humanized anti-human tlr7 antibody 7  (Cellular Technology Ltd)

Journal: The FASEB Journal

Article Title: Propofol directly binds to and inhibits TLR7

doi: 10.1096/fj.202200312r

Figure Lengend Snippet: FIGURE 4 The impact of propofol on TLR7 dimer formation and MyD88 interaction. (A) TLR7 protein was subjected to crosslinking experiment using glutaraldehyde with or without R837 and/or propofol (PPF). R837-induced dimer formation. Dimer and monomer formations were quantitated by densimetry. (B) HEK-TLR7 cells were stimulated with R837 in the presence or absence of PPF 50 μM. Then, cell lysates were immunoprecipitated (IP) with TLR7, followed by Western bloting (WB) of MyD88 (left). Densimetry analysis is shown. Immunoprecipitated TLR7 was also probed and shown on the right. ***p < .001 using one-way ANOVA with Bonferroni post hoc analysis.

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti- human TLR7 antibody (Cell Signaling technology; Danvers, MA) and Dynabeads Protein A immunoprecipitation kit (Thermofischer Scientific).

Techniques: Immunoprecipitation, Western Blot

Journal: The FASEB Journal

Article Title: Propofol directly binds to and inhibits TLR7

doi: 10.1096/fj.202200312r

Figure Lengend Snippet: FIGURE 3 Propofol-binding sites on TLR7. PDB5ZSF was used for the analysis. (A) R837-binding sites on TLR7 dimer (TLR7/TLR7*). R837 binds to the two sites (Site 1-1 and site 1-2). (B) Coverage map for TLR7 mass spectrometry analysis of photolabeled samples with propofol. Sequenced residues are shown in orange. Adducted residues are shown in red. (C) Propofol-binding sites on TLR7 dimer are shown. Based on the photolabeling and rigid docking experiments, five propofol-binding sites were identified. (D) R837-binding sites and propofol-binding sites are shown on TLR7 dimer together. R837 is shown in red arrow. Propofol is shown in blue circle.

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti- human TLR7 antibody (Cell Signaling technology; Danvers, MA) and Dynabeads Protein A immunoprecipitation kit (Thermofischer Scientific).

Techniques: Binding Assay, Mass Spectrometry

Journal: The FASEB Journal

Article Title: Propofol directly binds to and inhibits TLR7

doi: 10.1096/fj.202200312r

Figure Lengend Snippet: FIGURE 5 The role of hydroxyl group in propofol in TLR7 binding. Fropofol is a propofol derivative where the hydroxyl (-OH) group was replaced with fluoride. (a) The effect of propofol (PPF) and fropofol (FPL) on TLR7 activation was examined. HEK-TLR7 cells were stimulated with R837 (10 mg/ml) in the presence of PPF and FPL (10 μM, 100 μM). Data were shown as mean ± SD of eight replicates. Two-way ANOVA was done. ***p < .001. (B) Coverage map for TLR7 mass spectrometry analysis of photolabeled samples with propofol. Sequenced residues are shown in orange. Adducted residues are shown in red. (C) Fropofol-binding sites on TLR7 dimer are shown. Based on the photolabeling and rigid docking experiments, two fropofol-binding sites were identified. (D) R837-binding sites and fropofol-binding sites are shown on TLR7 dimer together. R837 is shown in red arrow. Fropofol is shown in purple circle.

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti- human TLR7 antibody (Cell Signaling technology; Danvers, MA) and Dynabeads Protein A immunoprecipitation kit (Thermofischer Scientific).

Techniques: Binding Assay, Activation Assay, Mass Spectrometry