Extraction of total protein and RNA were performed according to standard protocols (19 (link), 23 (link)). The primary antibodies used in this study included CPS1 (Proteintech, #18703-1-AP), AOX1 (Proteintech, # 19495-1-AP), OXCT1 (Proteintech, #12175-1-AP), NME4 (Bioworld, #BS71176) and β-Actin (CST, #4967). Oligonucleotide primers used for detection of human-TRDMT1, SMS, UAP1, WARS2, PLOR3G, NNT, GAPDH (21 (link)) were described in Supplementary Figure S3
Oxct1
Manufactured by Proteintech
Sourced in United States
OXCT1 is a protein involved in the metabolism of ketone bodies. It catalyzes the transfer of Coenzyme A from acetoacetyl-CoA to acetate, forming acetyl-CoA.
Automatically generated - may contain errors
Lab products found in correlation
Nf200, by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
Smi31, by Fortrea (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Ruvbl2, by Proteintech (1 mentions)
Hsd17b4, by Proteintech (1 mentions)
Eif3a, by Proteintech (1 mentions)
Txnrd1, by Proteintech (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
β actin, by Sangon (1 mentions)
5 protocols using oxct1
1
Protein, RNA, and Gene Expression Analysis
2
Antibody Panel for Cell Characterization
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The following antibodies were used: actin (Sigma A3853), ACO2 (Sigma, HPA001097), APP (Chemicon MAB348), ATF6 (Abcam 40256), CAII (Said Ghandour), GFAP (Chemicon MAB3402), Iba1 (Wako 019-19741), MAC3 (Pharmigen 01781D), NF200 (Sigma N4142), Olig2 (Charles Stiles/John Alberta, DF308), OXCT1 (Proteintech Europe 12175-1AP), SMI31 (Covance SMI-31P), VDAC (Rockland), isolectin IB4 coupled to Alexa 594 (Vectorlab). For generation of MCT1 (SLC16a1) antisera, rabbits were immunized with the intracellular peptide 221–236 of mouse MCT1 (CDANTDLIGGSPKGEKL). Anti-MCT1 antibodies were purified by affinity chromatography.
3
Western Blotting of Cellular Proteins
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Antibodies against WHSC1 (1:1000, 65127S; Cell Signaling Technology, Beverly, MA, USA), mTOR (1:1000, 2983T; Cell Signaling Technology, Beverly, MA, USA), P70S6K (1:1000, 9204S; Cell Signaling Technology, Beverly, MA, USA), S6 (1:1000, 9204S; Cell Signaling Technology, Beverly, MA, USA), p-mTOR (1:1000, 5536T; Cell Signaling Technology, Beverly, MA, USA), p-P70S6K (1:1000, 9204S; Cell Signaling Technology, Beverly, MA, USA), p-S6 (1:1000, 4858T; Cell Signaling Technology, Beverly, MA, USA), OXCT1 (1:1000, 12,175-1-AP; Proteintech, Wuhan, Hubei, China), RUVBL2 (1:1000, 10,195-1-AP; Proteintech, Wuhan, Hubei, China), TXNRD1 (1:1000, 11,117-1-AP; Proteintech, Wuhan, Hubei, China), HSD17B4 (1:1000, 15,116-1-AP; Proteintech, Wuhan, Hubei, China), PKM (1:1000, 10,078-2-AP; Proteintech, Wuhan, Hubei, China), EIF3A (1:1000, 26,178-1-AP; Proteintech, Wuhan, Hubei, China), TPM4 (1:1000, 13,741-1-AP; Proteintech, Wuhan, Hubei, China), and P4HB (1:1000, ab2792; Abcam, Cambridge, UK) were used in this study. Western blotting was performed as previously described.17 (link)
4
Western Blot Protein Extraction
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To extract total protein, cells were washed twice with cold PBS and lysed in 1 ml RIPA buffer (50 mM Tris,150 mM NaCl,1% NP-40,0.5% sodium deoxycholate) and debris was removed by centrifugation at 13,500 g at 4°C. Polyvinylidene fluoride membranes (Millipore) were incubated with specific antibodies against BDH1, OXCT1 (at dilutions of 1:500 and 1:1000, respectively; Proteintech, Chicago, IL) and β-actin (Sangon). Then, samples were incubated with HRP-coupled anti-mouse secondary antibodies (Sangon) and visualized using enhanced chemiluminescence (Pierce, Rockford, IL).
5
Immunohistochemical Analysis of BDH1 and OXCT1
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Xenograft tumor tissue samples were fixed in 10% formalin, embedded in paraffin and cut into 4 μm-thick sections by using routine methods. For immunohistochemical staining, all procedures were performed according to the manufacturer’s protocol. The BDH1 and OXCT1 antibodies (Proteintech) were diluted at 1:50 and 1:200, respectively. Color development was carried out using chromogen (3, 3′-diaminobenzidine) reagent and hematoxylin was used as a counterstain. Finally, the slides were examined using a light microscope.
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