Journal: Nature Communications

Article Title: The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development

doi: 10.1038/s41467-025-56440-6

Figure Lengend Snippet: a Immunoblot analysis of Pellino1 protein levels in lysates of BMDMs from WT mice stimulated with TLR agonists (100 ng/mL Pam3CSK4, 10 7 cells/mL HKLM, 1 μg/mL poly(I:C), 1 μg/mL LPS, 1 μg/mL ST-FLA, 50 ng/mL FSL-1, 2 μg/mL Imiquimod, 3 μg/mL ssRNA40, and 5 μM ODN1826) for the indicated times. TLR1/2, Pam3CSK4; TLR2, HKLM; TLR3, poly(I:C); TLR4, LPS; TLR5, ST-FLA; TLR2/6, FSL-1; TLR7, Imiquimod; TLR8, ssRNA40; and TLR9, ODN1826. b Expression levels of Pellino1 mRNA in BMDMs from WT mice stimulated with TLR agonists for 6 h were measured and normalized to those of GAPDH ( n = 4). c Representative immunohistochemical staining of Pellino1 in colon tissues obtained from WT male mice drinking normal or 3% DSS water for 7 days. Scale bar = 200 µm. d (Left) Immunoblot analysis of Pellino1 and COX-2 protein levels in lysates extracted from intestines of WT male mice drinking normal or 1.5% DSS water for 9 days (H 2 O, n = 3; DSS, n = 4). (Right) Quantification of Pellino1 and COX-2 protein levels. Pellino1 and COX-2 protein levels were normalized to Actin. e (Left) Immunofluorescence staining of Pellino1 (red), F4/80 (green), and DAPI (blue) in colon tissues of WT mice drinking normal or 1.5% DSS water for 9 days. DAPI stains the nuclei. Scale bar = 50 μm. (Right) Number of Pellino1 + F4/80 + cells ( n = 4). Quantification of cells in the field of view area. f Expression levels of Pellino1 mRNA in intestinal macrophages obtained from WT male mice drinking normal or 1.5% DSS water for 9 days were measured and normalized to those of GAPDH ( n = 6). Data were represented as mean ± SD in ( b , d , e , f ). All statistical comparisons were made using a two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: The following fluorochrome-labeled mAbs and staining reagents were used according to the manufacturer’s protocol: PE- or PE-Cy7-anti-B220 (clone RA3-6B2, eBioscience), PerCP-Cy5.5- or PE-Cy7-anti-CD3 (clone 145-2C11, eBioscience), FITC- or PE-Cy7-anti-CD4 (clone RM4-5, eBioscience), PE- or APC-anti-CD8 (clone 53-6.7, eBioscience), APC- or PE-Cy7-anti-CD11b (clone M1/70, eBioscience), PE-Cy7-anti-CD11c (clone N418, eBioscience), PerCP-Cy5.5-anti-CD19 (clone 1D3, eBioscience), PerCP-Cy5.5-anti-CD45 (clone 30-F11, eBioscience), PE-Cy7-anti-CX3CR1 (clone SA011F11, BioLegend), FITC-anti-Ly6G (clone 1AB, eBioscience), APC-anti-Ly6C (clone HK1.4, eBioscience), FITC-anti-MHC2 (clone M5/114.15,2, eBioscience), PerCP-Cy5.5-anti-GR-1 (clone RB6-8C5, eBioscience), PE-anti-F4/80 (clone BM8, eBioscience), FITC-anti-CD80 (clone 16-10A1, eBioscience), PE-Cy7-anti-CD86 (clone GL1, eBioscience), APC-anti-CD206 (clone MR6F3, eBioscience), FITC-anti-CD282 (TLR2) (clone 6C2, eBioscience), PE-anti-CD284 (TLR4) (clone HTA125, eBioscience), PE-Cy7-anti-CD369 (Dectin-1) (clone Bg1fpj, eBioscience), and APC-anti-CD36 (clone HM36, eBioscience).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Staining, Immunofluorescence, Two Tailed Test

Journal: Arthritis Research & Therapy

Article Title: Role of TLR4 activation and signaling in bone remodeling, and afferent sprouting in serum transfer arthritis

doi: 10.1186/s13075-024-03424-4

Figure Lengend Snippet: TLR4 expressed by microglial cells modulates mechanical allodynia. Male and female Tlr4 Avil , Tlr4 Gfap , Tlr4 Cx3cr1 Tlr4 Tmem119 , Tlr4 f/f mice ( n = 8/group) were injected on days 0 and 2 with K/BxN sera and serially scored for arthritis ( A , B , E , F ) and assessed for mechanical withdrawal ( C , D , G , H ). ( A , B , E , F ) All mice developed visually detectable paw swelling . (B) Overall, there were differences over the time course in males (F (4, 35) = 5.911, p < 0.001 one-way ANOVA), notably for Tlr4 Tmem119 (* p = 0.039) and Tlr4 Cx3cr1 ( # p = 0.0009) compared to Tlr4 f/f controls with Dunnett’s test. ( F ) Similarly for female mice (F(4,35) = 5.91, p = 0003) there were significant differences in Tlr4 Tmem119 (* p = 0.0028) and Tlr4 Cx3cr1 ( # p = 0.0027). ( C ) The withdrawal thresholds for Tlr4 Tmem and Tlr4 Cx3cr1 male mice partially returned to baseline with significant differences at the end of the time course (F (4, 490) = 14.94, p < 0.0001). ( D ) The hyperalgesic indexes were significantly reduced overall (one-way ANOVA, p = 0.0058) for Tlr4 Tmem119 (* p = 0.03) and Tlr4 Cx3cr1 ( # p = 0.02) male mice compared to Tlr4 . f/f controls with Dunnett’s test. ( G , H ) There were no differences in the withdrawal thresholds and the hyperalgesic indexes in the female mice (F(4, 35) = 0.65; p = 0.63, one-way ANOVA)

Article Snippet: Following isolation, cells were fixed and blocked with 2% normal mouse serum containing an anti–CD16/CD32 antibody (BD Bioscience; FcγR blocker) for 30 min on ice, followed by staining with an antibody mix of 1:100 PerCP-Cy5.5–conjugated CD11b antibody (BioLegend; RRID:AB_893232), 1:100 PECy7-conjugated rabbit anti-mouse TMEM119 antibody, 1:100 PE-conjugated anti-TLR4 antibody (MTS510 clone Thermo Fisher Scientific; RRID:AB_2562503), 1:100 APC-conjugated anti-TLR4 antibody (SA15-21 clone; BioLegend; RRID:AB_466263), 1:200 dilution of CTxB-FITC (Thermo Fisher Scientific), and 1:1000 Ghost dye-Red 780 fixable viability dye (Cell Signaling) for 45 min on ice.

Techniques: Injection

Journal: Arthritis Research & Therapy

Article Title: Role of TLR4 activation and signaling in bone remodeling, and afferent sprouting in serum transfer arthritis

doi: 10.1186/s13075-024-03424-4

Figure Lengend Snippet: Microglia from arthritic mice show increased TLR4 dimers and lipid rafts. Single cell suspensions from spinal cords were generated from naïve and WT K/BxN male mice injected with serum after 28 days and stained for microglia markers, TLR4 and lipid rafts. ( A ) Gating strategy and representative plots of microglia (CD11b + /TMEM119 +) from K/BXN serum injected and naïve mice showing cell population intensity (mean fluorescence intensity), for TLR4 monomers (MTS510 clone), total TLR4 (SA15-21 clone) and lipid rafts (CTxB binding). Quantification of microglia populations ( B ), TLR4 dimers ( C ) and change in lipid rafts ( D ) are shown ( n = 5–9 per group). There were no significant changes in the numbers of CD11b + /TMEM119 + cells at 28 days; however, the percentage of TLR4 dimers ( p < 0.015) and increase in the lipid rafts were significant ( p = 0.033, Students t test)

Article Snippet: Following isolation, cells were fixed and blocked with 2% normal mouse serum containing an anti–CD16/CD32 antibody (BD Bioscience; FcγR blocker) for 30 min on ice, followed by staining with an antibody mix of 1:100 PerCP-Cy5.5–conjugated CD11b antibody (BioLegend; RRID:AB_893232), 1:100 PECy7-conjugated rabbit anti-mouse TMEM119 antibody, 1:100 PE-conjugated anti-TLR4 antibody (MTS510 clone Thermo Fisher Scientific; RRID:AB_2562503), 1:100 APC-conjugated anti-TLR4 antibody (SA15-21 clone; BioLegend; RRID:AB_466263), 1:200 dilution of CTxB-FITC (Thermo Fisher Scientific), and 1:1000 Ghost dye-Red 780 fixable viability dye (Cell Signaling) for 45 min on ice.

Techniques: Generated, Injection, Staining, Fluorescence, Binding Assay

Journal: Current Issues in Molecular Biology

Article Title: TLR4 Downregulation Identifies High-Risk HPV Infection and Integration in H-SIL and Squamous Cell Carcinomas of the Uterine Cervix

doi: 10.3390/cimb46100670

Figure Lengend Snippet: TLR4 expression intensity and percentage. The analysis of TLR4 SI in NILM, H-SIL, and SCC samples showed a statistically significant difference between the 3 groups.

Article Snippet: Primary mouse monoclonal anti-TLR4 antibody has been used according to manufacturer instruction (clone 76B357.1, Novus Biologicals, Toronto, ON, Canada).

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: TLR4 Downregulation Identifies High-Risk HPV Infection and Integration in H-SIL and Squamous Cell Carcinomas of the Uterine Cervix

doi: 10.3390/cimb46100670

Figure Lengend Snippet: Differences in TLR4 expression in NILM, H-SIL, and CSCC.

Article Snippet: Primary mouse monoclonal anti-TLR4 antibody has been used according to manufacturer instruction (clone 76B357.1, Novus Biologicals, Toronto, ON, Canada).

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: TLR4 Downregulation Identifies High-Risk HPV Infection and Integration in H-SIL and Squamous Cell Carcinomas of the Uterine Cervix

doi: 10.3390/cimb46100670

Figure Lengend Snippet: ( A ) NILM showing negative immunostaining for TLR4. ( B ) H-SIL showing diffuse and intense immunostaining for TLR4.

Article Snippet: Primary mouse monoclonal anti-TLR4 antibody has been used according to manufacturer instruction (clone 76B357.1, Novus Biologicals, Toronto, ON, Canada).

Techniques: Immunostaining

Journal: Current Issues in Molecular Biology

Article Title: TLR4 Downregulation Identifies High-Risk HPV Infection and Integration in H-SIL and Squamous Cell Carcinomas of the Uterine Cervix

doi: 10.3390/cimb46100670

Figure Lengend Snippet: TLR4 staining patterns. ( A , B ) Double immunostaining for p16 (DAB–brown) and TLR-4 (Fast Red) in SCC. With double immunostaining, p16 staining was distributed at the periphery of the tumoral nests while TLR4 was more evident centrally, in more differentiated tumoral cells. ( C ) A case of H-SIL showing low TLR-4 expression (downregulation) is depicted. ( D ) A case of SCC showing moderate TLR4 expression.

Article Snippet: Primary mouse monoclonal anti-TLR4 antibody has been used according to manufacturer instruction (clone 76B357.1, Novus Biologicals, Toronto, ON, Canada).

Techniques: Staining, Double Immunostaining, Expressing

Journal: Current Issues in Molecular Biology

Article Title: TLR4 Downregulation Identifies High-Risk HPV Infection and Integration in H-SIL and Squamous Cell Carcinomas of the Uterine Cervix

doi: 10.3390/cimb46100670

Figure Lengend Snippet: Correlation expression of TLR4 and CK19. Inverse statistically significant correlation between TLR4-SI and CK19 percentage of expressions (R = −0.4906; p = 0.0001, ANOVA), with CK19 upregulation and TLR4 downregulation in the cervical carcinogenetic process.

Article Snippet: Primary mouse monoclonal anti-TLR4 antibody has been used according to manufacturer instruction (clone 76B357.1, Novus Biologicals, Toronto, ON, Canada).

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: TLR4 Downregulation Identifies High-Risk HPV Infection and Integration in H-SIL and Squamous Cell Carcinomas of the Uterine Cervix

doi: 10.3390/cimb46100670

Figure Lengend Snippet: CK19 upregulation and TLR4 downregulation in the cervical carcinogenetic process. ( A ) Immunohistochemical expression of CK19 SCC; ( B ) immunohistochemical expression of TLR4 in the same case of SCC.

Article Snippet: Primary mouse monoclonal anti-TLR4 antibody has been used according to manufacturer instruction (clone 76B357.1, Novus Biologicals, Toronto, ON, Canada).

Techniques: Immunohistochemical staining, Expressing

Journal: Current Issues in Molecular Biology

Article Title: TLR4 Downregulation Identifies High-Risk HPV Infection and Integration in H-SIL and Squamous Cell Carcinomas of the Uterine Cervix

doi: 10.3390/cimb46100670

Figure Lengend Snippet: Correlation between intensity of TLR-4 expression and the degree of inflammatory infiltration. A statistically significant correlation between TLR4 downregulation and inflammatory tumoral microenvironment has been demonstrated (R = 0.5341; p = 0.001, ANOVA).

Article Snippet: Primary mouse monoclonal anti-TLR4 antibody has been used according to manufacturer instruction (clone 76B357.1, Novus Biologicals, Toronto, ON, Canada).

Techniques: Expressing

Journal: Parasite immunology

Article Title: Brugia malayi infective larvae fail to activate Langerhans cells and dermal dendritic cells in human skin

doi: 10.1111/pim.12169

Figure Lengend Snippet: Real-time PCR: Assay identification numbers for genes tested

Article Snippet: Antibodies for characterizing LC consisted of the following: FITC-conjugated mouse anti-human E-cadherin (Biolegend, San Diego, CA, USA), APC-conjugated mouse anti-human CD207 (Langerin) (Biolegend), PE-Cy7- or APC-eFluor 780-conjugated mouse anti-human CD14 (eBioscience, San Diego, CA, USA), anti-TLR4-PE (eBioscience) and Pacific Blue-conjugated rat anti-human cutaneous lymphocyte antigen (CLA) (Biolegend).

Techniques: Real-time Polymerase Chain Reaction