Immunoblot Analysis of Cellular Proteins

Lung and cell culture protein lysates (30 μg/lane) were separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots were developed as previously described [14 (link), 15 (link)]. The blots were probed with primary antibodies against NAMPT (1:2000, Thermo Fisher Scientific, Rockford, IL, USA), B-cell lymphoma (Bcl)-2 (1:200, Santa Cruz Biotechnology, Dallas, Texas, USA), NF-κB p65, phospho-NF-κB p65, inhibitor of NF-κB (IκB)-α, extracellular signal-related protein kinase 1/2 (ERK1/2), phosho-ERK1/2, c-Jun N-terminal kinase (JNK), phospho-JNK, p38 protein kinase (p38), phospho-p38, and mitogen-activated protein kinase phosphatase-1 (MKP-1) (1:1000, Cell Signaling Technology, Danvers, MA, USA), proliferating cell nuclear antigen (PCNA) (1:1000, Abcam, Cambridge, MA, USA), and β-actin (1:10000, Sigma Chemical Company, St. Louis, MO, USA). The data are presented as the relative ratio of the target protein to the reference protein.