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Ex 527

Manufactured by R&D Systems
Sourced in China

EX 527 is a small molecule screening compound. It functions as a potent and selective SIRT1 inhibitor. The product is supplied as a solution in DMSO.

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2 protocols using ex 527

1

Signaling Pathway Modulation Assay

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Tumor necrosis factor alpha (TNF-α) was purchased from Research Diagnostics Inc. (Flanders, NJ). Interleukin 1- β (IL1-β), interleukin- 6 (IL-6), IL-6 soluble receptor and EX 527 were purchased from R&D Systems (Minneapolis, MN). Antibodies against total and phosphorylated extracellular-regulated protein kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated kinase, nuclear factor κB (NFκB) [including inhibitory κB (IκB) α and β], phospho-Hsp27, and IGFBP-4 were purchased from Abcam (Cambridge, MA). Antibodies against total and phosphorylated AKT were purchased from Novus Biologicals (Littleton, CO). SB203580, SP600125, rolipram and splitomycin were purchased from Enzo Life Sciences (Farmingdale, NY). Bay 11-7082 was purchased from Calbiochem (San Diego, CA). Resveratrol was purchased from Sigma-Aldrich (St Louis, MO). Reagents for SDS-PAGE, mini-gels and blocking buffer were purchased from Bio-Rad Laboratories (Richmond, CA) and tissue culture supplements and fetal bovine serum were purchased from Life Technologies (Grand Island, NY). A769662 was purchased from Santa Cruz Biotechnology (Dallas, TX).
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2

Activation and Modulation of Primary Astrocytes

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Primary astrocytes from neonatal Kunming mice were obtained as described previously (Kong et al., 1996; Li et al., 2017). In brief, the mouse cortex was dissociated with 0.05% trypsin/ethylenediaminetetraacetic acid and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The medium was changed after 3 days and every 3 days thereafter. An astrocyte culture with a purity of greater than 90% was obtained on day 9 by shaking at 250 r/min for 24 hours. On day 9, the astrocytes were activated by exposure to 1 ng/mL interleukin-1β (IL-1β) as previously described (Sticozzi et al., 2013; Li et al., 2017). The astrocytes (1 × 106) were pretreated with 0.1 or 0.26 U/mL batroxobin (Yang et al., 2011), 10 μM EX527 (test concentration data are shown in Additional Figure 1) or dimethyl sulfoxide (as a vehicle) for 30 minutes in serum-free media before stimulation with IL-1β (R & D Systems, Jiangsu, China). Astrocytes in the control group did not receive any treatment.
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