Hypoxylonol F Protects Renal Cells from Cisplatin Toxicity

The protective effect of hypoxylonol F (2) against cisplatin-induced renal cellular pathway was determined by Western blotting analysis [28 (link)]. LLC-PK1 cells were seeded into 6-well culture plates (4 × 10⁵ cells/well) and treated with hypoxylonol F (2) as described above, washed twice with cold phosphate-buffered saline. Cells were scraped from the plates and lysed in RIPA buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) supplemented with 1× EDTA-free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) according to the manufacturer’s instructions. A bicinchoninic acid protein assay was used for the quantitation of protein of samples, and 20 μg of protein were separated using 10% SDS PAGE gels for 90 min at 80 V. The proteins were transferred to PVDF transfer membranes, which were incubated with primary antibodies against phospho-p38, p38, phospho-JNK, JNK, phospho-ERK, ERK, cleaved caspase-3, and GAPDH (1:1000 dilution). Horseradish peroxidase-conjugated anti-rabbit IgG (1:2000 dilution) was used to detect the primary antibodies. Bound antibodies were visualized using ECL Advance Western Blotting Detection Reagents (GE Healthcare, Cambridge, UK). GAPDH was utilized as a loading control.