Use of fbp aldolase in the preparation of drugs for activating AMPK
A drug and application technology, applied in the direction of drug combination, application, microorganisms, etc., can solve the problems of inhibiting aldolase and the lack of universality of aldolase inhibition, and achieve the effect of overcoming difficulties
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Embodiment 1
[0099] Embodiment 1: shRNA suppresses the expression of ALDOA-C gene
[0100] 1. Experimental materials and main reagents
[0101] Cell line: mouse fibroblast MEF, which is an immortalized MEF, that is, after the fibroblasts are isolated from mouse embryos, they are transfected with SV40 T antigen to make the cells immortal. For the construction method, please refer to the literature Lei Y, Methods MolBiol. 2013; 1031:59-64. Generation and culture of mouse embryonic fibroblasts.
[0102] Human embryonic kidney cells HEK293T (cat.CRL-3216) were purchased from ATCC.
[0103] Vector pLVX-IRES (cat. #631849) was purchased from Clontech.
[0104] Transfection reagent: Lipofectamine 2000 (cat.11668-027), purchased from Invitrogen.
[0105] Doxycycline (cat. S4163) was purchased from Selleckchem.
[0106] Dulbecco's modified Eagle's medium (DMEM, Gibco, cat. 11965) was purchased from Thermofisher.
[0107] Primary antibody used in Western:
[0108] anti-ALDOA (cat.#8060), pur...
Embodiment 2
[0142] Example 2: shRNA targeting ALDOA-C can activate AMPK
[0143] 1. Experimental materials and main reagents
[0144] Primary antibody used in Western:
[0145] Rabbit 548anti-phospho-AMPKα-T172 (cat#2535), purchased from Cell Signaling Technology;
[0146] anti-AMPKα (cat#2532, 1:1000 for IB), purchased from Cell Signaling Technology;
[0147] anti-phospho-ACC-Ser79 (cat.#3661, 1:1000 for IB), purchased from Cell Signaling Technology;
[0148] anti-ACC (cat.#3662, 1:1000 for IB), purchased from Cell Signaling Technology.
[0149] Other primary and secondary antibodies used are as described in Example 1.
[0150] DMEM without glucose (Gibco, cat. 11966) was purchased from Thermofisher.
[0151] 2. Experimental method
[0152] (1) First, with reference to the method described in Example 1, prepare the following figure 1 ALDOA-C knockdown MEF cells are shown. Among them, the shRNA targeting GFP (Green Fluorescent Protein Green Fluorescent Protein) is used as the co...
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