Gapdh antibody

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.

Lung tissue homogenate were taken out for the detection of total protein concentration using a BCA Protein Quantitation kit (Pierce, Rockford, IL). Based on the samples with minimum concentration, other samples were adjusted to the same concentration and 5×SDS sample buffer of the same volume were added. The samples were mixed using vortex oscillation and denaturation at 100°C for 5–10 min. Then, the samples were conducted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to membrane, sealed, and incubated with primary antibody (Rabbit anti-mouse A20, nuclear factor-kappa B (NF-κB) p65, NF-κB p-P65 and GAPDH antibody, R&D Systems, Minneapolis, MN, USA), at 4°C overnight. After membrane cleaning, the samples were incubated with secondary antibody at room temperature for 1 h. Then, the cleaning was performed for 10 min repeated 3 times, and detected by electrogenerated chemiluminescence (ECL).