Journal: Frontiers in Immunology

Article Title: Coevolution of the Toll-Like Receptor 4 Complex with Calgranulins and Lipopolysaccharide

doi: 10.3389/fimmu.2018.00304

Figure Lengend Snippet: Calgranulin activation of toll-like receptor 4 (TLR4) is shared across amniotes. NF-κB activity for species-matched TLR4/MD-2/CD14 complexes in the presence of calgranulins from (A) human, (B) mouse, (C) opossum, and (D) chicken. A “+” in the table below each series indicates which components are included in that treatment. Lipopolysaccharide (LPS) is used as a positive control for expression and activation of the complex. Polymyxin B is included to control for endotoxin-mediated activation of the complex. Activity is normalized to LPS activity of positive control within each biological replicate (i.e., for human, each technical replicate is divided by the average LPS activation of hTLR4, hMD2, and hCD14 for that biological replicate). Points are the technical triplicates from three biological replicates. Bold lines are the mean of the biological replicates. Error bars are SEM of the biological replicates. A two-tailed t -test was used to assess significance of difference in mean between the indicated series (** p -value <0.01).

Article Snippet: Mammalian expression vectors containing human TLR4 and mouse TLR4 were a gift from Ruslan Medzhitov (Addgene plasmid #13085 and #13086) and human CD14 and ELAM-Luc were a gift from Doug Golenbock (Addgene plasmid #13645 and #13029).

Techniques: Activation Assay, Activity Assay, Positive Control, Expressing, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Coevolution of the Toll-Like Receptor 4 Complex with Calgranulins and Lipopolysaccharide

doi: 10.3389/fimmu.2018.00304

Figure Lengend Snippet: Amniote calgranulins have similar toll-like receptor 4 (TLR4) complex requirements. NF-κB activation of TLR4 from (A) human, (B) mouse, (C) opossum, and (D) chicken. NF-κB is normalized to lipopolysaccharide (LPS) activation of the control complex for that species. Points are the technical replicates from three biological replicates. Bold lines are the mean of the biological replicates. Error bars are SEM of the biological replicates. A “+” in the panel below indicates which components are included in the treatment. A two-tailed t-test was used to assess significance of difference in mean between the indicated series (** p -value <0.01).

Article Snippet: Mammalian expression vectors containing human TLR4 and mouse TLR4 were a gift from Ruslan Medzhitov (Addgene plasmid #13085 and #13086) and human CD14 and ELAM-Luc were a gift from Doug Golenbock (Addgene plasmid #13645 and #13029).

Techniques: Activation Assay, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Coevolution of the Toll-Like Receptor 4 Complex with Calgranulins and Lipopolysaccharide

doi: 10.3389/fimmu.2018.00304

Figure Lengend Snippet: Toll-like receptor 4 (TLR4) complex components exhibit lineage-specific coevolution. (A–J) show activation of NF-κB signaling by receptor complexes assembled from host (black) and guest (red) proteins. Protein names on the left and top of each sub-panel indicate the components included. S100 is used to denote human S100A9, mouse S100A9, opossum S100A9, or chicken MRP-126. Letters indicate which species components come from: human (H), mouse (M), opossum (O), or chicken (C). If the cross-species complex activates >2-fold above the buffer control, the corresponding box is filled gray. The icon to the top-left of each sub-panel indicates graphically which components are being combined. *indicates > 2-fold activation above buffer control, but low total activation (see Figure S5 in Supplementary Material).

Article Snippet: Mammalian expression vectors containing human TLR4 and mouse TLR4 were a gift from Ruslan Medzhitov (Addgene plasmid #13085 and #13086) and human CD14 and ELAM-Luc were a gift from Doug Golenbock (Addgene plasmid #13645 and #13029).

Techniques: Activation Assay, Control

Journal: Frontiers in Immunology

Article Title: Coevolution of the Toll-Like Receptor 4 Complex with Calgranulins and Lipopolysaccharide

doi: 10.3389/fimmu.2018.00304

Figure Lengend Snippet: Lipopolysaccharide (LPS) and calgranulin exhibit different complex requirements. (A–C) show NF-κB activation of mouse toll-like receptor 4 (TLR4) with different combinations of mouse and opossum MD-2 and CD14. Activation by: (A) LPS, (B) mouse S100A9, and (C) opossum S100A9. (D–F) show NF-κB activation of human TLR4 with different combinations of human and mouse MD-2 and CD14. Activation by: (D) LPS, (E) human S100A9, and (F) mouse S100A9. Letters indicate which species the component was taken from: human (H), mouse (M), or opossum (O). NF-κB is normalized to LPS activation of the control complex for that species. Points are the technical triplicates from three biological replicates. Bold lines are the mean of the biological replicates. Error bars are SEM of the biological replicates. A two-tailed t -test was used to assess significance of difference in mean between the indicated series (* p -value <0.05, ** p -value <0.01).

Article Snippet: Mammalian expression vectors containing human TLR4 and mouse TLR4 were a gift from Ruslan Medzhitov (Addgene plasmid #13085 and #13086) and human CD14 and ELAM-Luc were a gift from Doug Golenbock (Addgene plasmid #13645 and #13029).

Techniques: Activation Assay, Control, Two Tailed Test