Signaling Pathways Modulation in LLC-PK1 Cells

LLC-PK1 cells were seeded onto 6-well plates at 4 × 10⁵ cells/well and treated with the vehicle control (0.5% DMSO), A. argyi extract (50 μg/mL), compound 2, and NAC (10 mM) as the positive control. After incubation for 2 h, 25 mg/mL iodixanol was added to each well and incubated for 3 h. The cells were lysed with RIPA buffer and supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) immediately before use. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit according to the manufacturer’s instructions, and bovine serum albumin (BSA) was used as the standard protein. Equal amounts (20 μg/lane) of protein sample were separated via electrophoresis in 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto PVDF transfer membranes [64 (link)]. Proteins were analyzed with epitope-specific primary antibodies to JNK, phospho-JNK, p44/42 MAP Kinase (ERK), phospho-p44/42 (pERK), p38 MAP Kinase, phospho-p38, cleaved caspase-8, cleaved caspase-3, PPAR-γ, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and horseradish peroxidase (HRP) conjugated anti-rabbit secondary antibodies. Bound antibodies were detected using ECL Advance Western Blotting Detection Reagents and visualized on a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).