Figure 3

Effect of LPS treatment on gDAF RNA and protein expression in lymphoblasts. (a) Total RNA was extracted from lymphoblast cell lines from three controls (WT1 and WT2 and one MG patient without the SNP, WT-MG) and four MG patients with the c.-198C>G SNP (MG1 to MG4), with and without LPS stimulation (10 μg/ml) for 6 h. Relative expression (normalized to GUS1B) by qRT-PCR is shown expressed as fold change over non-LPS control for each cell line (normalized to 1). Data (mean±s.d.) from triplicate experiments. (b) Western blot analysis of DAF protein levels in control (WT1) and MG patient (MG1) derived cell lines. The cells were treated with 10 μg ml⁻¹ of LPS for 6 h. Protein was extracted under non-reducing conditions and quantified using BCA standard curve method (Materials and methods). For each sample, 30 μg of protein was loaded on an SDS-PAGE gel and p38 was used as a loading control to ensure equal loading of protein. DAF protein levels increased in control cell line but decreased in the MG patient cell line on treatment with LPS. (c) The bar graph represents quantitative DAF protein results by showing densitometry readings from the autoradiograph of DAF protein levels normalized to p38 protein levels. For the control cell line, DAF protein levels showed an increase of approximately two-folds as a result of LPS treatment. On the contrary, the MG SNP cell line showed a decrease of approximately two-fold when treated with LPS.