Ripa buffer with protease inhibitors

MEFs were cultured in complete MEF medium overnight and then treated for 24 h with 3 μM 4OH-PCB11 or vehicle (dimethyl sulfoxide, DMSO, 0.1% v/v). Cells were collected, washed with cold phosphate buffered saline (PBS), and lysed in a radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (Roche, Indianapolis, IN, USA). Protein concentrations were determined with the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific). Protein samples were diluted in 1X Laemmli Sample buffer with 5% 2-beta mercaptoethanol (2-BME) in order to load 30 μg of protein to each lane of a 5–14% gradient Mini-PROTEAN TGX gel (Bio-Rad, Hercules, CA, USA). Proteins were transferred to a nitrocellulose membrane and blocked for 1 h in Odyssey Blocking Buffer (LI-COR, Lincoln, NE, USA), then incubated overnight in a 1:1000 dilution of the target antibody (MnSOD, Millipore, Burlington, MA, USA; SIRT3, Cell Signaling, Danvers, MA, USA). β-actin was used as a loading control (Cell Signaling). After washing, the blots were incubated in a 1:10,000 dilution of LI-COR anti-mouse/anti-rabbit fluorescent antibodies for 40 min and visualized with the Odyssey Fc Imaging System (LI-COR).

RIPA buffer with protease inhibitors (Roche) was used to lyse cellular proteins, and an equal amount of proteins was electrotransferred onto a polyvinylidene difluoride membrane (Millipore). The main and secondary antibodies were used to incubate the protein, which was then identified using enhanced chemiluminescence methods.