Complete protease inhibitor

Untreated cells (control) or cells treated with the CT-Fe₂O₃-NPs were rinsed with PBS and scraped from the culture plates in the presence of RIPA buffer supplemented with Complete Protease Inhibitor (MedChemExpress, NJ). Cells were lysed using probe sonication (Fisher Scientific, CL-18) for 10 min at 20 kHz using a pulse of 1 s⁻¹ and 40% acoustic power. Samples were kept on ice during sonication to prevent overheating. Samples were then centrifuged at 15 000 rpm for 20 min, and the supernatants were collected and subjected to protein quantification. The PAGE (polyacrylamide gel electrophoresis) on 6–10% SDS gels was performed followed by semi-dry transfer to PVDF membranes using a Bio-Rad Trans-Blot Turbo Transfer System and detection using antibodies against t-ERK (1:1000), p-ERK (1:1000), NOS (1:200), and GAPDH (1:2,000) (Cell Signaling Technology, MA). Blots were scanned with both calorimetry to image molecular markers and chemiluminescence to capture the protein signal intensity using a Bio-Rad imager.

After the completion of in-life studies, mice treated with the different control, plain and drug-loaded nanoparticles were euthanized, and the kidneys were collected and rinsed with PBS. Approximately 100 mg of tissue samples were placed in RIPA buffer supplemented with Complete Protease Inhibitor (MedChemExpress, NJ). Tissues were homogenized using probe sonication (Fisher Scientific, CL-18) for 15 min at 30 kHz using a pulse of 5 S⁻¹ and 45% acoustic power under the ice. Samples were then centrifuged at 20,000 rpm for 15 min, and the supernatants were collected and subjected to protein quantification. The PAGE (polyacrylamide gel electrophoresis) on 8–10% SDS gels was performed, followed by semi-dry transfer to PVDF membranes using a Bio-Rad Trans-Blot (Turbo Transfer) machine and detection using antibodies against p-S6 (1:000), p-S6K (1:1000), t-S6 (1:2000), t-S6K (1:2000) and GAPDH (1:1000) (Cell Signaling Technology, MA). Blots were scanned with both calorimetries to image molecular markers and chemiluminescence to capture the protein signal intensity using a BioRad Geldoc imaging system.