Pepck

Manufactured by Cayman Chemical

PEPCK is an enzyme that plays a crucial role in gluconeogenesis, the metabolic pathway that generates glucose from non-carbohydrate precursors. It catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, a key step in this process.

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3 protocols using pepck

Endothelial Cell Protein Expression

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Unless otherwise specified, all reagents and chemicals were purchased from Sigma-Aldrich. Antibodies purchased were SIRT1, vascular cell adhesion molecule (VCAM)-1, and activated caspase 3 (Cell Signaling Technology), PEPCK (Cayman Chemical Company), F4/80 (Abcam), DBC1 (Bethyl Laboratories), p53 and p21 (Santa Cruz Biotechnology, Inc.), and actin (Sigma-Aldrich). Human aortic endothelial cells and EGM2 culture media were purchased from Lonza.

Escande C., Nin V., Pirtskhalava T., Chini C.C., Tchkonia T., Kirkland J.L, & Chini E.N. (2014). Deleted in Breast Cancer 1 Limits Adipose Tissue Fat Accumulation and Plays a Key Role in the Development of Metabolic Syndrome Phenotype. Diabetes, 64(1), 12-22.

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Protein Expression Analysis in Cellular Homogenates

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Cells and tissues were homogenized in RIPA buffer (EDM Millipore, 20-188) with phosphatase and protease inhibitor cocktails added. Proteins were separated on a 4–12% SDS polyacrylamide gel, transferred to nitrocellulose paper, and probed with antibodies against mouse and human PEPCK (Abnova, H00005105-M01) and PEPCK (Cayman Chemical, 10004943). Primary antibodies for AMP-activated protein kinase (AMPK; Cell Signaling, #2532), pAMPK (Cell Signaling, #2535), ACC (Cell Signaling, #3662), pACC (Cell Signaling, #3661), poly ADP ribose polymerase (PARP; Cell Signaling, #9532), and Actin (Sigma, A1978) were incubated at a 1:1000 dilution in 5% milk overnight, except Actin which was 1:10,000 dilution. HRP-conjugated secondary antibodies (Jackson Immunoresearch, 715-035-150 and 715-035-152) were used and proteins visualized using enhanced chemiluminescence (ECL; Thermo Scientific, 32106).

Montal E.D., Bhalla K., Dewi R.E., Ruiz C.F., Haley J.A., Ropell A.E., Gordon C., Haley J.D, & Girnun G.D. (2019). Inhibition of phosphoenolpyruvate carboxykinase blocks lactate utilization and impairs tumor growth in colorectal cancer. Cancer & Metabolism, 7, 8.

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Western Blotting of Liver Proteins

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For western blotting, liver lysates were collected in RIPA lysis buffer (Cell Signaling Technology; 9806) with protease/phosphatase inhibitors (Cell Signaling Technology; 5872) using a TissueLyser. Lysates were normalized to protein concentration, denatured, and run on NuPAGE precast 4–12%gels (ThermoFisher Scientific) using MOPS or MES buffers (ThermoFisher Scientific). Separated proteins were transferred to Immobilon PVDF membrane and blocked for one hour with 5% BSA in TBST. The antibodies used in this study were ALT1 (Abcam; ab154034, ALT2 (Abcam; ab202083), MPC1 (Cell Signaling Technology; D2L9I), MPC2 (Cell Signaling Technology; D4I7G), Vinculin (Cell Signaling Technology; 13901), PEPCK (Cayman Chemical; 10004943), PC (Cell Signaling Technology; 66470), CS (Cell Signaling Technology; 14309), LDH (Abcam; ab52488), OXPHOS cocktail (Abcam; ab110413). Blots were imaged using a LI-COR Odyssey system with Image Studio Lite software.

Martino M.R., Habibi M., Ferguson D., Brookheart R.T., Thyfault J.P., Meyer G.A., Lantier L., Hughey C.C, & Finck B.N. (2023). Disruption of Hepatic Mitochondrial Pyruvate and Amino Acid Metabolism Impairs Gluconeogenesis and Endurance Exercise Capacity in Mice. bioRxiv.

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