1. Preparing and counting the Namalwa cells
2. Electroporating the cells in 96-well Plates
3. Extract and check RNA
4. Real-time PCR
5. Analysis of siRNA knockdown
6. Representative Results
Figure 1. Overview of the IL-4 signaling pathway.
A) Binding of IL-4 to its receptor leads to dimerization and activation of STAT6. Activated STAT6 translocates to the nucleus and activates transcription of its target genes, such as CC17.
B) Without IL-4 induction, transcription of CCL17 will remain at low, constitutive levels.
C) After siRNA-mediated silencing of the STAT6 gene, treatment with IL4 should lead to little or no increase in CCL17 expression.
Figure 2. Induction of CCL17 by IL4 measured by different conditions by quantitative real time PCR.
A) Samples grown without IL-4 had a low constitutive level of expression regardless of gene expression.
B) As shown in green and blue, control samples responded normally to treatment with IL-4; with an 8-10 fold induction of CCL17. However, cells transfected with siRNAs targeting STAT6, shown in red and pink, had diminished CCL17 expression in response to IL-4, supporting the idea that STAT6 plays a role in the IL-4 induced expression of CCL17.