Alpha ketoglutarate alpha kg assay kit

Cells were plated at 3 × 10⁶ cells per dish in 10 cm dishes and treated with either 10 μM NCT-inactive or 10 μM NCT-inactive with 15 μM NCT-503 for 48 h. PHGDH activity was assessed using cell lysates in the PHGDH Activity Assay Kit (Cat # PK-CA577-K569, PromoCell, Heidelberg, Germany) per manufacturer’s protocol. Extracellular lactate levels were assessed using extracellular medium in the PicoProbe Lactate Fluorometric Assay Kit (Cat # K638, Biovision, Milpitas, CA) per manufacturer’s protocol. Total and serine-derived αKG levels were assessed using cell lysates in the Alpha Ketoglutarate (alpha KG) Assay Kit (Cat # ab83431, Abcam, Cambridge, United Kingdom) per manufacturer’s protocol.

IDH3 activity was measured using the Isocitrate Dehydrogenase Assay Kit (Colorimetric) (ab102528, Abcam), while L-lactate levels were assessed with L-lactate Assay Kit (Colorimetric/Fluorometric) (ab65330, Abcam). The quantification of NAD⁺ and NADH was conducted using the NAD⁺/NADH Assay Kit (Colorimetric) (ab65348, Abcam). Alpha Ketoglutarate levels were determined with an Alpha-Ketoglutarate (alpha KG) Assay Kit (ab83431, Abcam). Additionally, ATP levels were measured utilizing the ATP Assay Kit (S0027, Beyotime). The Deproteinizing Sample Preparation Kit—TCA (ab204708, Abcam) was employed to remove proteins from the samples. Details of the reagents and resources utilized are provided in Supplementary Table s5.

Nuclei were isolated from cells with nuclear expression of IDH-2 and PDHC or cells treated with DRN as per the experimental requirements. As per the requirement of the protocol, the samples were prepared using assay buffer or were deproteinated using 13% TCA and precipitated at 15,000×g for 5 min. The pH of the supernatant was adjusted with 2 N KOH to 7.5–8. The concentration of alpha-ketoglutarate was measured using an Alpha Ketoglutarate (alpha KG) Assay Kit (Abcam, ab83431). Acetyl-CoA was measured using a PicoProbe™Acetyl-CoA Fluorometric Assay Kit (BioVision, K317). Pyruvate, malate, succinate, and fumarate were measured using a Pyruvate Assay Kit (Sigma, MAK332-1KT), Malate Colorimetric Assay Kit (BioVision, K637), Succinate Assay Kit (Colorimetric) (Abcam, ab204718), and Fumarate Colorimetric Assay Kit (BioVision, K633), respectively.

The enzymatic activities of PDH, citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate (αKG) dehydrogenase, and succinate dehydrogenase were measured on 10 µg mitochondrial proteins using the Pyruvate Dehydrogenase (PDH) Assay Kit (Abcam), the Citrate Synthase Assay Kit (Sigma Aldrich), the Aconitase Assay Kit (Cayman Chemical), the Isocitrate Dehydrogenase Assay Kit (Sigma Aldrich), the Alpha Ketoglutarate (alpha KG) Assay Kit (Abcam), and the Succinate Dehydrogenase Activity Colorimetric Assay Kit (BioVision, Milpitas, CA), as per the manufacturer’s instructions. Results were expressed as nmoles NADH/mg mitochondrial proteins (PDH, isocitrate dehydrogenase, αKG dehydrogenase), nmoles citrate or isocitrate/mg mitochondrial proteins (citrate synthase, aconitase), and nmoles FADH₂/mg mitochondrial proteins (succinate dehydrogenase).