Preparation of Lipids, Proteins, and Cellular Components

NAD, NADH, AICAR, hexanoic acid, octanoic acid, octanal, betaine aldehyde, betaine, and 2-aminoadipic acid were obtained (Sigma). Cholesterol, sphingomyelin, DOPC, DOPE, DOPS, and phosphatidic acid (PA) were obtained (Avanti). Alexa 546-conjugated transferrin (Tf), Alexa 555-conjugated forms of EGF, dextran, and CT were also obtained (Invitrogen). Fugene 6 Transfection Reagent was from Promega. RNAiMax was from Invitrogen. Preparations of coatomer, ARF1, ARFGAP1, BARS, Golgi membrane, and cytosol have been described^{14 (link),16 (link)}. To generate recombinant ALDH7A1, the human cDNA was expressed in bacteria (BL21) using the pET-15b vector (Novagen), followed by isolation of the 6×-his tagged form of ALDH7A1 using a nickel column. AMPK was purified from cells by transfection of a flag-tagged construct into HeLa cells, followed by immunoprecipitation of the tagged construct from cell lysates using an anti-flag antibody (M2).

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Yang J.S., Hsu J.W., Park S.Y., Lee S.Y., Li J., Bai M., Alves C., Tseng W., Michelet X., Ho I.C, & Hsu V.W. (2019). ALDH7A1 inhibits the intracellular transport pathways during hypoxia and starvation to promote cellular energy homeostasis. Nature Communications, 10, 4068.

Publication 2019

AICAR hexanoic acid 2-aminoadipic acid Fugene 6 Transfection Reagent RNAiMax

2 aminoadipic acid Aicar Anti antibody Bacteria Bars Betaine Betaine aldehyde Cdna Cell Cholesterol Coatomer Cytosol Dextran Dopc Dops Fugene 6 Golgi Hela cells Hexanoic acid Human Immunoprecipitation Isolation Membrane Nadh Nickel Octanal Octanoic acid Phosphatidic acid Promega Sphingomyelin Transfection Transferrin Vector

Corresponding Organization : Harvard University

Other organizations : Kansas State University

Top 5 similar protocols

Variable analysis

independent variables

Transfection of a flag-tagged AMPK construct into HeLa cells

dependent variables

Immunoprecipitation of the tagged AMPK construct from cell lysates using an anti-flag antibody (M2)

control variables

Expression of the human ALDH7A1 cDNA in bacteria (BL21) using the pET-15b vector (Novagen)
Isolation of the 6x-his tagged form of ALDH7A1 using a nickel column

positive controls

Preparations of coatomer, ARF1, ARFGAP1, BARS, Golgi membrane, and cytosol

negative controls

Not mentioned

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