Ab134988

Whole-cell lysates and mitochondrial lysates were taken as small fractions from the samples during MS sample preparation steps, measured by BCA quantification, and mixed with 5× SDS-PAGE loading buffer to a final concentration of 1×. Samples of nondiabetic and diabetic mice were adjusted to the same protein concentration using 1× SDS-PAGE loading buffer based on the BCA quantification. Immunoblotting was performed with 4–15% gradient Tris-glycine SDS-PAGE gel and 0.20 μm PVDF membrane. Rabbit anti-CPT1B pAb (Abcam, ab134988, 1:1,000 dilution), rabbit anti-ACSM2A (Abcam, ab181865, 1:2,000 dilution), and rabbit anti-Aldh3a2 (Abcam, ab184171, 1:2,000 dilution) were used as primary antibodies while HRP-linked anti-rabbit IgG (Cell Signaling Technology, 7074 S, 1:5,000 dilution) was used as the secondary antibody.

Immunoblotting was performed with anti-CPT1A (1/1,000) (ab128568; Abcam), anti-CPT1B (1/1,000) (ab134988; Abcam), anti-CPT1C (1/1,000) (ab87498; Abcam), anti-CTP2 (1/1,000) (ab181114; Abcam), anti-heat-shock protein-60 (HSP60) (1/1,000) (ab46798; Abcam), anti-TOMM20 (1/1,000) (ab56783; Abcam), anti-NDUFS1 (1/500) (sc-50132; Santa Cruz Biotechnology), anti-UQCRC2 (1/1,000) (ab14745; Abcam), anti-GFAP (1/500) (G6171; Sigma), anti-NDUFB8 (1/1,000) (ab110242; Abcam), anti-NDUFA9 (1/1,000) (ab14713; Abcam), anti-SDHA (1/1,000) (ab14715; Abcam), anti-MTCO1 (1/1,000) (ab14705; Abcam), anti-COX IV (1/1,000) (ab16056; Abcam), anti-Iba1 (1/1,000) (019-19741; Wako), anti-MAP2 (1/1,000) (ab32454; Abcam), anti-OLIG2 (1/1,000) (ab109186; Abcam), anti-PDHA1 (1/1,000) (no. 3205; Cell Signaling), anti-phosphoSer²⁹³-PDHA1 (1/1,000) (no. 31866; Cell Signalling), anti-β-Tubulin III (1/300) (T2200; Sigma) and anti-β-actin (1/30,000) (A5441; Sigma).

Cells were lysed in RIPA buffer with Triton X-100 supplemented with protease/phosphatase inhibitor cocktail (Roche)^{77 (link)}. Protein concentrations were measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of proteins were resolved by SDS-PAGE. The antibodies against CPT1A (ab128568) and CPT1B (ab134988) were obtained from Abcam (Cambridge, MA). The antibodies for β-actin (#4970) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#5174) were from Cell Signaling Technology (Danvers, MA). The antibody against HADHA (#PA5-27348) was obtained from Thermo Fisher Scientific. Immunocomplexes were visualized by an enhanced chemiluminescence detection kit (Thermo Fisher Scientific) using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology).
Cellular proteins (100 μg) from MCF-7 and T47D cells treated with or without etomoxir were immunoprecipitated with 3 μg anti-CPT1A antibody (ab128568) and 20 μl Dynabeads™ Protein G (Thermo Fisher Scientific). The immunoprecipitates and original cell lysates (input control) were separated by SDS-PAGE and blotted with the same CPT1A antibody as described above.