Jun n terminal kinase jnk

Total tissue and cell lysates were assayed by Western blot according to our previous research 10. The antibodies Nrf2, Histione, extracellular signal‐regulated kinase (ERK), phosphor‐ERK, Jun N‐terminal kinase (JNK), phosphor‐JNK, p38, phosphor‐p38, anti‐rabbit‐HRP, anti‐goat‐HRP and anti‐mouse‐HRP were from Cell Signaling Technology (Danvers, MA, USA), and β‐actin, c‐fos, phosphor‐c‐fos, c‐jun, phosphor‐c‐jun, MMP‐1, procollagen type I, TGF‐β1, elastin and DLD‐E3 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Each experiment was repeated at least three times.

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.

Total protein was extracted from the cultured hGFs under the same conditions maintained for total RNA extraction, and an extraction buffer containing 2% sodium dodecyl sulfate (SDS) and 0.1 M dithiothreitol (DTT) was used. After pretreatment, 10% SDS-PAGE was performed to separate the proteins and transfer them to the polyvinylidene difluoride (PVDF) membrane (Bi-Rad Laboratories, Hercules, CA, USA). After blocking with bovine serum albumin (BSA), an antibody specific for each protein (nuclear factor kappa B (NF-κB), phospho NF-κB, p38 mitogen-activated protein kinase (MAPK), phospho p38 MAPK, Jun N-terminal kinase (JNK), and phospho JNK; Cell Signaling Technology, Danvers, MA, USA) was used as the primary antibody, and horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin G (IgG) antibody (Cell Signaling Technology) was used as the secondary antibody. Immunological detection was performed using enhanced chemiluminescence (ECL) Western blotting detection reagents (GE Healthcare Life Sciences Corp, Verdesian, MA, USA), and the activation of intracellular signal transduction molecules associated with the expression of inflammatory cytokines was investigated. β-actin (Cell Signaling Technology) was used as the internal standard.