tlr 4 expression  (Cusabio)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Mapping of the TLR4/MD2/RAGE-interacting motifs of HMGB1 or PTX3. (A, B) Schematic diagram of the domain of HMGB1 (A) and PTX3 (B) (top). GST-tagged truncates covering TLR4-, RAGE-, or MD2-binding domain of HMGB1 or PTX3 were constructed. Bottom panel (A, B) depicts GST-pull down of the indicated tagged receptors (HA-TLR4, Myc-RAGE, and His-MD2) with transiently expressed GST-tagged truncates. HEK293T cells transfected with the GST-tagged constructs in combination with HA-TLR4, Myc-RAGE or His-MD2 are indicated followed by GST-pull down. Representative western blots of WCLs and eluates after GST-pull down are shown, using GAPDH as a loading control in WCLs. Based on pulldown binding results, TLR4-, MD2-, and RAGE-targeted peptides were constructed and designated as H-T, H-R, P-T, or P-M peptides (indicated in panels A, B). (C) BMDMs were incubated with H-T, H-R, P-T, or P-M peptide for the indicated concentrations for 48 h, and then cell viability was measured with cell viability assay. Data shown are the means ± SD of three experiments (D) Cell-free immunoprecipitation assay using H-T, H-R, P-T, or P-M peptides. Mix 5 μg of alarmin protein (rHMGB1 or rPTX3) with 5 μg of each indicated tagged receptors protein (HA-TLR4, Myc-RAGE, and His-MD2), and mix as indicated to confirm the competitive inhibition effect of peptides was treated for 4 h. They were immunoprecipitated with α Flag for 2 h, separated by SDS-PAGE, and evaluated by immunoblotting. (E) Competition between rHMGB1 (top) or PTX3 (bottom) and the constructed peptides for their respective receptors. After treatment with rHMGB1 (top) or PTX3 (bottom) (10 μg/mL, 3 h) together with increasing amounts of the indicated peptides, BMDMs were used for immunoprecipitation (IP) with α TLR4 or α RAGE and IB with indicated antibodies. (F) Decrease of LPS-induced cytokine production by H-T, P-T, or P-M peptide and rHMGB1-induced cytokine production by H-R peptide. TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) together with increasing amounts of the indicated peptides-treated BMDMs were determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), bars denote mean ± SEM (∗∗∗ P < 0.001, ∗∗∗∗ P < 0.001), and significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Binding Assay, Construct, Transfection, Western Blot, Control, Incubation, Viability Assay, Immunoprecipitation, Inhibition, SDS Page, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Extracellular HMGB1 and PTX3 contribute to inflammatory responses by interacting with TLR4 or RAGE in murine macrophages. (A, B) Release of HMGB1 (A) or PTX3 (B) from murine macrophages after stimulation with LPS. LPS-induced HMGB1 (A) or PTX3 (B) release from murine macrophages. Immunoblots (IB) for HMGB1 and PTX3 in the supernatant and cell lysates in LPS-treated BMDMs at indicated time points after LPS (100 ng/mL). (C, D) Interaction between HMGB1 (C) or PTX3 (D) with its receptors. HEK293T cells were transfected with constructs, and cell lysates were immunoprecipitated with a flag-specific antibody and were analyzed by immunoblotting with the indicated antibodies. BMDMs were incubated with the rFlag-HMGB1 (C) or rFlag-PTX3 (D) 10 μg/mL for 3 h, and cell lysates were immunoprecipitated with α TLR4 or α RAGE (C, right), and α TLR4 or α MD-2 (D, right). Immunoprecipitants and WCL were analyzed by immunoblotting with the indicated antibodies. (E) TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) or recombinant alarmins (rHMGB1 or rPTX3, each (2, 5 μg/mL))-treated BMDMs determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Inhibitory effects of TMR-Lipo on TLR4/RAGE-mediated responses. (A) LPS-primed BMDMs (100 ng/mL LPS with 12 h) were treated with TMR-Lipo (0.1, 1, 5 μmol/L) for 12 h, followed by co-IP with α TLR4, α RAGE, or α MD2 and IB with indicated antibodies. (B) Schematic representation of TLR4 and RAGE signaling pathway and therapeutic targeting of TMR-Lipo (C) BMDMs were treated with TMR-Lipo as indicated concentrations (2 h), followed by treatment with LPS (100 ng/mL, 8 h). The amounts of HMGB1, PTX3, MyD88, IRAK, TRAF6, NF- κ B, KRAS, and p-p38 were measured by IB in the whole cell lysates. GAPDH was used as a loading control. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant. (D) BMDMs were treated with TMR-Lipo (0.1, 0.5, 5, 20, or 100 μmol/L) in the presence LPS for 18 h. The secretion levels of TNF- α , IL-6, IL-12p40 and IL-10 were measured by ELISAs. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: TMR-Lipo-Abs protects mice from CLP-induced polymicrobial sepsis. (A–C) Schematic design of mid-grade (A) or high-grade (B, C) sepsis mouse model induced by cecal ligation and punctuation (CLP) depending on the position of the cecal ligation (top). The survival of CLP mice treated with the indicated therapy was monitored for 10 days (A) or 72 h (B, C); mortality was measured for n = 10 mice per group (bottom). Statistical differences compared with the PBS or TMR-Lipo-Abs-treated mice are indicated (log-rank test). The data are representative of three independent experimental replicates with similar results. (D) Serum cytokine levels. The data shown represent at least three independent experiments ( n ≥ 3), significance was measured by Ordinary one-way ANOVA (TNF- α , ∗∗ P = 0.0010 and ∗∗∗ P = 0.0002, IL-6, ∗∗ P = 0.0057 and ∗∗∗ P = 0.0004 and IL12p40, both ∗∗∗∗ P < 0.0001). n.s = not significant. (E) IB identification and comparison of HMGB1 and PTX3 expression in spleen, lung, and liver cells of CLP mice after different treatment as a DAMP indicator for tissue damage. (F) Representative H&E staining of the lung, spleen, and liver from 3 mice per group. Scale bar, 100 μm.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Ligation, Comparison, Expressing, Staining

Journal: PLOS Pathogens

Article Title: Hepatitis B virus e antigen induces atypical metabolism and differentially regulates programmed cell deaths of macrophages

doi: 10.1371/journal.ppat.1012079

Figure Lengend Snippet: (A) THP-1 macrophages transduced by a lentiviral vector that expressed nonspecific shRNA (shNS), TLR2 shRNA (shTLR2) or TLR4 shRNA (shTLR4) were not treated (mock) or treated with LPS, HBsAg or HBeAg. The incubation media were then harvested for the measurement of IL-1β levels by ELISA. (B) Kupffer cells isolated from control mice were treated with nonspecific siRNA (siNS), TLR2 siRNA (siTLR2) or TLR4 siRNA (siTLR4) followed by the treatment with LPS, HBsAg or HBeAg. The incubation media were then harvested for the quantification of mouse IL-1β (mIL-1β) by ELISA. (C) Human MDMs treated with siNS, siTLR2 or siTLR4 were treated with LPS, HBsAg or HBeAg. The incubation media were then harvested for quantification of IL-1β by ELISA. (D) THP-1 macrophages without or with TLR2 or TLR4 silencing were not treated (mock) or treated with HSA, LPS, HBsAg or HBeAg for three days. The viability of cells was then analyzed by the CCK8 assay. (E) THP-1 macrophages without (shNS) or with TLR4 silencing (shTLR4) were not treated (mock) or treated with HBeAg. Cells were then stained with propidium iodide and with the anti-annexin V antibody for the analysis of apoptosis by flow cytometry. (F) THP-1 macrophages without (shNS) or with TLR4 silencing (shTLR4) were treated with HSA, LPS, HBsAg or HBeAg and then lysed for immunoblot analysis. (G) THP-1 macrophages without (shNS) or with TLR4 silencing (shTLR4) were treated with HSA, LPS, HBsAg or HBeAg and then lysed for immunoblot analysis. (H) THP-1 macrophages without (shNS) or with TLR4 silencing (shTLR4) were treated with HSA or HBeAg and then subjected to Seahorse assay for the OCR. (I) HEK293 cells were transfected with the expression plasmid of Flag-tagged TLR4, lysed and treated with GST-HBeAg, and then incubated with glutathione beads for the pull-down experiment. The protein sample was then analyzed by immunoblot using the anti-Flag, anti-GST and anti-TLR4 antibodies. Input represents the total cell lysates prior to the GST-pulldown experiment. GAPDH was used as the loading control. (J) THP-1 macrophages with the treatment of either GST, HBeAg-GST or HBcAg were subjected to the proximity ligation assay (PLA) for the co-localization analysis. Red dots represent positive colocalization signals. DAPI was used to stain the nuclei (blue color). The chart to the right is the average number of PLA signals per cell after a total of 50 cells were analyzed. In (A-E), N.S., not significant; ***, p <0.001.

Article Snippet: 1x10 6 HEK293T cells were seed in each well of a 6-well plate and transfected with the DNA plasmid (4 μg) that expressed the Flag-tagged human TLR4 (Sino Biological, #HG10146-NF) using lipofectamine following the manufacturer’s instructions.

Techniques: Plasmid Preparation, shRNA, Incubation, Enzyme-linked Immunosorbent Assay, Isolation, CCK-8 Assay, Staining, Flow Cytometry, Western Blot, Transfection, Expressing, Proximity Ligation Assay

Journal: iScience

Article Title: Exercise-acclimated microbiota improves skeletal muscle metabolism via circulating bile acid deconjugation

doi: 10.1016/j.isci.2023.106251

Figure Lengend Snippet:

Article Snippet: Taqman Gene Expression Assay TLR-4 , Thermo Fisher Scientific , ID#Mm00445273_m1.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Protein Extraction, Gene Expression, Software, Mass Spectrometry, Real-time Polymerase Chain Reaction