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 Clinical Investigation

Article Title: Selective disruption of TLR2-MyD88 interaction inhibits inflammation and attenuates Alzheimer’s pathology

doi: 10.1172/jci96209

Figure Lengend Snippet: Figure 1. Monitoring TLR2, TLR4, and MyD88 levels in the CNS of individuals clinically diagnosed with NCI, MCI, or AD. (A) PFC homogenates (25 μg) from NCI (light blue), MCI (dark blue), and AD (gray) individuals were immunoblotted for TLR2, TLR4, and MyD88. Actin was used to normalize the signals obtained by densitometric measurement (ImageJ). Coomassie was used to verify protein loading. Twelve NCI, eleven MCI, and ten AD samples were run in three independent experiments. (B) MyD88 levels were significantly elevated in AD subjects relative to levels in both NCI and MCI (***P < 0.001; Krus- kal-Wallis test) subjects. (C) TLR2 levels were significantly higher in AD compared with MCI subjects. *P < 0.05; Kruskal-Wallis test. (D) TLR4 levels did not differ significantly across the 3 groups. (E) MyD88 (0.371, P = 0.033) and (F) TLR2 (0.463, P = 0.007) were positively correlated with the Braak score by Krus- kal-Wallis test. (G) No such correlation was found between TLR4 (–0.012, P = 0.947) and the Braak score. (H) MyD88 was negatively correlated with MMSE scores (–0.538, P = 0.001) and the (I) GCS index (–0.475, P = –0.005). However, the negative correlation was not significant for TLR2 with (J) the MMSE (–0.278, P = 0.117) or (K) the GCS (–0.177, P = 0.326). TLR4 was also not negatively correlated with (L) the MMSE (–0.173, P = 0.336) or (M) the GCS (0.047, P = 0.794). Statistical significance was determined by Spearman’s rank-order test in G–M. Hippocampal sections of NCI and AD brains were double labeled with Iba-1 (microglia) and TLR2, TLR4, or MyD88. Cells positive for TLR2 (N, cortex; O, CA1), MyD88 (P, cortex; Q, CA1), and TLR4 (R, cortex; S, CA1) were counted in 2 sections (2 images/slide) of each of 4 different cases. †P < 0.001 versus NCI; 2-sample t test. Data represent the mean ± SEM.

Article Snippet: The TLR2 full-length construct (pLenticMyc-DDK/Tlr2) was purchased from Origene. cTLR2 (640–784 amino acids) tagged with cMyc was subcloned into a lentivector using the TOPO TA Cloning Kit (K5310-00; Life technologies, Thermo Fisher Scientific).

Techniques: Labeling

Journal: Journal of Clinical Investigation

Article Title: Selective disruption of TLR2-MyD88 interaction inhibits inflammation and attenuates Alzheimer’s pathology

doi: 10.1172/jci96209

Figure Lengend Snippet: Figure 2. Design of a peptide for disruption of TLR2 and MyD88 interaction. (A) A rigid-body, in silico docked pose of mouse TLR2 (blue) and MyD88 (green) (electrostatic energy = –7.750 kcal/mol; desolvation energy = –24.99 kcal/mol; VDW energy = 105.25 kcal/mol; total energy = –22.216 kcal/mol) shows strong interaction between amino acids 245 and 250 of the CD loop of MyD88 and the BB loop of TLR2. Therefore, the peptide corresponding to this domain of MyD88 (TIDM) was used to dissociate the interaction between TLR2 and MyD88. (B) TLR2-MyD88 interaction was complexed with the WT TIDM peptide (electrostatic energy = –4.516 kcal/mol; desolvation energy = –24.027 kcal/mol; VDW energy = 16.724 kcal/mol; total energy = –26.871 kcal/mol). (C) Generation of a cMyc-tagged cTLR2 recombinant protein. Amp, ampicillin resistance. The in vitro binding affinity of increasing doses of WT TIDM (D) and mTIDM (E) with cTLR2 was examined using SPR analyses (n = 2 replicates/dose in 3 independent experiments). (F) Plot of the binding response values ver- sus the concentrations of WT TIDM (circles) and mTIDM (squares) peptides. (G) Melting curve of cTLR2 protein (black) alone and with WT TIDM peptides (green). Thermal shift analyses showed a 4.96°C shift of the melting temperature (ΔTm) (n = 2 replicates/dose in 3 independent experiments). (H) Melting curve of cTLR2 protein (black) alone and with mTIDM peptides (red) indicated a ΔTm of 0.87°C (n = 2 replicates/dose in 3 independent experiments). μRIU, micro refractive index units. Data represent the mean ± SEM.

Article Snippet: The TLR2 full-length construct (pLenticMyc-DDK/Tlr2) was purchased from Origene. cTLR2 (640–784 amino acids) tagged with cMyc was subcloned into a lentivector using the TOPO TA Cloning Kit (K5310-00; Life technologies, Thermo Fisher Scientific).

Techniques: Disruption, In Silico, Recombinant, In Vitro, Binding Assay, Refractive Index

Journal: Journal of Clinical Investigation

Article Title: Selective disruption of TLR2-MyD88 interaction inhibits inflammation and attenuates Alzheimer’s pathology

doi: 10.1172/jci96209

Figure Lengend Snippet: Figure 3. Selective disruption of TLR2 and MyD88 interaction by WT TIDM. In silico analyses of interactions of WT TIDM with TLR1, TLR4, TLR5, TLR6, TLR7, and TLR9. Rigid-body interaction analyses were performed using the pyDock in silico analysis tool. Complexes of TLR1–WT TIDM (A), TLR4–WT TIDM (B), TLR5–WT TIDM (C), TLR6–WT TIDM (D), TLR7–WT TIDM (E), and TLR9–WT TIDM (F) are shown. (G) BV-2 microglial cells preincubated with WT TIDM and mTIDM peptides for 1 hour were stimulated with 1 μM fibrillar Aβ1-42 (fAβ) under serum-free conditions. After 1 hour, cellular extracts were immunoprecipitated (IP) with an anti-MyD88 antibody, followed by Western blotting of immunoprecipitates for TLR2. As a control, cellular extracts were immunoprecipitated with normal IgG. Input was also immunoblotted (IB) with TLR2 and MyD88. (H) Bands were scanned, and values (TLR2/input) are presented relative to the control (n = 2 replicates/condition in 3 independent experiments). ***P < 0.001; 2-sample t test. Results were analyzed by 2-sample t test. (I) BV-2 microglial cells preincubated with WT TIDM and mTIDM peptides for 1 hour were stimulated with LPS under serum-free condition. After 1 hour, cellular extracts were immunoprecipitated with an anti-MyD88 antibody, followed by Western blotting of immunoprecipitates for TLR4. As a control, cellular extracts were immunoprecipitated with normal IgG. Input was also immunoblotted with TLR4 and MyD88. (J) Bands were scanned, and values (TLR4/input) are presented relative to the control (n = 2 replicates/condition in 3 independent experiments). ***P < 0.001; 2-sample t test. (K) BV-2 microglial cells were transduced with pLenti-cMyc-cTlr2 lentivirions, and 48 hours after transduction, cells were treated with WT TIDM and mTIDM for 1 hour, followed by stimulation with fibrillar Aβ1-42. After 1 hour, cellular extracts were immunoprecipitated with anti-MyD88 antibody, followed by Western blotting of immunoprecipitates for cMyc. Immunodepleted (ID) fractions were also immunoblotted for cMyc as a control. (L) Bands were scanned and values (cMyc/input) presented relative to the control (n = 2 replicates/condition in 3 independent experiments). *P < 0.05 and **P < 0.01; 2-sample t test. Data represent the mean ± SEM.

Article Snippet: The TLR2 full-length construct (pLenticMyc-DDK/Tlr2) was purchased from Origene. cTLR2 (640–784 amino acids) tagged with cMyc was subcloned into a lentivector using the TOPO TA Cloning Kit (K5310-00; Life technologies, Thermo Fisher Scientific).

Techniques: Disruption, In Silico, Immunoprecipitation, Western Blot, Control, Transduction