E ab 48018

The proteins were separated in a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene fluoride membrane (Millipore, MA, USA) by wet transfer at 100 V. For immunodetection, the membrane was blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with the primary antibody overnight at 277.15 K, and then incubated with the secondary antibody for 2 h at room temperature. The primary antibodies included anti-Tau (1:100; sc-32274, Santa Cruz Biotechnology Inc.), anti-HK1 (1:1,000; 19662-1-AP, Proteintech, Wuhan, China), anti-VDAC I (1:1,000; AF1027, Beyotime Biotechnology), anti-Bax (1:200; sc-7480, Santa Cruz Biotechnology Inc.), anti-PARP-1 (1:200; sc-8007, Santa Cruz Biotechnology Inc.), anti-caspase-3 (1:1,000; 9662S, Cell Signaling Technology, MA, USA), and anti-β-actin (E-AB-48018, Elabscience, Wuhan, China). The secondary antibodies included anti-mouse secondary antibody (HS201-01, TransGen Biotech, Beijing, China) at a 1:1,000 dilution or anti-rabbit secondary antibody (HS101-01, TransGen Biotech) at a 1:1,000 dilution. The signals were detected via chemiluminescence (Tanon, Shanghai, China).

The lysates of 16-HBE cells and lung tissues following cigarette smoke exposure were prepared using RIPA buffer (Beyotime, P0013B) containing cOmplete™ Protease Inhibitor Cocktail (Roche Diagnostics GmbH, 4693132001-20) and phosphatase inhibitors (Roche Diagnostics GmbH, 04-906-837-001). The total protein concentration was measured using a BCA protein assay kit (ThermoFisher, USA). Proteins were isolated using 10% SDS-PAGE and transferred onto PVDF membranes. They were incubated overnight with anti-RAGE (sc-74473, Santa Cruz Biotechnology, USA), anti-phospho-ERK (ABP0035, abbkine, Wuhan), anti-ERK (ABP0085, abbkine, Wuhan), anti-β-actin (E-AB-48018, elabscience, Wuhan), and anti-GAPDH (5174S, CST, USA) antibodies at 4 ℃. Following washing, the PVDF membranes were incubated with an enzyme-linked secondary antibody for 1 h at room temperature. Quantitative analysis was performed using density measurement alongside ImageJ software.