Ppargc1a

Twenty-five µg of protein extract was separated by SDS-PAGE (8% gels) and transferred to nitrocellulose membranes (GE Healthcare, Freiburg, Germany) by semidry electroblotting. The primary antibodies used were against SLC7A11 (rabbit mAb, clone D2M7A, 1:1000, Cell Signaling, Frankfurt, Germany), HIST1H3B (rabbit pAb, PA5-111876, 1:1000, Thermo Fisher Scientific), HK2 (rabbit mAb, clone C64G5, 1:1000, Cell Signaling), PPARGC1A (mouse mAb, clone 1C1B2, 1:3000, Proteintech, Manchester, UK), ALDH6A1 (rabbit pAb, 20452-1-AP, 1:6000, Proteintech), MARC2 (rabbit pAb, 24782-1-AP, 1:1000, Proteintech), SLC47A1 (rabbit mAb, clone D4C62, 1:500, Cell Signaling), and GAPDH (rabbit mAb, clone 14C10, 1:10,000, Cell Signaling). Secondary horseradish peroxidase-conjugated antibodies against rabbit or mouse were purchased from Jackson ImmunoResearch (Suffolk, UK) and used at a concentration of 1:5000. Protein bands were detected by enhanced chemiluminescence in an LAS-4000 chemiluminescence detection system (GE Healthcare, Munich, Germany).

Slides were baked for 60 min at 60 °C, deparaffinized with xylene for 5 min, rehydrated in ethanol (100%, 95%, 70%, and 50%). For antigen retrieval, slides were immersed in 10 mM sodium citrate and subjected to high heat and pressure for 20 min. After cooling, slides were rinsed with PBS for 10 min, and 3% H₂O₂ for 10 min. Slides were blocked in normal goat serum (ABC Kit, Vector Laboratories, Inc.) and 0.3%Triton x-100 in PBS and incubated overnight at 4 °C with primary antibodies: PPAR gamma (Bioss, catalogue no. bs-4590R, 1:200, Woburn, MA), PPARGC1A (Proteintech, catalogue no. 66369-1-Ig, 1:200, Rosemont, IL). YWHAZ (Bioss, catalogue no. bsm-215397M, 1:200, Woburn, MA), ZFYVE27 (Bioss, catalogue no. bs-11777R, 1:200, Woburn, MA). After PBS rinses, slides were incubated with biotinylated secondary antibodies diluted in the blocking solution for 2 hours at RT. The slides were developed using ImmPACT^TM DAB chromogen in ImmPACT^TM DAB diluent (ImmPACT^TM DAB peroxidase substrate kit, catalogue no. SK-410, Burlingame CA) and counterstained with hematoxylin. Slides were then dehydrated in ethanol (50%, 70%, 95%, 100%), immersed in xylene and coverslipped with DEPEX mounting medium (Electron microscopy sciences, cat# 13514, Hatfield, PA).