DV NS1 protein up-regulates TLR2 and TLR6 of PBMC.

<p>Mock-infected, His-tag-treated, DV2-infected, DV NS1 protein-treated, LPS-treated PBMC were lysed and equal total protein was loaded into SDS-PAGE gel. PBMC of two donors were used. The presence of TLR6 protein in the cell lysate was detected using rabbit anti-TLR6 antibody and goat anti-rabbit HRP conjugated antibody on the Western blot (A). Actin was used as loading control. The band intensity was measured using GelQuantNET software program. The intensities of the TLR6 bands were normalized against the intensity of the corresponding actin bands and were plotted in the graph (B). The presence of TLR2 protein in the cell lysate was detected using rabbit anti-TLR2 antibody and goat anti-rabbit HRP conjugated antibody on the Western blot (C). Actin was used as loading control. The intensities of the TLR2 bands were normalized against the intensity of the corresponding actin bands and were plotted in the graph (D). The relative intensities of the bands were indicated on the charts. PBMC were untreated (E & J), mock-infected (F & K), DV2-infected (G & L), His-tag-treated (H & M) and DV NS1 protein-treated (I & N). The PBMC were harvested at day 3 post-treatment. TLR6 was stained red using primary rabbit anti-TLR6 antibody and secondary goat anti-rabbit DyLight 633 antibody (E to I). TLR2 is stained red using primary mouse anti-TLR2 antibody and secondary goat anti-mouse DyLight 633 (J to N). CD14 was stained green using anti-CD14 antibody conjugated with FITC (E to N). Cell nuclei are stained blue with DAPI. Representative images from triplicate experiments obtained using separate PBMC from three donors are shown.