Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Recombinant sTLR proteins are monomeric, properly folded, and biologically functional. A, 100 μg of each purified sTLR protein as indicated was loaded for 7.5% SDS-PAGE and stained with Coomassie blue dye. B, each purified sTLR protein as indicated was analyzed by size exclusion chromatography using a Superdex 200 column. C, soluble TLRs were incubated in microtiter plate wells coated with either the anti-TLR1 mAb (clone GD2F4) or the anti-TLR2 mAb (T2.5) as indicated. Binding of soluble TLR1, TLR1P315L, or TLR2 was detected using HRP-conjugated anti-HA and anti-FLAG mAbs. D, SW620 cells were co-transfected with full-length TLR1, TLR2, an IL-8 promoter-driven luciferase reporter gene, and a Renilla luciferase transfection control. 48 h post-transfection, the cells were stimulated with 10 ng/ml Pam3CSK4 with or without 1 μg/ml soluble TLR1 or TLR2 as indicated. Firefly luciferase activities were normalized to that of the Renilla luciferase control. These values were normalized to that of empty CMV vector whose value was taken as 1. Error bars represent the S.D. of three independent events. mAu, milliabsorbance units.

Article Snippet: Cells were co-transfected with 50 ng each of TLR1 and TLR2 together with 75 ng of a firefly luciferase reporter gene driven by an NF-κB promoter and 25 ng of a Renilla luciferase (pRL-null) transfection control (Promega).

Techniques: Recombinant, Functional Assay, Purification, SDS Page, Staining, Size-exclusion Chromatography, Incubation, Binding Assay, Transfection, Luciferase, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Either LBP or soluble CD14 enhances cellular responses to Pam3CSK4 and OspA. HEK 293F cells were co-transfected with vectors expressing full-length TLR1 and TLR2 or empty CMV control vector as indicated together with an NF-κB-promoter driven luciferase reporter gene and a Renilla luciferase reporter gene. About 48 h post-transfection, cells were stimulated with 1 ng/ml Pam3CSK4, OspA, or the non-acylated Ac2CSK4 control in the presence of 0.1 μg/ml LBP, sCD14, or human serum albumin (HSA) as indicated (left side). In one set of experiments, agonists were preincubated with proteins for 1 h at 37 °C prior to addition to transfected cells (right side). Cell values on the y axis represent the level of constitutive reporter activation normalized to the empty CMV vector control (value of 1). Error bars represent the S.D. of three independent values.

Article Snippet: Cells were co-transfected with 50 ng each of TLR1 and TLR2 together with 75 ng of a firefly luciferase reporter gene driven by an NF-κB promoter and 25 ng of a Renilla luciferase (pRL-null) transfection control (Promega).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activation Assay