FIG. 1.

Unmethylated CpG motifs in the plasmids and presence of the Toll-like receptor-9 (TLR9) in the NIH3T3 cell line. (A) Analysis of the presence and distribution of CpG dinucleotides was performed by means of EMBOSSES CpGPlot. The CpG motifs of plasmid DNA are presented by black arrows, the position of the motifs within the plasmid are indicated on the top of the arrows in nucleotide (nt) numbers. The boxes in bold represent the sequences of VP2 and VP3 of Mouse polyomavirus (MPyV). Sequences of the phGfΔG plasmid were arbitrarily divided into three regions. Dotted lines in the expression vectors, ph2pΔ and ph3pΔG, indicate sequences deleted by cloning. (B) The methylation state of phGfΔG plasmid (lanes 1, 2), ph2pΔG plasmid (lanes 3, 4), or ph3pΔG plasmid (lanes 5, 6) was tested by Hpa II digestion. HpaII digested (lanes 2–4) and control non-digested (lanes 1, 3, 5) plasmids were resolved by 0.8% agarose electrophoresis. (C) Confocal section of NIH3T3 cells (untreated or mock-transfected cells) stained with antibody against the TLR9 receptor (green) and by DAPI. (D) Levels of the TLR9 protein in cells nucleofected with phGfΔG, ph2pΔG, or ph3pΔG plasmids and in mock-transfected cells (4 h post-transfection) were analyzed in Western blots using anti-TLR9 antibody. Antibody against β-actin was used as loading control.