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Data was presented as fold change in gene expression level in the target sample normalized to GAPDH housekeeping gene and relative to the control sample.
After blocking with 5% non-fat milk for 2 h at room temperature, membranes were incubated with primary antibodies (1:1,000 for GAPDH and 1:800 for others) overnight at 4[degrees]C.
Immunoblotting were performed using a mouse monoclonal LC3 (#12741), p62 (#5114) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) thermo scientific AM4300 which purchased from Cell Signalling Co, Ltd.
During standard PCR reactions, the most intense bands for GAPDH and E.
Antibodies used in the present study included: TNF-[alpha] (1:1000 dilution; 500-P72, PeproTech Inc., USA), NF-[kappa]B p65 (1:1000 dilution; 3034, Cell Signaling, USA), p-NF-[kappa]B p65 (1:1000 dilution; 3033S, Cell Signaling), I[kappa]B[alpha] (1:1000 dilution; 4812S, Cell Signaling), p-I[kappa]B[alpha] (1:1000 dilution; 2697S, Cell Signaling), p-IKK (1:1000 dilution; 2697P, Cell Signaling), iNOS (1:500 dilution; ab3523, Abcam, UK), IL-1[beta] (1:1000 dilution; 500-P80, PeproTech Inc.), IL-6 (1:1000 dilution; 500-P73, PeproTech Inc.), and GAPDH (1:5000 dilution; ab8245, Abcam).
The [C.sub.q] varied according to the gene analyzed: for example, GAPDH and Cyc[C.sub.q] [approximately equal to] 21; whereas, [alpha]-tub and Ubi[C.sub.q] [approximately equal to] 29, showing a wide variation in gene expression (Figure 2).
The membranes were blocked for 60 min with Tris base buffer containing 0.1% Tween 20 (TBST) and 5% nonfat milk and incubated overnight at 4[degrees]C with primary antibodies' against Hdac1 (10197-1-AP, Proteintech, USA, 1:1000)/Hdac2 (12922-3-AP, Proteintech, 1:1000)/Hdac3 (10255-1-AP, Proteintech, 1:500)/Vangl2 (21492-1-AP, Proteintech, 1:1000)/Scrib (C0613, Santa Cruz, USA, 1:100)/Rac1 (D2115, Santa Cruz, 1:200) and Gapdh (CW0100A, CWBIO, 1:500).
Several natural compounds, such as the flavonoids tiliroside [6, 7], 7-hydroxy-4', 6-dimethoxyisoflavone , 3', 4', 5', 5, 7-pentamethoxyflavone , quercetin, guajaverin  and isosakuranetin , besides chalepin and other synthetic derivatives of coumarin [6, 11] and the xanthonoid mangiferin  have shown promising results for the inhibition of the GAPDH enzyme (Figure 1).
Eight commonly used reference genes were selected: nicotinamide adenine dinucleotide dehydrogenase, gapdh, cytochrome c oxidase subunit 1 (cox1), 40S rihosomal protein S27 (rps27), translation initiation factor 5 A (tif5a), 40S rihosomal protein S4 (rps4), act, and 18S.
GDF15 expression in Hep G2 hepatocytes over the course of 24 hours stimulation with IL-6, tumor necrosis factor [alpha] (TNF[alpha]), or lipopolysaccharide (LPS) determined by qPCR and normalised to GAPDH. Data from 3 independent experiments are shown.
The reference genes' (gapdh, [beta]-actin, and 28S rRNA) [C.sub.q] results for each group were tested for stability using NormFinder 20 .
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- Gap/Overlap Analysis Tool
- gap0 period
- gap0 phase
- gap1 period
- gap1 phase
- gap2 period
- gap2 phase
- Gapar Aitiev
- Gapar Aitievich Aitiev
- gape at
- gape at (someone or something)
- gape at her
- gape at him
- gape at it
- gape at me
- gape at one
- gape at somebody
- gape at someone
- gape at something
- gape at them
- gape at us
- gape at you
- Gapeev, Aleksandr
- Gapeev, Aleksandr Aleksandrovich