Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiulcerogenic Activity of Li-Zhong Decoction on Duodenal Ulcers Induced by Indomethacin in Rats: Involvement of TLR-2/MyD88 Signaling Pathway

doi: 10.1155/2020/6538156

Figure Lengend Snippet: List of primers used in qRT-PCR.

Article Snippet: The sections were incubated with rabbit anti-TLR2 antibody (1 : 100 dilution; Bioss antibodies, Beijing, China) or rabbit anti-MyD88 antibody (1 : 100 dilution; Bioss antibodies, Beijing, China) overnight at 4°C and then with HRP-conjugated goat anti-rabbit secondary antibody (1 : 100 dilution; Bioss antibodies, Beijing, China).

Techniques: Sequencing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiulcerogenic Activity of Li-Zhong Decoction on Duodenal Ulcers Induced by Indomethacin in Rats: Involvement of TLR-2/MyD88 Signaling Pathway

doi: 10.1155/2020/6538156

Figure Lengend Snippet: Effects of LZD on levels of TLR-2 (a) and MyD88 (b) mRNA expression during prevention of IND‐induced duodenal ulceration. Levels of TLR-2 and MyD88 mRNA were determined by qRT-PCR assay. β -Actin mRNA was used as internal control for equal loading. Results are expressed as mean ± SEM, n = 3. Asterisks and pound signs indicate significant differences: ∗∗ P < 0.01 versus normal control (NC); ## P < 0.01 versus negative control (IND).

Article Snippet: The sections were incubated with rabbit anti-TLR2 antibody (1 : 100 dilution; Bioss antibodies, Beijing, China) or rabbit anti-MyD88 antibody (1 : 100 dilution; Bioss antibodies, Beijing, China) overnight at 4°C and then with HRP-conjugated goat anti-rabbit secondary antibody (1 : 100 dilution; Bioss antibodies, Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Negative Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiulcerogenic Activity of Li-Zhong Decoction on Duodenal Ulcers Induced by Indomethacin in Rats: Involvement of TLR-2/MyD88 Signaling Pathway

doi: 10.1155/2020/6538156

Figure Lengend Snippet: Effects of LZD on levels of TLR-2 and MyD88 protein expression during prevention of IND induced duodenal ulceration. (a) Representative immunoblots for TLR-2 and MyD88 proteins as measured by western blot analysis using specific antibodies. Loading control was monitored by β -actin immunoblotting. Quantitative analysis derived from densitometric scans of western blots of TLR-2 (b) and MyD88 (c) proteins of duodenal mucosa. Results are expressed as mean ± SEM, n = 3. Asterisks and pound signs indicate significant differences: ∗∗ P < 0.01 versus normal control (NC); ## P < 0.01 versus negative control (IND).

Article Snippet: The sections were incubated with rabbit anti-TLR2 antibody (1 : 100 dilution; Bioss antibodies, Beijing, China) or rabbit anti-MyD88 antibody (1 : 100 dilution; Bioss antibodies, Beijing, China) overnight at 4°C and then with HRP-conjugated goat anti-rabbit secondary antibody (1 : 100 dilution; Bioss antibodies, Beijing, China).

Techniques: Expressing, Western Blot, Derivative Assay, Negative Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiulcerogenic Activity of Li-Zhong Decoction on Duodenal Ulcers Induced by Indomethacin in Rats: Involvement of TLR-2/MyD88 Signaling Pathway

doi: 10.1155/2020/6538156

Figure Lengend Snippet: Localization of TLR-2 and MyD88 in duodenal tissues after IND administration and effect of LZD thereon. (a) Representative pictures showing the immunohistochemical analysis of TLR-2 and MyD88 in sections of duodenal mucosa obtained from rats in normal control, negative control, ESO, and LZD groups. The black arrows denote the immunopositive expression parts of TLR-2 and MyD88. Magnification 200x. (b, c) Quantitative analysis of the immunohistochemical signals as measured by IOD values. Results are expressed as the mean ± SEM ( n = 3). Asterisks and pound signs indicate significant differences: ∗∗ P < 0.01 versus normal control (NC); ## P < 0.01 versus negative control (IND).

Article Snippet: The sections were incubated with rabbit anti-TLR2 antibody (1 : 100 dilution; Bioss antibodies, Beijing, China) or rabbit anti-MyD88 antibody (1 : 100 dilution; Bioss antibodies, Beijing, China) overnight at 4°C and then with HRP-conjugated goat anti-rabbit secondary antibody (1 : 100 dilution; Bioss antibodies, Beijing, China).

Techniques: Immunohistochemical staining, Negative Control, Expressing

Journal: International journal of immunopathology and pharmacology

Article Title: Corilagin relieves atherosclerosis via the toll-like receptor 4 signaling pathway in vascular smooth muscle cells.

doi: 10.1177/03946320241254083

Figure Lengend Snippet: Figure 2. Effect of ox-LDL on the TLR4 signaling pathway in MOVAS cells. (a) mRNA expression of TLR4, TIRAP and MyD88 was measured by qRT-PCR. *p < .05 compared with other concentrations of ox-LDL groups or control group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (b) *p < .05 compared with either time point or control groups determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5).

Article Snippet: Afterwards, the cells were subjected to a 2-h incubation at 37°C with either primary anti-TLR4 antibody (1:200 dilution; Cat No. BA1717; Boster Biological Technology, Pleasanton, CA, USA) or anti-MyD88 antibody (1:400 dilution; Cat No. PB9148; Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: International journal of immunopathology and pharmacology

Article Title: Corilagin relieves atherosclerosis via the toll-like receptor 4 signaling pathway in vascular smooth muscle cells.

doi: 10.1177/03946320241254083

Figure Lengend Snippet: Figure 3. Effect of corilagin on the TLR4 signaling pathway in MOVAS cells stimulated by ox-LDL. (a) mRNA expression of TLR4, TIRAP, MyD88, TRAF6, p38, NEMO and IRF5 was measured by qRT-PCR. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (B. C) Protein abundance was measured by western blotting. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group, as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (D, E) Abundance of IL-6 and MCP-1 in cell culture supernatant was measured by ELISAs. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (F) Effect of corilagin on the MOVAS cells proliferation. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (G, H) apoptosis ratio of MOVAS were detected by Annexin V staining. No significant difference was found among four groups determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5).

Article Snippet: Afterwards, the cells were subjected to a 2-h incubation at 37°C with either primary anti-TLR4 antibody (1:200 dilution; Cat No. BA1717; Boster Biological Technology, Pleasanton, CA, USA) or anti-MyD88 antibody (1:400 dilution; Cat No. PB9148; Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control, Quantitative Proteomics, Western Blot, Cell Culture, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Change in Oxidative Stress and Mitochondrial Dynamics in Response to Elevated Cold-Inducible RNA-Binding Protein in Cardiac Surgery-Associated Acute Kidney Injury

doi: 10.1155/2022/3576892

Figure Lengend Snippet: CPB induced mitochondrial dynamics disorder and increased inflammatory factor secretion. (a–f) Expression of IL-6, IL-1 β , and TNF- α in the serum at 0 and 4 hours after CPB. Compared to the CPB group, C23 administration decreased the expression of IL-6, IL-1 β , and TNF- α by 54.0%, 67.5%, and 45.2% at 4 hours, respectively. (g) Western blot analysis of renal Bax, Bcl-2, and cleaved-caspase-3 expression. (h) CPB upregulated the NADPH oxidase via the TLR-4/MyD88 pathway, which promoted the expressions of mitochondrial fission-related proteins Fis1 and Drp1 and reduced the expression of fusion-related protein Mfn2. ∗ P < 0.05 vs. sham; # P < 0.05 vs. CPB.

Article Snippet: The other antibodies used in this study included TLR-4 (19811-1-AP, Proteintech, 1 : 1000), MyD88 (BA2321, Boster, 1 : 500), and anti- β -actin (4967, Cell Signaling Technology, 1 : 1000).

Techniques: Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Change in Oxidative Stress and Mitochondrial Dynamics in Response to Elevated Cold-Inducible RNA-Binding Protein in Cardiac Surgery-Associated Acute Kidney Injury

doi: 10.1155/2022/3576892

Figure Lengend Snippet: Putative mechanism of CIRP in cardiac surgery-associated acute kidney injury. During cardiac surgery, CIRP is secreted into the circulation in response to hypothermia and hemodynamics change. Extracellular CIRP promotes the expression of NADPH oxidase in renal tubular epithelial cells via the TLR-4/MyD88 pathway and aggravates intracellular oxidative stress. ROS accumulation induces mitochondrial dynamics disorder, which ultimately increases apoptosis and promotes AKI. CIR: cold-inducible RNA-binding protein; NADPH: nicotinamide adenine dinucleotide phosphate; ROS: reactive oxygen species.

Article Snippet: The other antibodies used in this study included TLR-4 (19811-1-AP, Proteintech, 1 : 1000), MyD88 (BA2321, Boster, 1 : 500), and anti- β -actin (4967, Cell Signaling Technology, 1 : 1000).

Techniques: Expressing, RNA Binding Assay

Journal: Pharmacological Research - Modern Chinese Medicine

Article Title: The pharmaceutical applications of total flavonoids extract from Isatis tinctoria L. leaves

doi: 10.1016/j.prmcm.2022.100122

Figure Lengend Snippet: Fig. 8. Effects of TFE from Folium isatidis on the genes expression of TLR4 (a), TRAF6 (b), TRIF (c), I 𝜅B (d) and p65 (e) in lungs tissues of LPS induced ALI mice ( # compared with the control, ∗ compared with LPS, ∗ P < 0.05, ∗ ∗ / ## P < 0.01).

Article Snippet: Antibodies against TLR4, RAF6, TRIF, NF- κB p65, p-NF- κB p65, I κB α, p-I κB α and β-actin were urchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and econdary antibodies were obtained from Wuhan Boster Biological Techology., Ltd (Wuhan, China).

Techniques: Expressing, Control

Journal: Pharmacological Research - Modern Chinese Medicine

Article Title: The pharmaceutical applications of total flavonoids extract from Isatis tinctoria L. leaves

doi: 10.1016/j.prmcm.2022.100122

Figure Lengend Snippet: Fig. 9. Effects of TFE from Folium isatidis on the proteins expression of TLR4 (a), TRAF6 (b), TRIF (c), I 𝜅B (d), p-I 𝜅B (e), p65 (f) and p-p65 (g) in lungs tissues of LPS induced ALI mice ( # compared with the cntrol, ∗ compared with LPS, ∗ P < 0.05, ∗ ∗ / ## P < 0.01).

Article Snippet: Antibodies against TLR4, RAF6, TRIF, NF- κB p65, p-NF- κB p65, I κB α, p-I κB α and β-actin were urchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and econdary antibodies were obtained from Wuhan Boster Biological Techology., Ltd (Wuhan, China).

Techniques: Expressing

Journal: Pharmacological Research - Modern Chinese Medicine

Article Title: The pharmaceutical applications of total flavonoids extract from Isatis tinctoria L. leaves

doi: 10.1016/j.prmcm.2022.100122

Figure Lengend Snippet: Fig. 10. Regulation of TFE from Folium isatidis on TLR4-TRIF-NF- 𝜅B signaling pathway in LPS induced ALI mice.

Article Snippet: Antibodies against TLR4, RAF6, TRIF, NF- κB p65, p-NF- κB p65, I κB α, p-I κB α and β-actin were urchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and econdary antibodies were obtained from Wuhan Boster Biological Techology., Ltd (Wuhan, China).

Techniques:

Journal: PLoS ONE

Article Title: Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

doi: 10.1371/journal.pone.0015985

Figure Lengend Snippet: (A) Transcriptional activation of MyD88, IL-1R1AcP and TNF-α in HLA-DR positive primary monocytes treated with 200 ng SEB/ml (optimum dose), mAbs (10 µg/ml, optimum dose) directed against MHC-class II molecule (anti-DR-DP-DQ or LB3.1) or unrelated control antibody OKT3 was examined by semi-quantitative RT-PCR. Data shown is one of 3 similar experiments; (B) Agonists binding to TLR4 or HLA class II molecules on CD14 + monocytes induced transcriptional up regulation of MyD88. Real time RT-PCR was used to determine relative expression of MyD88 normalized to the expression of β-actin. Expression levels are determined as means +/− SD. Data presented as one of 3 similar experiments. Significance compared to untreated control (*) was assigned as P≤0.0001. (C) MHC class II molecule dependence of SEB- induced TNF- α gene expression. Pretreatment of monocytes in ice with anti-DR-DP-DQ at optimum dose (10 µg/ml) followed by SEB stimulation resulted in reduced TNF- α gene expression. Transcriptional activation of TNF- α expression normalized to the expression of β-actin. Expression levels of TNF- α are expressed as means +/− SD. Significance was assigned (*) or (**) as P values ≤0.003 comparing untreated vs treatment groups with anti-DR-DP-DQ, SEB or SEB vs anti-DR-DP-DQ+SEB respectively. (D) Confocal images show expression of HLA-DR (green) and intracellular MyD88 (red) proteins in CD14 + monocytes treated with HLA class II- ligands or control antibody OKT3 for 16 h; Scale bar = 5 µm; (E) intracellular expression of MyD88 protein in activated monocytes. Primary monocytes (CD14 + , CD3 - ) were activated as described earlier, permeabilized and labeled with primary MyD88 antibody followed by PE-labeled secondary antibody and analyzed by flow cytometry. Histogram represents a MHC class II ligand-induced increase in expression of MyD88 protein compared to non-MHC class II ligand (OKT3).

Article Snippet: Primary anti-MyD88 antibody was obtained from AnaSpec, Inc. (San Jose, CA) and Alexis Biochemicals (San Diego, CA).

Techniques: Activation Assay, Control, Quantitative RT-PCR, Binding Assay, Expressing, Gene Expression, Labeling, Flow Cytometry

Journal: PLoS ONE

Article Title: Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

doi: 10.1371/journal.pone.0015985

Figure Lengend Snippet: SEB engagement of HLA-class II on human monocytes induced intracellular up regulation of MyD88, TNF-α, and IL-1β compared to untreated control and additionally activated NF-kB. (A) Confocal images show intracellular up regulation of MyD88 protein in primary monocytes stimulated with SEB for 1 h compared to untreated monocytes; Scale bar = 10 µm. (B) Activation of NF-kB in primary monocytes treated with SEB. Data are presented in the figure as percentage increase over the untreated control and represent one of three experiments using separate donors. Significance was assigned as follows; p50 (p = 0.009) and p65 (p = 0.004). (C) Transcriptional activation of IL-1β, and TNF-α mRNA was measured by real time RT-PCR in isolated primary monocytes after stimulation of total mononuclear cells with SEA or SEB.

Article Snippet: Primary anti-MyD88 antibody was obtained from AnaSpec, Inc. (San Jose, CA) and Alexis Biochemicals (San Diego, CA).

Techniques: Control, Activation Assay, Quantitative RT-PCR, Isolation

Journal: PLoS ONE

Article Title: Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

doi: 10.1371/journal.pone.0015985

Figure Lengend Snippet: CD14 + cells were treated with SEB for different times or left untreated as indicated. Cells were lysed, membrane fraction and cytoplasm fraction were isolated by centrifugation. Cytoplasmic fractions were run by electrophoresis and blotted using an anti-human MyD88 antibody. (A) Up regulation of MyD88 (62 kDa) dimer and 31 kDa monomer after SEB stimulation. Monocytes were treated with SEB (200 ng/ml) for 1 h or left untreated. (B) Phosphotyrosine antibody recognized MyD88 (25 kDa) band in cytoplasmic extracts of monocytes stimulated with SEB. (C) MyD88 protein up regulation in monocytes after SEB stimulation. Monocytes (CD14 + ) were treated with SEB (200 ng/ml) for 30 min or 2 h or left untreated. After treatments, cytoplasmic fractions were isolated, separated by electrophoresis and probed with anti-MyD88 or anti-β-actin antibody. (D) SEB stimulation of monocytes induced phosphorylation of MyD88. Part of the cytoplasmic fractions isolated after SEB stimulation as described in (C) was immune-precipitated with an agarose-bound anti-human MyD88, separated by electrophoresis and blotted against an anti-phosphotyrosine antibody (α-PY).

Article Snippet: Primary anti-MyD88 antibody was obtained from AnaSpec, Inc. (San Jose, CA) and Alexis Biochemicals (San Diego, CA).

Techniques: Membrane, Isolation, Centrifugation, Electrophoresis, Phospho-proteomics

Journal: PLoS ONE

Article Title: Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

doi: 10.1371/journal.pone.0015985

Figure Lengend Snippet: Primary monocytes or human monocytic cell line U937 were treated with SEB or left untreated as indicated. Cells were lysed, membrane fraction and cytoplasm fraction were isolated by centrifugation. Cytoplasmic fractions were run by electrophoresis and blotted using an anti-human MyD88 antibody or anti-IRAK4, or anti-TRAF6 antibody. (A) Up regulation of MyD88 (62 kDa) dimer and 31 kDa monomer, TRAF6, IRAK4 after SEB stimulation in monocytes (B) Up regulation of MyD88 (62 kDa) dimer and 31 kDa monomer, TRAF6, IRAK4 after SEB stimulation in U937 cells.

Article Snippet: Primary anti-MyD88 antibody was obtained from AnaSpec, Inc. (San Jose, CA) and Alexis Biochemicals (San Diego, CA).

Techniques: Membrane, Isolation, Centrifugation, Electrophoresis

Journal: PLoS ONE

Article Title: Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

doi: 10.1371/journal.pone.0015985

Figure Lengend Snippet: Primary B cells (CD19 + ) were cultured in the presence of SEB (200 ng/ml), CpG (10 µg/ml), or LPS (1 µg/ml), fixed, permeabilized and labeled with anti-human MyD88 antibody. Confocal images show intracellular up regulation of MyD88 protein in primary monocytes stimulated with SEB, LPS or CpG compared to untreated cells; Scale bar = 5 µm.

Article Snippet: Primary anti-MyD88 antibody was obtained from AnaSpec, Inc. (San Jose, CA) and Alexis Biochemicals (San Diego, CA).

Techniques: Cell Culture, Labeling

Journal: PLoS ONE

Article Title: Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

doi: 10.1371/journal.pone.0015985

Figure Lengend Snippet: B cells or T2 cells were treated with SEA (200 ng/ml) or SEB (200 ng/ml) or kept untreated. Cells were lysed, membrane fraction and cytoplasm fraction were isolated by centrifugation. Cytoplasmic fractions were run by electrophoresis and blotted using an anti-human MyD88 antibody. (A) Detection of MyD88 after SEA and SEB stimulation in B cells or T2 cells. (B) Intracellular up regulation of MyD88 (red) in T2 cells after CpG and LPS stimulation; Scale bar = 10 µm.

Article Snippet: Primary anti-MyD88 antibody was obtained from AnaSpec, Inc. (San Jose, CA) and Alexis Biochemicals (San Diego, CA).

Techniques: Membrane, Isolation, Centrifugation, Electrophoresis

Journal: Scientific Reports

Article Title: The parasitic worm-derived immunomodulator, ES-62 and its drug-like small molecule analogues exhibit therapeutic potential in a model of chronic asthma

doi: 10.1038/srep19224

Figure Lengend Snippet: ( a ) Total and differential neutrophil cell counts in the BALF of the indicated number of individual mice from the PBS-, OVA- and ES1-groups at day 69, where *p < 0.05 relative to the OVA group. ( b ) Mean values of eotaxin2 in the BALF of the indicated number of individual mice from the PBS-, OVA- and ES1-groups at day 69, demonstrating that the mean ± SEM levels in the OVA, but not ES1, group are significantly (*p < 0.05) different to those in PBS-treated mice. ( c ) ES-62 reduces the levels of OVA-specific IgG1 in serum where *p < 0.05 for the mean ± SEM of mean values of the indicated number of ES1 versus OVA mice. ( d ) qRT-PCR analysis of mRNA levels of periostin, muc5ac and muc5b where data are presented as the mean of mean 2 −ΔΔCt values for individual mice and where ***p < 0.001 for ES1 versus OVA groups of mice. ( e ) qRT-PCR analysis of mRNA levels of tlr4 and myd88 where data are presented as the means ± SEM of mean 2 −ΔΔCt values for 3 individual mice in each group. ( f ) Immunofluorescence analysis of MyD88 (green) and nuclear (DAPI; blue) staining in day 69 lung sections from the PBS-, OVA- ES1- and ES4 groups.

Article Snippet: Lung expression of MyD88 and Collagen VI was visualised by staining tissue sections (7 μm) with a polyclonal anti-human/mouse MyD88 antibody (eBioscience; 1:500 dilution) and FITC-labelled anti-rabbit IgG secondary antibody (1:200 dilution) or a rabbit polyclonal anti-Collagen VI antibody (1:200 dilution), biotinylated anti-rabbit IgG antibody (1:200 dilution) and streptavidin-AF647 (1:200 dilution), with DAPI (diluted to 1:10000 in PBS) used as a counterstain.

Techniques: Quantitative RT-PCR, Immunofluorescence, Staining