Ab128568

Western blotting analysis was performed as described previously.17 Proteins were fractionated using NuPAGE 4% to 12% Bis‐Tris gels (Invitrogen) and transferred to Protran nitrocellulose transfer membrane (Whatman, Japan). The membrane was blocked using PBS containing 5% nonfat milk for 30 minutes at room temperature and incubated with the primary antibody (anti‐ABCA1: 1:2000, NB400‐105; anti‐ABCG1 (anti‐ATP binding cassette G1): 1:2000, NB400‐132 [Novus Biologicals, CO], anti–carnitine palmitoyltransferase Ia: 1:2000, ab128568 [Abcam, UK]; anti–β‐actin: 1:3000, A5441 [Sigma‐Aldrich]) overnight at 4°C. The membrane was washed in PBS containing 0.05% Tween‐20, and then it was incubated with the secondary antibody (anti‐rabbit IgG horseradish peroxidase linked: 1:2000; anti‐mouse IgG horseradish peroxidase linked: 1:2000 [GE Healthcare, UK]) for 30 minutes at room temperature. The membrane was washed in PBS containing 0.05% Tween‐20 and incubated with ECL Western Blotting Detection Reagent (GE Healthcare) for 5 minutes; then, the signal was detected using an LAS‐4000 system (Fuji Film, Japan).

The tumor tissues fixed in 4% paraformaldehyde (at room temperature for 24 h) were embedded in paraffin and then sectioned into 5-µm-thick slices. Immunohistochemistry was performed using an Instant SABC-POD kit (cat. no. SA1020, Boster Biological Technology) according to the manufacturer's instructions. Briefly, after dewaxing, rehydration and antigen retrieval, the tumor sections were blocked and were then separately incubated with primary antibodies against pan-cytokeratin (AE1/AE3; cat. no. ab27988, Abcam, dilution: 1:200) and CPT1A (cat. no. ab128568, Abcam, dilution: 1:200) overnight at 4°C. After washing, the sections were incubated with biotin-conjugated secondary antibody (cat. no. SA1020, Boster Biological Technology) at 37°C for 30 min. After washing, the sections were incubated with SAB at 37°C for 30 min and stained with DAB (cat. no. AR1027, Boster Biological Technology) at room temperature for 5 min. Following DAB visualization, the sections were immersed into water and then re-stained using hematoxylin at room temperature for 3 min. The AE1/AE3-positive cells or the CPT1A-positive cells were finally observed under a light microscope (Nikon Corporation).

Isolation of mitochondria from mouse heart and colon was performed by differential centrifugation (46 (link)). Twenty micrograms of isolated mitochondria or 50 μg of total protein extracts was resuspended in 4× Laemmli buffer. Proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) using 12% precast gels (Invitrogen) and then transferred onto polyvinylidene difluoride membranes (GE Healthcare). Immunodetection was performed according to standard techniques using enhanced chemiluminescence Immun-Star HRP Luminol/Enhancer (Bio-Rad). The following antibodies were used: SDHA (ab14715, Abcam), Ndufs3 (ab14711, Abcam), VDAC/Porin (ab128568, Abcam), ATP synthase subunit alpha (Molecular Probes A21350, Thermo Fisher Scientific), UQCRC1 CIII subunit core1 (Molecular Probes A21362, Thermo Fisher Scientific), COX I (459600, Invitrogen), TFAM (rabbit polyclonal antisera against TFAM generated using recombinant mouse protein), and GAPDH (ab8245, Abcam).