Journal: Immunology

Article Title: Differential regulation of Toll-like receptor signalling in spleen and Peyer's patch dendritic cells

doi: 10.1111/j.1365-2567.2010.03317.x

Figure Lengend Snippet: Gene-specific primers and probe combinations assayed by quantitative real-time polymerase chain reaction

Article Snippet: Flow cytometry Using a FACSCalibur (BD Biosciences, San Jose, CA) flow cytometer, the surface phenotype of cells was determined using four-colour staining with the following antibodies: fluorescein isothiocyanate-conjugated (-FITC) or allophycocyanin-conjugated CD11c (HL3), phycoerythrin-conjugated (-PE) CD80 (16-10A1) (BD-Pharmingen, Oxford, UK); TLR2-PE (6C2), TLR4/MD2-PE-Cy7 (MTS510) (eBioscience, San Diego, CA) and major histocompatibility class II (MHCII)-FITC (M5/114) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Sequencing

Journal: Immunology

Article Title: Differential regulation of Toll-like receptor signalling in spleen and Peyer's patch dendritic cells

doi: 10.1111/j.1365-2567.2010.03317.x

Figure Lengend Snippet: A greater percentage of spleen dendritic cells (DCs) express surface Toll-like receptor 2 (TLR2) and TLR4 than Peyer's patch (PP) DCs. (a) AutoMACS-separated CD11c+ cells from spleen and PP were gated on the expression of CD11c and MHCII as in Fig. 1(a). The expression of surface TLR2 and TLR4-MD2 was then determined on the double-positive population. Values in the upper right of the histograms indicate the mean fluorescence intensity of cells in the indicated gate, n = 3. Bars represent mean ± SEM of percentage of cells falling in the indicated gate of the CD11c+ MHCII+ population, n = 3. (b) autoMACS-separated, flow cytometrically sorted DCs were analysed for gene expression of tlr 1–9. Expression was determined as fold induction compared with β-actin housekeeper. There was no statistical difference between spleen and PP in the messenger RNA levels of any of the genes investigated. Bars represent mean ± SEM, n = 4 or n = 5. Significance determined by unpaired Student's t-test, ***P < 0·001, *P < 0·05.

Article Snippet: Flow cytometry Using a FACSCalibur (BD Biosciences, San Jose, CA) flow cytometer, the surface phenotype of cells was determined using four-colour staining with the following antibodies: fluorescein isothiocyanate-conjugated (-FITC) or allophycocyanin-conjugated CD11c (HL3), phycoerythrin-conjugated (-PE) CD80 (16-10A1) (BD-Pharmingen, Oxford, UK); TLR2-PE (6C2), TLR4/MD2-PE-Cy7 (MTS510) (eBioscience, San Diego, CA) and major histocompatibility class II (MHCII)-FITC (M5/114) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Fluorescence, Gene Expression

Journal: International journal of molecular sciences

Article Title: Targeting TLR2/Rac1/cdc42/JNK Pathway to Reveal That Ruxolitinib Promotes Thrombocytopoiesis.

doi: 10.3390/ijms232416137

Figure Lengend Snippet: Figure 8. Biophysical validation reveals TLR2 as a new protein target of ruxolitinib. (A) Venn di‑ agram shows the common targets of Ruxolitinib and thrombocytopenia. The intersecting part rep‑ resents the common targets between Ruxolitinib and thrombocytopenia; (B) PPI network for identi‑ fying core targets of Ruxolitinib against thrombocytopenia through the screening conditions of De‑ gree > 47, BC > 0.002858932, CC > 0.507867733; (C) TLR2 and ligands (ruxolitinib) by molecular dock‑ ing; (D). Representative immunoblot images and biochemical quantification of TLR2 after treatment with Ruxolitinib (5, 10, and 20 µM) in Meg‑01 cells for 5 day (E) The DARTS assay for target valida‑ tion. TLR2 protein stability was increased upon Ruxolitinib (200 µM) treatment in Meg‑01 lysates. Pronase was added using several dilutions (1:500, 1:1000, or 1500) from 50 µg/mL stock for 10 min at 40 ◦C; (F) The DARTS assay demonstrated the dose‑dependent binding of Ruxolitinib to TLR2. Treatment with pronase (1:1000) was conducted for 10 min at 40 ◦C; (G) Meg‑01 cells were treated with ruxolitinib (20 µM), C29 (50 µM), ruxolitinib (20 µM) + C29 (50 µM) for 5 days. FCM analysis of the expression of CD41 and CD42b. (H) The histogram shows the percentage of CD41+/CD42b+

Article Snippet: The primary antibodies were as follows: FOS (Proteintech, USA, 66590‐1‐lg), EGR1 (Proteintech, USA, 22008–1‐AP), RUNX1 (Proteintech, USA, 25315‐1‐AP), TLR2 (Pro‐ teintech, USA, 17236‐1‐AP), Rac1/cdc42 (CST, USA, 4651S), JNK (Abmart, China, T55490), p‐JNK (Abmart, China, T55541), NF‐E2 (Proteintech, USA, 11089‐1‐AP), and GAPDH (Pro‐ teintech, USA, 60004‐1‐lg).

Techniques: Biomarker Discovery, Western Blot, Binding Assay, Expressing

Journal: International journal of molecular sciences

Article Title: Targeting TLR2/Rac1/cdc42/JNK Pathway to Reveal That Ruxolitinib Promotes Thrombocytopoiesis.

doi: 10.3390/ijms232416137

Figure Lengend Snippet: Figure 10. Schematic illustration of the role of ruxolitinib in MK differentiation and platelet pro‑duction. Ruxolitinib induces the expression of various cytokines and TLR2, activates the Rac1/cdc42/JNK signaling pathway, and leads to the expression of AP‑1, EGR1, RUNX1, and NF‑E2. As a result, the activation of AP‑1, EGR1, RUNX1, NF‑E2 promote the expression of genes related to MK differentiation and thrombopoiesis. These genes contribute to MK maturation and platelet for‑ mation and promote the recovery of bone marrow and spleen MKs and accelerate platelet production in RI‑mice. PPF: proplatelet‑forming MK.

Article Snippet: The primary antibodies were as follows: FOS (Proteintech, USA, 66590‐1‐lg), EGR1 (Proteintech, USA, 22008–1‐AP), RUNX1 (Proteintech, USA, 25315‐1‐AP), TLR2 (Pro‐ teintech, USA, 17236‐1‐AP), Rac1/cdc42 (CST, USA, 4651S), JNK (Abmart, China, T55490), p‐JNK (Abmart, China, T55541), NF‐E2 (Proteintech, USA, 11089‐1‐AP), and GAPDH (Pro‐ teintech, USA, 60004‐1‐lg).

Techniques: Expressing, Activation Assay

Journal: Journal of Inflammation Research

Article Title: Therapeutic Effect of C-C Chemokine Receptor Type 1 (CCR1) Antagonist BX471 on Allergic Rhinitis

doi: 10.2147/JIR.S254717

Figure Lengend Snippet: Relative quantification of NF-kB p65, TLR4, and TLR2 protein expression levels. ( A ) Western blot analysis for NF-kB p65, TLR4, and TLR2 relative expression from groups indicated. ( B ) summary graph for relative expression of NF-kB (n=8 per group). ( C ) summary graph for relative expression of TLR4 (n=8 per group). ( D ) summary graph for relative expression of TLR2 (n=8 per group). All values are represented as mean ± SEM. * p<0.05; ** p<0.01; and *** p<0.001 versus the vehicle control group.

Article Snippet: Samples were subsequently treated with anti β-actin antibody (BM3873, Boster Biological Technology, China), NF-kB p65 monoclonal antibody (A10609, Abclonal, USA.), TLR4 antibody (BA1717, Boster Biological Technology, China), or TLR2 antibody (BM4001, Boster Biological Technology, China), and were incubated overnight at 4°C.

Techniques: Quantitative Proteomics, Expressing, Western Blot, Control