Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: (a) Chemical structures of CU-CPT8m and 6 (negative control), concentration-response curve and dose-dependent cytotoxicity of CU-CPT8m in HEK-Blue TLR8 cell line. Data was normalized to a DMSO control (data are mean ± SD ; n = 3 independent experiments). (b) ITC thermogram of CU-CPT8m titrated into hTLR8 to determine binding affinity and stoichiometry (representative of one independent experiment). The raw data are presented on top and the integrated peak areas are shown and fitted below. Mean K d = 0.22 μM; stoichiometric binding N = 0.5. (c) Specificity test for CU-CPT8m (1 μM) with TLR-specific agonists used to selectively activate different HEK-Blue TLR-overexpressing cells in the presence or absence of 1 μM CU-CPT8m (data are mean ± SD ; n = 3 independent experiments). (d) TNF-α and IL-8 mRNA level in R848 treated HEK-Blue TLR8 cells in the presence and absence of 1 μM CU-CPT8m or the negative control, 6 (10 μM). Data are the average quantification of two independent experiments. (e) Dose-dependent response of CU-CPT8m on TLR8-mediated TNF-α production in THP-1 cells with indicated concentration of CU-CPT8m or 6 . Data are mean ± SD ; n = 3 independent experiments. (f) Dose-dependent response of CU-CPT8m or 6 on TLR8-mediated TNF-α production in PBMC cells induced by 1 μg/mL R848. Data are mean ± SD ; n = 3 independent experiments.

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Negative Control, Concentration Assay, Control, Binding Assay

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: (a) Front (top) and side (bottom) views of the unliganded (left, PDB ID 3W3G), TLR8/ CU-CPT8m (middle) and TLR8/R848 (right, PDB ID 3W3N) complexes. TLR8 and its dimerization partner TLR8* are colored green and cyan, respectively. The distances between the C-termini of the two protomers of TLR8 dimer (TLR8/ CU-CPT8m ) is similar to that of the unliganded dimer (right). Superimposition of the TLR8 structure complexed with CU-CPT8m onto the corresponding unliganded TLR8 segment (a.a. 32–816) produces root-mean-square deviation (RMSD) values of 2.4 Å. The ligand molecules are illustrated by space-filling representations. The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. (b) Close-up view of antagonist binding site of unliganded TLR8 (left) and TLR8/ CU-CPT8m (right). Water molecules are indicated by red filled circles. (c) Schematic representation of interactions between CU-CPT8m and the TLR8 protein. The hydrophobic pocket and hydrogen bonds are shown as dashed gray arcs and dashed red lines, respectively.

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Binding Assay

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: LRR8, LRR11-13, LRR15-16, and LRR17-18 are colored yellow, green, blue, and purple, respectively. In the bottom panel, the antagonist and agonist are illustrated by yellow and orange circles. Interactions between ligands and protruding loop regions are shown by dashed arrows. TLR8 utilized LRR11-13 in common for both agonist and antagonist binding on one side of the interface, while on the other side LRR17*-18* and LRR15*-16* for agonist and antagonist binding, respectively. Binding of agonist (e.g. R848 ) brings two TLR8 C-termini to a closer distance to initiate downstream signaling; while binding of antagonists (e.g. CU-CPT8m , CU-CPT9b ) at the antagonist binding site stabilizes inactive TLR8 dimer with C-termini further apart, preventing TLR8 from activation.

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Binding Assay, Activation Assay

Journal: Nature chemical biology

Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

doi: 10.1038/nchembio.2518

Figure Lengend Snippet: (a) Chemical structure of CU-CPT9a and CU-CPT9b. (b) Close-up view of antagonist binding site (left) and its schematic representation of TLR8/ CU-CPT9b (right). The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. Water molecules mediating the ligand recognition are indicated by red filled circles and hydrogen bonds by dashed lines. (c) Dose-dependent dimerization of TLR8. Elution profiles of gel filtration chromatography of TLR8 with CU-CPT9b (left) and R848 (right) at various concentrations. Retention volume and normalized absorbance at 280 nm (A 280 ) are shown on the left, and retention volume of TLR8 peak is plotted against its molar ratio (ligand/TLR8) on the right (representative of one independent experiment).

Article Snippet: TLR8 antibody (Novus Biologicals; NBP2-24972) was added in PBS containing 1% FBS, then incubated for 10 min.

Techniques: Binding Assay, Filtration, Chromatography

Journal: Cell reports

Article Title: Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling

doi: 10.1016/j.celrep.2021.109142

Figure Lengend Snippet:

Article Snippet: Human IgG Isotype Control [Biotin] , Novus Biologicals , Cat# NBP1-96855.

Techniques: Purification, Control, Recombinant, Phagocytosis Assay, Immunoprecipitation, Knock-Out, Software

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: List of antibodies and antibody-blocking peptides used in the study.

Article Snippet: BAK antibody blocking peptide , ELISA detection , N/A , N/A , Novus Biologicals , NBP1-77152PEP , N/A.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Peptide ELISA, Activity Assay