Fig 1: The role of α5β1 integrin, TLR2, and CD36 in S. aureus internalization into bMECs modulated by NaO. bMECs were treated with 0.25 or 1 mM NaO for 24 h, then incubated with a specific blocking (A) anti-α5β1 integrin (10 μg/ml), (B) anti-TLR2 (5 μg/ml), or (C) anti-CD36 (0.25 μg/ml) antibody for 30 min, 1 h, or 45 min, respectively, and further challenged with S. aureus for 2 h. The number of internalized bacteria is represented by the ratio CFU/bMEC. Each bar shows the mean of triplicates ± SE of three independent experiments. The symbol “*”indicates significant changes (P < 0.05) compared to control cells (untreated cells and w/o blockade).
Fig 2: The NCL alarmin activity resides in its GAR/RGG motif.A Recombinant NCL and its mutants. NCL mutants were generated by C-terminal deletion and the new C-termini are bracketed (i.e., 274, 477, 522, 609, 649, 670 and 698). Two head-to-head repeats in the GAR/RGG motif were highlighted in red boxes and two head-to-tail repeats were identified in cyan boxes. Deletion of these four repeats yields another mutant (Δ653-698). Recombinant NCL and its mutants were expressed in 293T cells with C-terminal HA tags. B Monocytes were stimulated with coated NCL-HA and its mutants (40 μg/ml) for 24 h to measure TNFα and IL-1β production. Triplicate experiments were performed to obtain data as mean ± SD. Data were analyzed by one-way ANOVA. C NCL, NCL-HA, NCL(649)-HA, and BSA (10 μg/ml) were coated to incubate with TLR2-10xHis (0.375–6.0 μg/ml). Bound TLR2 was detected using a mouse anti-His antibody (2.6 μg/ml). D TLR2 (2 μg/ml) was coated to incubate with NCL-HA, NCL(649)-HA, or NCL(522)-HA (0–20 μg/ml). Bound NCL-HA and its mutants were detected using a mouse anti-HA antibody (1 μg/ml). E TLR2 (2 μg/ml) was coated, pre-incubated with TLR2 or TLR4-blocking antibodies (5 μg/ml), and incubated with NCL, NCL-HA or BSA (10 μg/ml). Bound proteins were detected using a rabbit anti-NCL antibody (1 μg/ml). F Binding of NCL-HA and its mutants to TLR2. NCL-HA, its mutants, and control BSA were plate-coated (10 μg/ml) to incubate with TLR2 (2 μg/ml). Bound TLR2 was detected using the mouse anti-His antibody. In these experiments, HRP-conjugated secondary antibodies were used. Triplicate experiments were performed to obtain data as mean ± SD. Data was analyzed by one-way ANOVA. *p < 0.05, ***p < 0.001, ****p < 0.0001. n.s., not significant.
Fig 3: Heat map of gene expression (qRT PCR) upon PRP-1 treatment. The clustrogram performs non-supervised hierarchical clustering of the entire dataset to display a heat map with dendrograms indicating co-regulated genes across groups or individual samples; it represents the average of Ct values displayed across the genes of each sample. S2 stands for samples treated with 1 μg/ml of PRP-1 and S1 stands for 10 μg/ml of PRP-1. TLR2 and TLR6 were highly expressed in S2, while TLR1 was highly expressed in S1 and moderately in S2 group. c-Myc and MUC5B were highly expressed in S2. GAPDH and ACTB were housekeeping genes.
Fig 4: IL-8 concentrations were measured by ELISA in the supernatant of unstimulated, stimulated, neutralized, and immunomodulated HEK-BlueTM cell cultures. Statistical differences with regard to: # p < 0.0001 HEK-BlueTMNull1 and unstimulated HEK-BlueTMhTLR2, *** p < 0.0001 Anti-TLR2 and LTF, δ p < 0.0001 Pam2CSK4 HEK-BlueTMhTLR2. Abbreviations: Anti-TLR2, Anti-hTLR2-IgA; IL-8, interleukin 8; LTF, lactoferrin; No stim., unstimulated; Pam2, Pam2CSK4; Pam3, Pam3CSK4; TLR2, Toll-like receptor 2.
Fig 5: Screening and survival analysis of the overlapping genes. (A) Venn diagram of genes regulated by regulation of Toll-like receptor signaling pathway and TCGA-DEGs, with 8 intersection genes in the middle. (B-G) OS analysis on (B) IL1B, (C) JUN, (D) TLR2, (E) TLR4, (F) TLR8 and (G) TREM1 in lung cancer. The black line indicates low expression and the gold line indicates high expression. TCGA, The Cancer Genome Atlas; DEGs, differentially expressed genes; OS, overall survival; TREM1, triggering receptor expressed on myeloid cells 1.
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