Western Blot Analysis of Proteins

The separation of investigated proteins was performed by SDS-PAGE, followed by transfer to a PVDF membrane, which was blocked by 5% skimmed milk solution (Szatmári-Tóth et al., 2020 (link); Shaw et al., 2021 (link)). The primary antibodies were utilized in the following dilutions: anti-β-actin (1:5000, A2066), anti-UCP1 (1:750, R&D Systems, Minneapolis, MN, United States, MAB6158), anti-OXPHOS (1:1,000, Abcam, Cambridge, MA, United States, ab110411), anti-SLC7A10 (1:500, Abnova, Taipei City, Taiwan, H00056301-B01P), anti-PGC1α (1:1,000, Novus Biologicals, Centennial, CO, United States, NBP1-04676) anti-GPT2 (1:2000, Thermo Fisher Scientific, PA5-62426), and anti-SHMT1 (1:2000, Thermo Fisher Scientific, PA5-88581). The following species corresponding secondary antibodies were used: HRP-conjugated goat anti-mouse (1:5000, Advansta, San Jose, CA, United States, R-05071-500) or anti-rabbit (1:5000, Advansta, R-05072-500) IgG. The expression of the visualized immunoreactive proteins were quantified by densitometry using the FIJI ImageJ software (National Institutes of Health (NIH), Bethesda, MD, United States) as previously described (Szatmári-Tóth et al., 2020 (link)).