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. 2013 May 31;8(5):e64544. doi: 10.1371/journal.pone.0064544

Figure 3. ChIP analysis of DDX21 gene, luciferase reporter assay and overexpression of C/EBPβ.

Figure 3

(A) qPCR analysis to quantify the relative amounts of DNA obtained by ChIP for the potential regulatory sites of DDX21. DDX21_I1 depicts the regulatory site in the first intron of the DDX21 gene. DNA quantities (expressed as percentages of input) were compared for specific IPs versus IgG IP samples. One representative qPCR result of the different sites is shown. Error bars indicate SD (n = 3), p<0.05. (B) Luciferase reporter assays after transfection of HEK293T cells with the reporter plasmid containing the regulatory site of the intron region of DDX21 and expression vector to overexpress the C/EBPβ isoforms LAP* and LAP. Bars represent relative luciferase activity for the respective vectors. Error bars indicate SD (n = 3), p<0.05. (C) Flow cytometric analysis of transduced Mac-1 cells and untreated controls three days after lentiviral infection. The percentage of GFP-positive cells represents the percentage of infected cells. (D) Western Blot analysis of the C/EBPβ isoforms LAP* and LAP and of DDX21 in the transduced Mac-1 and SR786 cells three days after infection. Each lane of the Western Blot contains 20 µg protein extract. Tubulin was used as loading control. Mac-1/SR786 = uninfected cells, pRRL = empty virus, pRRL-LAP* = virus containing the C/EBPβ isoform LAP* sequence, pRRL-LAP = virus containing the C/EBPβ isoform LAP sequence.