Use of FBPaldolase in preparation of AMPK activating drugs
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
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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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