Anti mouse anti rabbit fluorescent antibodies

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).

HepG2 cells were plated 1 × 10⁶ and treated with IC50 and IC90 doses for 1 h, 3 h, 6 h, and 24 h, and the cells were collected. The protein concentrations were analyzed using the Pierce BCA protein assay kit and standard curve (23225, Thermo Fisher, United States). Protein samples were then diluted in 1X Laemmli sample buffer with 5% 2-beta mercaptoethanol (2-BME) and evenly loaded with 25 µg of protein per lane in a 5–14% gradient Mini-PROTEAN TGX gel (456-1034 or 456-1084, Bio-Rad, United States). The gels were run for 60 min at 90 mV and transferred to nitrocellulose blot paper and run for 60 min at 100 mV at 4°C. The blots were blocked for 1 h in Odyssey Blocking Buffer (LI-COR, Lincoln, NE, United States) then incubated in a 1:1000 dilution of the target antibody GPx4 overnight (ab125066, Abcam, MA, United States). The loading control antibody lamin-A/C was incubated for 3 h (4777, Cell Signaling, MA, United States). After washing, the blots were incubated in a 1:10,000 dilution of LI-COR anti mouse/anti-rabbit fluorescent antibodies for 40 min. Blots were stored in 1X PBS. Imaging was visualized by using an Odyssey Fc Imaging System (LI-COR, Lincoln, NE, United States). Blots were exposed for 2 minutes each in the 700 and 800λ channels. Analysis of densitometry was completed with LI-COR software.