Gtx81126

Cells and tissues were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific), followed by sonication and centrifugation (10 min at 12,000 rpm at 4°C). Protein concentration was determined by the BCA method. Proteins were separated on Bolt TM 4-12% Bis-Tris Plus gels (Invitrogen, Thermo Fisher Scientific), blotted onto a nitrocellulose membrane (926-31092, LI-COR) and detected using the following primary antibodies: anti-ACOX1 (ab184032, Abcam), anti-ACAA1 (12319-2-AP, Proteintech), anti-EHHADH (GTX81126, Genetex), anti-HSD17B4 (ab128565, Abcam), anti-HMGCR (AMAb90618, Atlas Antibodies), anti-citrate synthase (GTX628143, Genetex), and anti-α-tubulin (32-2500, Thermo Fisher). Anti-MVK and anti-PMVK antibodies were a gift from Dr. Hans Waterham (AMC, Amsterdam) (Hogenboom et al. 2004b,a) . Proteins were visualized with goat anti-mouse and goat anti-rabbit secondary antibodies IRDye 800CW and IRDye 680RD (LI-COR, 926-32 210, 926-68 070, 926-32 211, 926-68 071) . Band intensities were quantified using the Fiji distribution of ImageJ 1.x (Schindelin et al. 2012) .

Livers were collected after euthanasia and fixed by immersion in 10% formalin (Thermo Fisher Scientific) for 24 hours, washed in PBS and transferred to 70% EtOH in water until they were embedded in paraffin blocks. Serial sections (4 µm thick) were cut with a microtome.
Hematoxylin & Eosin (H&E) and periodic acid Schiff (PAS) staining were performed following standard protocols, and the sections were analyzed by light microscopy. EHHADH immunofluorescence to detect hepatic peroxisomes was performed as previously described (Ranea-Robles et al. 2021a) using a specific anti-EHHADH antibody (dilution 1:50, GTX81126, Genetex). Microscopy images were taken with a Nikon Eclipse 80i microscope and the NIS-Elements BR 5.20.01 software (Nikon). Images were analyzed with ImageJ (Schindelin et al. 2012) (link).