Hrp conjugated β actin

Lung tissues and PASMCs were lysed with RIPA lysis buffer. Lysates were centrifuged at 12,000 rpm at 4 °C for 15 min. Supernatants were collected, and the protein concentration was measured using a BCA kit. Equal amounts of protein were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The following primary antibodies were used.
Polyclonal antibodies against PCNA, BAX, caspase 3, PARP, HRP-conjugated β-actin, and Survivin were purchased from Proteintech (USA); anti-Bcl2 antibody was obtained from Boster (China); anti-MMP9 antibody was obtained from Affinity (USA); anti-p38/p-p38 MAPK, anti-JNK/p-JNK, anti-ERK/p-ERK, and normal rabbit IgG antibodies were acquired from Cell Signaling Technology (USA); normal mouse IgG was obtained from Santa Cruz Biotechnology (USA). Western blots were visualized using the ChemiDocTM XRS+ imaging system.

SMMC7721 cells were prepared by trypsin/DNase II digestion and incubated at seeding density of 2.5 × 10⁶ cells/well in 6-well plates for 24 h with or without drug treatment. After drug treatment, cells were lysed with 30 μL RIPA regent (Sangon, Shanghai, China), with the presence of 1% thioglycol. The samples were boiled at 100 °C for 5 min and then stored at −20 °C. A total of 30 μg of each sample was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Immobilon^®, Merck Millipore, Cork, Ireland). Subsequently, the membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with the primary antibody phosphorylated form (p-) and total protein of the extracellular signal-regulated kinase 1 and 2 (ERK1/2, 1:3000, Cell Signaling Technology, Beverly, MA, USA), p38 (1:3000, Cell Signaling Technology, Beverly, MA, USA), Jun-N-terminal kinase (JNK, 1:3000, Cell Signaling Technology, Beverly, MA, USA), HRP-conjugated β-actin (Proteintech, Wuhan, China) at 4 °C overnight, followed by 1.5 h incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Sangon, Shanghai, China) at room temperature.