Anti acox1

Tumor or liver tissues and cells were lysed by 1% SDS lysis buffer and boiled at 95 °C for 10 min, then centrifuged at 12,000 rpm for 10 min at room temperature. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). The protein (35 μg) was separated by SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 2h by 5% fat free milk in TBST at room temperature, followed by incubation overnight at 4 °C with the primary antibodies for anti-Glut1 (Abcam, Cambridge, UK), anti-Bcl2 antibody (Abcam), anti-Pparg antibody (Proteintech, Wuhan, Hubei, China), anti-Acaa1 (Proteintech), anti-Acadm (Proteintech,), anti-CD36 (Bioworld, Nanjing, China), anti-Hadh (Proteintech), anti-RXRA (Proteintech), anti-ACOX1 (Proteintech), and anti-β-actin (Sigma), respectively. The membranes were washed with TBST and incubated for 1 h with the corresponding HRP-conjugated second antibodies. Bands were visualized with ECL Reagents (Merck, Billerica, MA, USA).

Fresh CDX/PDX tumors and tissue samples were fixed in 4% PFA and were embedded in paraffin to prepare formalin-fixed paraffin-embedded blocks, following which the blocks were cut into 5-μm paraffin sections. The paraffin sections were sequentially subjected to dewaxing (dewaxing agent), dehydration (gradient alcohol), microwave thermal repair (sodium citrate buffer solution), and blocking (10% goat serum in PBS). The following primary antibodies were used and incubation was performed overnight at 4°C: anti-CD147 (1:500, Abcam), anti-HIF-1α (1:200, Abcam), anti-PPARα (1:100, Abcam), anti-GLUT1 (1:500, Abcam), anti-HK2 (1:100, Abcam), anti-PKM2 (1:200, CST), anti-LDHA (1:200, Proteintech), anti-ACOX1 (1:200, Proteintech), anti-CPT1A (1:100, Proteintech), anti-CPT2 (1:200, Proteintech), and anti-Ki67 (Proteintech, 1:5000). The next day, the sections were incubated with HRP-conjugated anti-rabbit/mouse IgG (ZSGB-BIO, China) and reactions were detected by performing DAB staining (ZSGB-BIO, China). The nuclei were subjected to staining procedures with hematoxylin and were differentiated in 1% acid alcohol, following which the sections were rinsed with running water. Finally, the slides were sealed with neutral gel. Images were acquired by using the Axio Scope A1 microscope (Zeiss, Germany).