Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Primer used for Real-time qPCR.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Sequencing

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Cell surface level of TLR4 in BV-2 cells treated with chestnut leaf or spiny bur extracts and LPS. BV-2 cells were pre-treated (or not) with spiny bur (SB) or leaf (L) extracts (0.5 mg/mL) for 3 h, incubated with or without LPS (0.5 μg/mL) for a total of 24 h, then subjected to flow cytometric analysis of the immunostaining for TLR4 receptor. ( a ) Representative plots of untreated BV-2 cells ( b ) Representative plots of BV-2 cells not activated with LPS ( c ) Representative plots of LPS-stimulated BV-2 cells ( d ) Relative quantification is expressed as MFI, median fluorescence intensity. Results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Incubation, Immunostaining, Fluorescence, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 gene expression in BV-2 cells treated with chestnut extracts. BV-2 cells were treated with spiny bur or leaf extracts (0.5 mg/mL) for 3 h, then stimulated (or not) with LPS (0.5 µg/mL) for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4, as explained in the Materials and Methods section. The relative mRNA content of TLR4 was normalised to ( a ) untreated control and ( b ) untreated LPS control. Relative quantification is obtained as reported in the Materials and Methods section; results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 and CD14 gene expression in BV-2 cells treated with different chestnut leaf extract fractions. BV-2 cells were treated for 3 h with 50 μg/mL of fractions at different polarity (L Fr.), then stimulated with 0.5 μg/mL LPS for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4 ( a ) and CD14 ( b ), as reported in the Materials and Methods section. The relative mRNA contents of TLR4 and CD14 were normalised to LPS-treated cells (red bars). Relative quantification is obtained as described in the Materials and Methods section, and results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation

Journal: Molecules

Article Title: Improvement of Colonic Immune Function with Soy Isoflavones in High-Fat Diet-Induced Obese Rats

doi: 10.3390/molecules24061139

Figure Lengend Snippet: SIF modulated TLR4/NF-κB signaling pathway related genes expression. ( a ) mRNA relative expressions(fold of chow) of TLR2, TLR4, myD88 and NF-κB P65 in colon were assessed using qRT–PCR; ( b , c ) western blots and quantification shown the protein level of TLR4 and NF-κB p65 in colon. Error bars indicate SD. * p < 0.05, ** p < 0.005. The dashed is the chow group.

Article Snippet: Standard WB procedures were carried out with those antibodies: rabbit anti-TNF alpha polyclonal antibody (bs-2081R, bioss, Beijing, China), IL10 antibody (GTX632359, Gene Tex, CA, USA), rabbit anti-occludin polyclonal antibody (bioss, bs-10011R), anti-NF-κB (CST, mAb #8242, Danvers, MA, USA), TLR4 antibody (NB100-56566SS, Novus, CO, USA), mouse anti-β-actin (BM0627, boster, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Abdominal paracentesis drainage attenuates intestinal inflammation in rats with severe acute pancreatitis by inhibiting the HMGB1-mediated TLR4 signaling pathway

doi: 10.3748/wjg.v27.i9.815

Figure Lengend Snippet: Effects of abdominal paracentesis drainage on intestinal toll-like receptor 4 signaling pathway in rats with severe acute pancreatitis. A: Representative immunohistochemistry images of toll-like receptor 4 (TLR4) in intestinal tissue (bar = 100 μm); B: TLR4 mRNA measurement by real-time polymerase chain reaction (PCR); C: Immunoblotting of TLR4, myeloid differentiation factor 88 (MyD88) and p65 protein expression and p65 phosphorylation level from intestinal samples; D-F: Densitometry analysis of TLR4, MyD88 and ratio of p65 to phosphorylated p65. Data are expressed as means ± SD, n = 6 real-time PCR results per group; means ± SD, n = 3 immunoblotting results per group. a P < 0.05 vs sham group; b P < 0.05 vs severe acute pancreatitis group. SAP: Severe acute pancreatitis; APD: Abdominal paracentesis drainage; MyD88: Myeloid differentiation factor 88; TLR4: Toll-like receptor 4; p-p65: Phosphorylated p65; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Subsequently, the antibody was blocked by 5% bovine serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 30 min, and then incubated with rabbit anti-TLR4 at a 1:200 dilution overnight at 4 ℃.

Techniques: Immunohistochemistry, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: World Journal of Gastroenterology

Article Title: Abdominal paracentesis drainage attenuates intestinal inflammation in rats with severe acute pancreatitis by inhibiting the HMGB1-mediated TLR4 signaling pathway

doi: 10.3748/wjg.v27.i9.815

Figure Lengend Snippet: Effects of pancreatitis-associated ascitic fluid intraperitoneal injection with or without anti-high mobility group box protein 1 neutralizing antibody on intestinal toll-like receptor 4 signaling pathway in cerulein-treated rats. A: High mobility group box protein 1 in serum; B: Toll-like receptor 4 (TLR4) mRNA measurement by real-time polymerase chain reaction; C: Immunoblotting of TLR4, myeloid differentiation factor 88 (MyD88) and p65 protein expression and p65 phosphorylation level from intestine samples; D: Representative immunohistochemical images of TLR4 in intestinal tissue (bar = 100 μm); E-G: Densitometry analysis of TLR4, MyD88 and ratio of p65 to phosphorylated p65. Data are expressed as means ± SD, n = 6 enzyme-linked immunosorbent assay results per group; means ± SD, n = 3 immunoblotting results per group. a P < 0.05 vs control group; b P < 0.05 vs pancreatitis-associated ascitic fluid injection group. HMGB1: High mobility group box protein 1; CN: Control; PI: Pancreatitis-associated ascitic fluid (PAAF) injection; PIH: PAAF and 200 μg anti-HMGB1 neutralizing antibody; PIC: PAAF + control lgY; TLR4: Toll-like receptor 4; MyD88: Myeloid differentiation factor 88; p-p65: Phosphorylated p65; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Subsequently, the antibody was blocked by 5% bovine serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 30 min, and then incubated with rabbit anti-TLR4 at a 1:200 dilution overnight at 4 ℃.

Techniques: Injection, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay

Journal: World Journal of Gastroenterology

Article Title: Abdominal paracentesis drainage attenuates intestinal inflammation in rats with severe acute pancreatitis by inhibiting the HMGB1-mediated TLR4 signaling pathway

doi: 10.3748/wjg.v27.i9.815

Figure Lengend Snippet: Possible mechanisms responsible for the protective effects of abdominal paracentesis drainage treatment on severe acute pancreatitis-induced intestinal inflammation and accompanying apoptosis. TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6; HMGB1: High mobility group box protein 1; IKK: IkappaB kinase; NF-kB: Nuclear factor kB; TLR4: Toll-like receptor 4.

Article Snippet: Subsequently, the antibody was blocked by 5% bovine serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 30 min, and then incubated with rabbit anti-TLR4 at a 1:200 dilution overnight at 4 ℃.

Techniques:

Journal: Cell reports. Medicine

Article Title: A CD36-dependent non-canonical lipid metabolism program promotes immune escape and resistance to hypomethylating agent therapy in AML.

doi: 10.1016/j.xcrm.2024.101592

Figure Lengend Snippet: Figure 3. OxLDL and PA synergize to suppress T cell activity via CD36-mediated innate immune signaling in AML cells (A and B) THP-1 cells (A) or MV4-11 cells (B) stimulated in CM or LDM supplied with different lipids (palmitate [PA], 20 mM; OxLDL, 25 mg/mL) for 3 days were further used for T cell proliferation assay (n = 3). (C–E) Immunoblot analysis of TLR4 (C and E) or LYN (D) precipitated proteins. (C and E) AML cells were treated with different reagents (PA, 20 mM; OxLDL, 25 mg/mL; PP1, 10 mM) for 5 h before immunoprecipitation. (F) MV4-11 cells treated with LDM supplied with different reagents (OxLDL, 25 mg/mL; TAK-242, 10 mM; PP1, 10 mM) for 3 days were further used for T cell proliferation assay (n = 3). (G–I) AML cells labeled with a chemical probe (alk-C16) for 4 h in the absence (G and H) or presence of SSO (50 mM) (I) were used for palmitoylation assay.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated with the the following antibodies overnight 4 C with constant rotation: p-p65 (Cell Signaling Technology; 3033S), p65 (Cell Signaling Technology; 8242S), actin (Cell Signaling Technology; 3700S), Lamin-B1 (Santa Cruz Biotechnology; sc-374015), MYD88 (Cell Signaling Technology; 4283), CD36 (abcom; ab133625), ZDHHC6 (abcom; ab121423), TLR4 (CUSABIO Technology; CSB-PA001434), LYN (Cell Signaling Technology; 2796S).

Techniques: Activity Assay, Proliferation Assay, Western Blot, Immunoprecipitation, Labeling

Journal: Frontiers in Immunology

Article Title: CEACAM 1, 3, 5 and 6 -positive classical monocytes correlate with interstitial lung disease in early systemic sclerosis

doi: 10.3389/fimmu.2022.1016914

Figure Lengend Snippet: Antibodies used for Mass Cytometry.

Article Snippet: TLR4 , HTA125 , 158Gd , Fluidigm.

Techniques: Cytometry