Mouse anti acox1 sc 517306

The total protein of adipocyte samples was extracted by using RIPA lysis buffer supplemented with phenylmethylsulfonyl fluoride (Servicebio, Wuhan, China) (100:1). Proteins in cell lysates were separated by 10% SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Millipore, Boston, MA, USA). The membranes were blocked with 5% nonfat milk for 1 h and then incubated with mouse anti-ACAA1 (sc-514051, Santa Cruz, Dallas, TX, USA), and mouse anti-ACOX1 (sc-517306, Santa Cruz, Dallas, TX, USA) antibodies at 4 °C overnight, followed by incubation with a secondary antibody conjugated with horse radish peroxidase (HRP) (GB23303, Servicebio) for 1 h at room temperature. Mouse anti-actin (Servicebio,) was used as the internal control. Signals were enhanced by ECL Plus (Solarbio, Beijing, China), and images were captured and analyzed by Photoshop CS6 and AlphaView 3.0 (Alpha Innotech, San Jose, CA, USA).

Carotid endarterectomy and lower extremity tissue were also homogenized in cold mammalian cell lysis kit (MCL1-KT; Millipore Sigma). Total protein for both Min and Max-diseased was determined using Bradford Protein Assay and loaded onto Bis-Tris gel (NW00082BOX; Thermo Fisher Scientific) and transferred to polyvinylidene fluoride membranes for Western blotting. Protein was detected with mouse anti-PPARα (SAB4502260; Millipore Sigma), mouse anti-ACOX1 (sc-517306, Santa Cruz), and mouse anti-CPT1a (66039–1-Ig; Proteintech). Rabbit anti-beta actin (ab8227, Abcam) or rabbit anti-GAPDH (G9545, Sigma) was used for Western blot loading controls. Band densitometry analysis was performed using ImageJ software as previously described (27 (link)). Band densities were expressed as ratios relative to the protein loading control.