Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-beta.

doi: 10.1073/pnas.1013776108

Figure Lengend Snippet: Fig. 2. TLR2 and TLR8 cellular expression. Costaining for TLR2 (green) and TLR8 (red) is shown in unstimulated monocytes and in both infected and uninfected cells (bystander) within 30 min of incubation with Bb (10:1 mul- tiplicity of infection). TLR2 fluorescent signals are visualized in unstimulated and bystander cells, greatly increasing in Bb-infected monocytes. TLR8 signals are of very low intensity in uninfected monocytes and, like TLR2, increase in Bb-infected monocytes. Black arrows point to degraded GFP-Bb. TLR2 coloc- alizes with TLR8 even at locations that do not coincide with internalized GFP- Bb (white arrows and region of interest). Numerical values shown in white correspond to Mander’s colocalization coefficient (M2), indicative of the proportion of the green signal (TLR2) overlapping with the signal in the red channel (TLR8). DIC, differential interference contrast.

Article Snippet: Following different time points of stimulation (0 min, 30 min, 60 min, 90 min, 3 h, and 4 h), slides were fixed with 4% (vol/vol) paraformaldehyde for 10 min at room temperature (RT); permeabilized with 0.2% saponin for 10 min; blocked with Connaught Medical Research Laboratories (CMRL) 10% FCS for 2 h at RT; incubated overnight at 4 °C with a rabbit anti-human IRF7 polyclonal or mouse anti-human IRF3 monoclonal antibody (Santa Cruz Biotechnology, Inc.), a rabbit anti-TLR8 polyclonal antibody (IMGENEX), or a rabbit anti-TLR2 polyclonal antibody (Rockland Immunochemicals) (1:100 dilution for all); and subsequently incubated with Texas RedR-X–conjugated goat anti-rabbit antibody (1:200 dilution; Invitrogen) for 1 h at RT or goat anti-mouse DyLight 594 (Thermo Scientific).

Techniques: Expressing, Infection, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-beta.

doi: 10.1073/pnas.1013776108

Figure Lengend Snippet: Fig. 3. Phagocytosed Bb colocalizes with TLR2 and TLR8 in human mono- cytes. A composite of representative confocal micrographs shows three consecutive Z-stack planes of GFP-Bb–stimulated human monocytes. Extra- cellular Bb interacts with TLR2 at the plasma membrane of the monocyte (A), whereas recently internalized (bacterial coil) and fully degraded (fluorescent bleb) Bb colocalizes with TLR2 (B) and TLR8 (C). Mander’s colocalization coefficient values (M2), shown in the upper right corner of each figure, are indicative of the proportion of the green signal overlapping with the signal in the red channel. Red depicts TLR2 signals in A and B and TLR8 in C, green depicts GFP-Bb, and white represents red and green pixels that colocalize. (Scale bar, 5 μm.) Colocalization through the Z-stack images illustrated in A and B (TLR2) and in C (TLR8) is shown in Movie S1, Movie S2, and Movie S3.

Article Snippet: Following different time points of stimulation (0 min, 30 min, 60 min, 90 min, 3 h, and 4 h), slides were fixed with 4% (vol/vol) paraformaldehyde for 10 min at room temperature (RT); permeabilized with 0.2% saponin for 10 min; blocked with Connaught Medical Research Laboratories (CMRL) 10% FCS for 2 h at RT; incubated overnight at 4 °C with a rabbit anti-human IRF7 polyclonal or mouse anti-human IRF3 monoclonal antibody (Santa Cruz Biotechnology, Inc.), a rabbit anti-TLR8 polyclonal antibody (IMGENEX), or a rabbit anti-TLR2 polyclonal antibody (Rockland Immunochemicals) (1:100 dilution for all); and subsequently incubated with Texas RedR-X–conjugated goat anti-rabbit antibody (1:200 dilution; Invitrogen) for 1 h at RT or goat anti-mouse DyLight 594 (Thermo Scientific).

Techniques: Clinical Proteomics, Membrane