cleaved caspase‐3 (Asp175) (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti‐human HGF (200 μg/mL), anti‐human/mouse/rat ERK (Erk1/Erk2; 0.2 μg/mL), anti‐phospho‐Erk1/Erk2 (T202/Y204; 0.1 μg/mL), GAPDH antibodies (1:1000; R&D Systems, Minneapolis, MN, USA), anti‐cyclin A (H432), anti‐cyclin B1 (GNS1), anti‐cyclin D1 (A12), or anti‐cyclin E (HE12) antibodies (1:200; Santa Cruz Biotechnology, Dallas, Texas, USA). The membranes were then washed three times and incubated for 1 h at room temperature with species‐specific HRP‐conjugated secondary antibodies. Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate (an enhanced chemiluminescent substrate) (Pierce Biotechnology, Rockford, IL, USA). Each experiment was carried out independently at least three times.