Oxphos rodent wb antibody cocktail

Western blots were performed following standard procedures and employed antibodies specific for TRF2 (Novus Biologicals, NB110‐57130), RAP1 (Cell Signaling, D9H4), Lamin B1 (Santa Cruz Biotechnology, sc‐374,015), and Rhodamine Anti‐GAPDH (BioRad). Briefly, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (MiniComplete, Roche). Following protein quantitation, 20–40 μg of protein were separated under reducing conditions using SDS‐PAGE and transferred to PVDF membranes. Proteins were blotted with antibodies specific to the desired protein and visualized on a ChemiDoc MP gel documentation system (BioRad). Mitochondrial electron transport chain proteins were identified using the antibody mix and instructions included in the OxPhos Rodent WB Antibody Cocktail (ThermoFisher) according to the manufacturer's protocol.

Separation of isolated proteins was achieved by SDS-PAGE with 12% gels (TGX Stain-Free FastCast Acrylamide Solutions, CAT# 1610185, Bio-Rad) and 20 µg total protein per lane. Total protein was detected for semi-quantification using Stain-Free imaging according to the manufacturer’s instructions (Bio-Rad). Wet transfer was conducted to a PVDF membrane. Membranes were incubated with primary antibodies UCP1^{63 (link)}, OXPHOS Rodent WB Antibody Cocktail (CAT# 45-8099, Thermo Fisher), DAT (dopamine transporter) (CAT# ab184451, Abcam), MAO-A (monoamine oxidase A) (CAT# ab126751, Abcam), or SERCA2 (sarco/endoplasmic reticulum Ca²⁺-ATPase) (CAT# 4388, Cell Signaling) overnight at 4 °C. Incubation with a secondary antibody (Polyclonal Goat Anti-Rabbit Ig/HRP, CAT# P0448, Dako; Polyclonal Goat Anti-Mouse Ig/HRP, CAT# P0447, Dako) was performed for 1 h at room temperature, before bands were detected with Clarity Max Western ECL substrate (CAT# 1705062, Bio-Rad) and a ChemiDoc Touch Imaging System (Bio-Rad). Semi-quantitative analysis was performed in Image-Lab (Bio-Rad). Target protein abundance was normalized to total protein.