Journal: Molecular Neurobiology

Article Title: MyD88 Deficiency, but Not Gut Microbiota Depletion, Is Sufficient to Modulate the Blood–Brain Barrier Function in the Mediobasal Hypothalamus

doi: 10.1007/s12035-022-02802-w

Figure Lengend Snippet: Antibodies. List of primary and secondary antibodies used for immunofluorescence with dilution and catalogue number

Article Snippet: TLR4 , 1:200 , SPC-200, Stress Marq Biosciences.

Techniques: Immunofluorescence

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A qPCR showed increased TLR4 expression in the 216911KO cell line. ** p < 0.01 by student T tests. B The diagram presented two pir-hsa-216911 binding sites in 3ʹUTR of TLR4. C The dual luciferase assay revealed that pir-has-216911 could bind both TLR4 binding sites TLR4S1 and TLR4S2. Plasmids carrying TLR4S1 and TLR4S2 generate a higher Firefly/Renilla Luciferase ratio in the 216911KO cell line, which pir-hsa-216911 was absent. **p < 0.01; ***p < 0.001 by student T tests. D TCGA-LIHC survival analysis found low expression of TLR4 to be unfavorable to the survival of HCC patients. E Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway. TLR4, p-p65, and NLRP3 levels were elevated in the 216911KO cell line, which pir-hsa-216911 was absent. F Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway through silencing TLR4. After silencing TLR4 with siRNA, pir-hsa-216911 absent could not elevate p-p65, and NLRP3 levels were elevated in the 216911KO cell line.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Expressing, Binding Assay, Luciferase, Western Blot

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A The diagram presented the xenograft modeling process. B Euthanized mouse to display the subcutaneous xenografted tumors. C 216911KO group resulted in much smaller tumors in size compared to the control group. D 216911KO group resulted in much smaller tumors in weight compared to the control group. The histogram represents the mean tumor weight of each group. ***p < 0.001 by student T tests. E Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway in vivo. Both TLR4 and p-p65 levels were elevated in tumors formed in the 216911KO group compared to the control group.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Control, Western Blot, In Vivo

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A The diagram presented the clinical HCC tumor tissue sampling process. B The immunohistochemistry demonstrates that TLR4 was downregulated in high pir-hsa-216911 tumor samples. The image shows typical IHC results. The box plot presents all IHC scores. *p < 0.01 by LSD test. C The immunohistochemistry demonstrates that c-GSDMD activation was suppressed in high pir-hsa-216911 tumor samples. The image shows typical IHC results. The box plot presents all IHC scores. * p < 0.01 by LSD test. D The tumor/adjacent tissue pir-hsa-216911 ratio was higher in HCC patients accompanied by steatosis. ** p < 0.01 by student T tests.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Sampling, Immunohistochemistry, Activation Assay

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: Correlation analysis between pir-hsa-216911 elevation, TLR4 expression, and GSDMD activation.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Expressing, Activation Assay

Journal: Frontiers in Immunology

Article Title: Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction

doi: 10.3389/fimmu.2022.1078880

Figure Lengend Snippet: Polarization-related signaling pathway of macrophages was activated after T.cp -MIF treatment. The macrophages binded with TLR4, and T.cp -MIF activated NF-κB signaling pathway 24 hours after induction. At this time, macrophages showed M1 type polarization, while PI3K/AKT/mTOR signaling pathway is activated at 48 hours, making macrophages show M2 type polarization.

Article Snippet: After blocking, the corresponding primary antibodies such as TLR4 (Wuhan Sanying Biotechnology Co., Ltd.), NF-κB (Wuhan Sanying Biotechnology Co., Ltd.), p-NF-κB (Cell signaling technology, USA), PI3K (Wuhan Sanying Biotechnology Co., Ltd.), p-PI3K (Abcam, UK), Akt (Wuhan Sanying Biotechnology Co. Ltd.), and β-actin (Hangzhou Huaan Biotechnology Co., Ltd.) was separately added to the box (antibody concentrations were diluted according to the instructions), incubated at 4°C for over 12 h. After incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

Techniques:

Journal: Frontiers in Immunology

Article Title: Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction

doi: 10.3389/fimmu.2022.1078880

Figure Lengend Snippet: Effect of T.cp -MIF on the expression of macrophage polarization-related proteins in different time. (A–D) The protein expression levers of TLR4, NF-κBp65, PI3K and AKT were analyzed by western blot and normalized against β-actin expression. * indicated P <0.05. Results are showed as the mean of three times of independent experiments ( x̄ ±SD, n=3).

Article Snippet: After blocking, the corresponding primary antibodies such as TLR4 (Wuhan Sanying Biotechnology Co., Ltd.), NF-κB (Wuhan Sanying Biotechnology Co., Ltd.), p-NF-κB (Cell signaling technology, USA), PI3K (Wuhan Sanying Biotechnology Co., Ltd.), p-PI3K (Abcam, UK), Akt (Wuhan Sanying Biotechnology Co. Ltd.), and β-actin (Hangzhou Huaan Biotechnology Co., Ltd.) was separately added to the box (antibody concentrations were diluted according to the instructions), incubated at 4°C for over 12 h. After incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

Techniques: Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction

doi: 10.3389/fimmu.2022.1078880

Figure Lengend Snippet: The effect of TAK-242 on the expression of macrophage polarization-related proteins. (A–D) The cells were pretreated with TAK-242 (the TLR4 inhibitor) for 6 h, and the expression levels of TLR4, NF-κBp65, p-NF-κBp65, PI3K, p-PI3K, Akt and p-Akt were detected by western blot. * indicated P <0.05. Results are showed as the mean of three times of independent experiments ( x̄ ±SD, n=3).

Article Snippet: After blocking, the corresponding primary antibodies such as TLR4 (Wuhan Sanying Biotechnology Co., Ltd.), NF-κB (Wuhan Sanying Biotechnology Co., Ltd.), p-NF-κB (Cell signaling technology, USA), PI3K (Wuhan Sanying Biotechnology Co., Ltd.), p-PI3K (Abcam, UK), Akt (Wuhan Sanying Biotechnology Co. Ltd.), and β-actin (Hangzhou Huaan Biotechnology Co., Ltd.) was separately added to the box (antibody concentrations were diluted according to the instructions), incubated at 4°C for over 12 h. After incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

Techniques: Expressing, Western Blot

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: International Journal of Molecular Sciences

Article Title: Effect of Calcium-Sulphate-Bicarbonate Water in a Murine Model of Non-Alcoholic Fatty Liver Disease: A Histopathology Study

doi: 10.3390/ijms231710065

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: TLR4 , Rabbit , Atlas Antibodies , HPA049174 , 1:200 , IHC/IF , [ ] .

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

doi: 10.1084/jem.20210862

Figure Lengend Snippet: Primary antibodies

Article Snippet: TLR4 , BioRad , MCA2154T.

Techniques:

Journal: Microbiome

Article Title: Ochratoxin A induces liver inflammation: involvement of intestinal microbiota

doi: 10.1186/s40168-019-0761-z

Figure Lengend Snippet: OTA promotes liver inflammation. a Relative mRNA expressions of TLR4, MYD88, IKBα, IL-6, and TNF-α in the liver after OTA oral gavage ( n = 6, mean with SEM). b Relative protein abundances of TLR4, MYD88, p-IKBα, p-IKBα/IKBα, and p-p65 in the liver after OTA oral gavage ( n = 6, mean with SEM). c Effect of OTA on levels of liver cytokines, including IL-1β, IL-6, TNF-α, IL-8, IL-10, and IFN-γ ( n = 6, mean with SEM). d Representative images of H&E-stained liver sections in CON and OTA group (magnification × 400, scale bar 100 μm, n = 6). e Statistical analysis of the percentage of inflammatory cells in different groups shown in d ( n = 6, mean with SEM). f Effect of OTA on serum levels of AST, ALT, ALP, and LDH ( n = 6, mean with SEM). g Serum LPS level with or without OTA treatment ( n = 6, mean with SEM). h Effect of OTA on serum levels of IL-1β, IL-6, TNF-α, and IL-10 ( n = 6, mean with SEM). Data in a , b , c , e , f , g , and h were analyzed with unpaired t test, * P < 0.05; ** P < 0.01, *** P < 0.001, P <0.0001

Article Snippet: Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA).

Techniques: Staining

Journal: Microbiome

Article Title: Ochratoxin A induces liver inflammation: involvement of intestinal microbiota

doi: 10.1186/s40168-019-0761-z

Figure Lengend Snippet: OTA fails to promote liver inflammation in antibiotics-treated ducks. a Liver LPS level in different groups ( n = 6, mean with SEM). b Relative mRNA expressions of TLR4, MYD88, IKBα, IL-6, and TNF-α in the liver of antibiotics-treated ducks with or without OTA ( n = 6, mean with SEM). c Levels of IL-1β, IL-6, TNF-α, IL-8, IL-10, and IFN-γ in the liver of antibiotics-treated ducks with or without OTA ( n = 6, mean with SEM). d Representative images of H&E-stained liver sections (magnification × 400; scale bar 100 μm; n = 6). e Statistical analysis of the percentage of inflammatory cells in different groups shown in d ( n = 6, mean with SEM). f Effect of OTA on serum levels of AST, ALT, ALP and LDH in antibiotics-treated ducks ( n = 6, mean with SEM). g Serum LPS level with or without OTA treatment in antibiotics-treated ducks ( n = 6, mean with SEM). h Effect of OTA on serum levels of IL-1β, IL-6, TNF-α, and IL-10 ( n = 6, mean with SEM). Data were analyzed with unpaired t test. n.s., not significant

Article Snippet: Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA).

Techniques: Staining

Journal: Microbiome

Article Title: Ochratoxin A induces liver inflammation: involvement of intestinal microbiota

doi: 10.1186/s40168-019-0761-z

Figure Lengend Snippet: OTA-originated microbiota induces liver inflammation. a Liver LPS level in FMT (CON) and FMT (OTA) ducks. b Relative mRNA expressions of TLR4, MYD88, IKBα, IL-6, and TNF-α in the liver after FMT ( n = 6, mean with SEM). c Relative protein abundance of TLR4, MYD88, p-IKBα, p-IKBα/IKBα, and p-p65 in the liver after FMT ( n = 6, mean with SEM). d Effect of FMT on the liver levels of IL-1β, IL-6, TNF-α, and IL-10 ( n = 6, mean with SEM). e Representative H&E-stained liver sections. f Statistical analysis of the percentage of inflammatory cells in different groups shown in e ( n = 6, mean with SEM). g Serum levels of AST, ALT, ALP, and LDH in different groups ( n = 6, mean with SEM). h Serum LPS level in different FMT groups ( n = 6, mean with SEM). i Serum levels of IL-1β, IL-6, and TNF-α after FMT ( n = 6, mean with SEM). FMT (CON): ducks received the CON group fecal microbiota. FMT (OTA): ducks received the OTA group fecal microbiota. Data were analyzed with unpaired t test, n = 6. * P < 0.05; ** P < 0.01, *** P < 0.001, **** P <0.0001

Article Snippet: Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA).

Techniques: Quantitative Proteomics, Staining