Effect of Lipopolysaccharide and TNFα on Neuronal Ascorbic Acid Uptake

Figure 3

Effect of exposure of SH-SY5Y cells and mouse brain with TNFα on different aspects of SVCT2 functional expression. (a) SH-SY5Y cells were serum-deprived overnight and exposed to TNFα (20 ng/ml). After 48 h, AA uptake was performed. (b) SH-SY5Y cells were pretreated with TNFα for 48 h, and the total protein was prepared and used to perform western blotting to determine hSVCT2 protein expression levels. (c) Total RNA isolated from TNFα pretreated SH-SY5Y cells was utilized to determine the level of hSVCT2 mRNA expression by RT-qPCR. (d) SLC23A2 full-length promoter activity was determined in TNFα pretreated SH-SY5Y cells. (e) Protein samples from mouse brain tissue of TNFα (15 μg/mouse; 72 h) exposed and control mice were isolated to perform western analysis to determine mSVCT2 protein expression levels. (f) Total RNA isolated from TNFα-injected and control mouse brain was used to determine the mSVCT2 mRNA expression by RT-qPCR. (g) Total RNA samples prepared from TNFα-administered and control mouse brain were subjected to determination of the mSVCT2 hnRNA expression levels by RT-qPCR. Values are of at least 3-5 separate investigations using different batches of SH-SY5Y cells or at least 3-4 pairs of mice. ; ; .

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