Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Fold changes in the microarray for all 35 genes were found to be differentially regulated by C. pneumoniae infection and satisfied the comparison filtering criteria

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Microarray, Infection, Comparison, Binding Assay, Coagulation

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: C. pneumoniae stimulates TLR2 expression in rVSMCs. (A) Validation of TLR2 gene expression profiling using quantitative real-time RT-PCR normalized to GAPDH expression. Data shown are mean values of PCR replicates from individual groups. (B) TLR2 mRNA expression at the indicated time points after C. pneumoniae infection. rVSMCs infected with C. pneumoniae (5 × 105 IFU) for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h or control cells were lysed to prepare the total RNA. cDNA was amplified for 30 cycles, and PCR products were separated by agarose gel electrophoresis. Relative TLR2 mRNA expression levels determined by standard PCR (n = 3 replicates per group). (C) C. pneumoniae infection stimulates TLR2 protein expression in rVSMCs. Cells were infected with C. pneumoniae for 0 h, 12 h, or 24 h. Cell lysates were separated by SDS-PAGE, and blots were probed with anti-TLR2 and anti-β-actin antibodies, followed by donkey anti-goat IgG-HRP and goat anti-mouse IgG-HRP antibodies, and developed with enhanced chemiluminescence (ECL).

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Infection, Control, Amplification, Agarose Gel Electrophoresis, SDS Page

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Confocal microscopy analysis of the TLR2 expression pattern in C. pneumoniae-infected rVSMCs. rVSMCs were grown in coverslips and infected with C. pneumoniae for 60 h and then were fixed, permeabilized, and stained using specific antibodies. C. pneumoniae inclusions were observed using a mouse polyclonal antibody to C. pneumoniae. Fluorescence micrographs were stained with TLR2-specific antibody. Cell nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole).

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Confocal Microscopy, Expressing, Infection, Staining, Fluorescence

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Effects of TLR2 on rVSMC migration induced by C. pneumoniae infection. The TLR2-neutralizing antibody was added 1 h before C. pneumoniae infection. (A) Wound healing assay. “Scratch wounds” were created by scraping the confluent cell monolayer with a sterile pipette tip, and then cells were infected with C. pneumoniae at an infectious dose of 5 × 105 IFU. Photographs were taken of the same wounded area of each well at 0 h and 24 h. The scratched regions were photographed under an inverted Nikon microscope (×100 magnification) at 24 h after C. pneumoniae infection. Migration velocity is presented as a ratio of the cellular recoverage area to the whole wound area. *, P < 0.05 versus control; **, P < 0.05 versus C. pneumoniae infection group. (B) Transwell migration assay. Cell morphology was observed by staining with 0.1% crystallin violet. The number of cells that had migrated through the pores was quantified by counting nine independent visual fields using a microscope (×200 magnification). *, P < 0.05 versus control; **, P < 0.05 versus the C. pneumoniae infection group.

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Migration, Infection, Wound Healing Assay, Sterility, Transferring, Microscopy, Control, Transwell Migration Assay, Staining

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: The TLR2-neutralizing antibody suppresses Akt phosphorylation induced by C. pneumoniae infection. rVSMCs cultured for 24 h in 6-well plates were incubated with the TLR2-neutralizing antibody (10 μg/ml) and then infected with C. pneumoniae. Equal amounts of protein lysates were subjected to SDS-PAGE, and blots were probed with anti-Akt, anti-phospho-Akt (Ser 473), and anti-β-actin antibodies, followed by the corresponding horseradish peroxidase-conjugated secondary antibodies, and developed with ECL. P-Akt indicates phosphorylated Akt. *, P < 0.05 versus control; **, P < 0.05 versus the C. pneumoniae infection group.

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Phospho-proteomics, Infection, Cell Culture, Incubation, SDS Page, Control

Journal: Cells

Article Title: Role of TLR4 Receptor Complex in the Regulation of the Innate Immune Response by Fibronectin

doi: 10.3390/cells9010216

Figure Lengend Snippet: TLR4 mediates IL-8 expression in response to FnIII-1c and LPS in dermal fibroblasts. Monolayers of human dermal fibroblasts in 10% FBS/DMEM were treated for 24 h with ( A ) FnIII-1c or FnIII-13 (1-20 µg/mL), ( B ) LPS (1-100 ng/mL), ( C ) LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of the designated amounts of blocking antibody to TLR4 or TLR2. IgG served as control. ( D ) TNF-α (25 ng/mL), LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of increasing amounts of the TLR4 inhibitor, TAK-242. The wells without antibodies ( C ) or inhibitors ( D ) were set as 100%. IL-8 concentration in conditioned medium was determined by ELISA. The data represent the mean ± S.E. of triplicate assays from three separate experiments.

Article Snippet: Recombinant human CD14, human TNF-α, human IL-1α, anti-human MD-2 antibody, and neutralizing antibodies: anti-human CD14, anti-human TLR2 and anti-human TLR4 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Blocking Assay, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 2. Real-time RT-PCR analysis of TLR2 and TLR4 mRNA expression in colon extracts in TNBS-induced colitis and their down-regulation after VIP treatment. Colitis was induced by rectal instillation of TNBS in 50% ethanol, and 1 nmol VIP was injected i.p. on alternate days. Controls received PBS or 50% ethanol. mRNA was extracted from colons at days 1, 3, 5, and 7. TLR2 (A) and TLR4 (B) mRNA expressions were measured by real-time RT-PCR, values were rectified by mRNA expression of -actin for each sample, and arbitrary units were calculated with respect to control expression, being 1 (2– Ct). Each result is the mean

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Quantitative RT-PCR, Expressing, Injection, Control

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 3. Western blot analysis of TLR2 and TLR4 in colon extracts in TNBS-induced colitis down-regulation after VIP treatment. Colon extracts from control and ethanol-, TNBS-, and TNBS/VIP-treated animals were prepared and electrophoresed as described in Materials and Methods. Specific bands for TLR2 (A) or TLR4 (B) were quantified by densitometry and rectified with respect to -actin protein levels in each case. Each result is the mean SEM of at least four mice. , P 0.05; , P 0.01; , P 0.001, versus control animals, and *, P 0.05; **, P 0.01; ***, P 0.001, versus TNBS-treated animals. A representative experiment of three others is shown in the upper panels. O.D. optical density.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Western Blot, Control

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 4. Immunohistochemical analysis of TLR2 and -4 at day 5 in gut sections. Immunohistochemical study was performed to study TLR2 and TLR4 expressions in colon mucosa as described in Materials and Methods. Mono- nuclear cells positive to antibody against TLR2 within the lamina propria in control (untreated animals; A), TNBS (B), and TNBS VIP-treated animals (C; original 400). Gut crypts showing positive staining for TLR2 and TLR4 in control (untreated animals; D and G), TNBS (E and H), and TNBS VIP-treated animals (F and I). Original magnification, 200. Arrows indicate the positive staining. VIP reduced the TNBS-induced expression of TLR2 and TLR4 in epithelial cells from intestinal crypts and mononuclear cells.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Immunohistochemical staining, Control, Staining, Expressing

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 5. Flow cytometry analysis of TLR2 expres- sion in TNBS-induced colitis. Effect of VIP treat- ment. Colitis was induced as described in Materials and Methods, and mice were treated i.p. with 1 nmol VIP on alternate days over 7 days. Cell sus- pensions from mesenteric lymph node extracted from control and treated animals were collected at different time-points and processed for immunoflu- orescence staining by using a PE-conjugated TLR2 mAb. (A) Aliquots of control lymph node cells were incubated with isotype control antibodies or anti- bodies to TLR2 (IgG2b), as described. (B) Total cells subjected to single immunofluorescence stain- ing were analyzed by FACSCalibur. (C and D) Double immunofluorescence staining for TLR2 and a subpopulation marker (C, FITC-conjugated CD11c; D, FITC-conjugated CD11b). (E) Lympho- cytes were identified by a FSC versus SSC gate and subjected to TLR2 immunofluorescence staining (F). Each result is the mean SEM of two separated experiments (three mice/group/experiment). *, P 0.05; **, P 0.01; ***, P 0.001 (ANOVA), with respect to TNBS-treated mice. , P 0.01; , P 0.001 (ANOVA), with respect to con- trol animals.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Flow Cytometry, Control, Staining, Incubation, Marker

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 7. Representative example at day 5 of the TLR2 and -4 expressions in CD11c and CD11b cells and lymphocytes after TNBS-induced colitis and VIP effect. Flow cytometry analysis of CD11c and CD11b cells and lymphocytes from mesenteric lymph nodes of control and treated animals. Cell suspensions were processed for double immunofluorescence staining for TLR2 and TLR4 and a subpopulation marker (CD11c and CD11b). Lymphocytes were identified by a FSC versus SSC gate and subjected to TLR2 and TLR4 immunofluorescence staining. Cell suspensions were incubated with isotype control antibodies or antibodies to TLR2 (IgG2b) or TLR4 (IgG2a), as described. The values shown in right quadrants and histograms indicate the percentage of CD11c/CD11b or lymphocytes and TLR2/4-positive cells. The results presented are representative of two independent experiments using three mice per group.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Flow Cytometry, Control, Staining, Marker, Incubation

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 9. Hypothesis planned to explain the two possible VIP modes of action. Based on our observations, we hypothesized that TLR2 and -4 modulations, induced after VIP treatment, may be explained through two possible ways that are not mutually exclusive. The first is an indirect through the reduction of TLR2 and -4, caused by the VIP-mediated decrease of inflammatory mediators such as IL-1 and IFN-, which synergize with bacterial products contributing to the amplification of TLR (blue arrows). The second possible mechanism, which remains to be elucidated in this model, is a primary (black arrows) through the VIP-mediated decrease of NF- B, which would cause a down-regulation of TLR expression. IRAK, IL-1 receptor-activated kinase; TRAF6, TNF receptor-associated factor 6; IKK, I B kinase.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Expressing

Journal: European journal of immunology

Article Title: TLR2 on CD4+ and CD8+ T cells promotes control of Mycobacterium tuberculosis infection.

doi: 10.1002/eji.202350715

Figure Lengend Snippet: Figure 1. Deletion of exon 3 of Tlr2 in CD4+ and CD8+ T cells in Tlr2fl/flxCd4cre/cre mice. (A) Location of LoxP sites around exon 3 of Tlr2 in Tlr2fl/fl

Article Snippet: Blots were either incubated with polyclonal goat anti-mouse TLR2 Ab (R&D Systems; AF1530) or β-actin (R&D Systems; MAB8929), followed by HRP-conjugated donkey anti-goat IgG(H+L) mAb (Jackson) and bands detected by chemiluminescence using Pierce ECL Plus substrate (Fisher).

Techniques:

Journal: PLoS ONE

Article Title: TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

doi: 10.1371/journal.pone.0167237

Figure Lengend Snippet: P . acnes surface proteins were extracted from a 5-day culture bacterial pellet and separated by electrophoresis in a 4–12% NuPAGE LDS BisTris gel (50 μg) with detection by Coomassie blue staining (A). Separated proteins were transferred onto nitrocellulose membranes, which were incubated with recombinant TLR2 (B) and TLR4 (C) (0.1 μg/ml). TLR binding activity was detected with specific biotinylated antibodies against TLR2 and TLR4, respectively, as described in the Materials and Methods. Lane 1 contains the molecular mass markers. Lanes 2 to 7 contain proteins from strains 6919, RON, CHR, GUE, TRI, and PIE, respectively. Arrows indicate the positions of the 24.5- and 27.5-kDa bands of interest.

Article Snippet: Unbound antibodies were removed by washing as described above, and the membrane was incubated with biotinylated human antibodies against TLR2 or TLR4 (R&D Systems, Abingdon, UK) diluted to 0.1 μg/ml in TBS/T supplemented with 5% BSA, for 20 h at 4°C, with gentle mixing.

Techniques: Electrophoresis, Staining, Incubation, Recombinant, Binding Assay, Activity Assay

Journal: PLoS ONE

Article Title: TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

doi: 10.1371/journal.pone.0167237

Figure Lengend Snippet: Surface protein extracts were electrophoretically separated in 10% SDS-PAGE gels (13 x 13 cm), with detection by Coomassie blue staining. Proteins of interest were excised from the gel, eluted and pooled as described in the Materials and Methods. HaCaT cells were treated for 2 h with human anti-TLR2 (dashed bar) or with goat anti-IgGa antibodies (dark gray bar) and transfected by incubation for 24 h with NF-κB transcription factor (A) and CXCL8 (-173 bp) (B) inserted into a construct upstream from the luciferase gene, after which, an internal control (the Renilla luciferase expression plasmid) was added to the transfection mixture. Cell were stimulated with eluted CAMP factor 1 (50 μg/ml) for 24 h at 37°C. Relative NF-κB and CXCL8 promoter activities were determined as the ratio of firefly and Renilla luciferase activities. Pretreated HaCaT cells were stimulated by incubation with eluted CAMP factor 1 (50 μg/ml) for 24 h at 37°C, CXCL8 mRNA levels were determined by RT-qPCR (C), and CXCL8 production was measured by ELISA (D). Control experiments were performed with cells alone (dark bar) and with stimulated cells not previously treated with CAMP factor 1 (light gray bar). Data are presented as the mean ± standard deviation of two independent experiments. Statistical significance is indicated by * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Unbound antibodies were removed by washing as described above, and the membrane was incubated with biotinylated human antibodies against TLR2 or TLR4 (R&D Systems, Abingdon, UK) diluted to 0.1 μg/ml in TBS/T supplemented with 5% BSA, for 20 h at 4°C, with gentle mixing.

Techniques: SDS Page, Staining, Transfection, Incubation, Construct, Luciferase, Control, Expressing, Plasmid Preparation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLoS ONE

Article Title: TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

doi: 10.1371/journal.pone.0167237

Figure Lengend Snippet: Comparison of P . acnes inflammatory capabilities with phylogroup and CAMP factor 1 gene nucleotide sequence

Article Snippet: Unbound antibodies were removed by washing as described above, and the membrane was incubated with biotinylated human antibodies against TLR2 or TLR4 (R&D Systems, Abingdon, UK) diluted to 0.1 μg/ml in TBS/T supplemented with 5% BSA, for 20 h at 4°C, with gentle mixing.

Techniques: Comparison, Binding Assay, Sequencing

Journal: PLoS ONE

Article Title: TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

doi: 10.1371/journal.pone.0167237

Figure Lengend Snippet: Phylogenetic analysis illustrating the relationship between CAMP factor 1 and 19 protein reference sequences from P . acnes (GenBank accession numbers ALT42318.1, ADE00273.1, AEE72567.1, AEW83915.1, AEW81649.1, AFU41158.1, AGJ79688.1, AID36048.1, ALT40080.1, ALT44585.1, AAS92206.1, ALT35489.1, AAT83098.1, AEH29671.1, ALT33229.1, AER06043.1, AAX14380.1, ALU23622.1, ALD69860.1), CAMP 5 factor Protein (AAV84920.1) and sequences of the 27 P . acnes strains used in this study. Phylogenetic trees were constructed by the maximum likelihood method, using PhyML3.0 , and the tree was rooted on the CAMP factor 5 protein sequence. Bootstrapping was applied to the trees (500 datasets) and bootstrap values are shown at nodes. The bar indicates the number of substitutions per site. Sequences were classified into four groups (A, B1 to B3). Squares correspond to phylotype IA 1 (red), IA 2 (yellow), IB (green), II (purple); and to CAMP1-TLR2 binding intensity with no binding as—(light gray), weak binding as +/- and + (dark gray), strong binding as ++ and +++ (black).

Article Snippet: Unbound antibodies were removed by washing as described above, and the membrane was incubated with biotinylated human antibodies against TLR2 or TLR4 (R&D Systems, Abingdon, UK) diluted to 0.1 μg/ml in TBS/T supplemented with 5% BSA, for 20 h at 4°C, with gentle mixing.

Techniques: Construct, Sequencing, Binding Assay

Journal: Journal of Investigative Dermatology

Article Title: Differential Expression and Function of Toll-like Receptors in Langerhans Cells: Comparison with Splenic Dendritic Cells

doi: 10.1046/j.0022-202x.2003.22116.x

Figure Lengend Snippet: Figure 1 Murine Langerhans cells express TLR2, TLR4, TLR9, and PR105 but not TLR7. (a) mRNA samples from Langerhans cells, splenic DC, and keratinocytes were reverse transcribed. The resultant cDNA was amplified using the primers described in Materials and Methods. The size of products is as follows: TLR2, 922 bp; TLR4, 556 bp; TLR7, 656 bp; TLR9, 542 bp; CD14, 1327 bp; RP105, 787 bp; G3PDH, 640 bp. NC denotes negative control. (b) Protein samples from Langerhans cells, splenic DC, and 2D6 were immunolabeled with anti-TLR2 antibody. The apparent size of TLR2 is 92 kDa. (c) Langerhans cells and splenic DC were analyzed for expression of TLR4, RP105, and CD14 (bold lines) by flow cytometry. Thin lines indicate isotype control staining. The results shown in the figure are representative of three independent experiments.

Article Snippet: The proteins in the gels were electrotransferred on to PVDF membranes (Millipore, Bedford, Massachusetts) and probed with 1:200 diluted goat anti-mouse TLR2 polyclonal IgG antibody (Santa Cruz, California).

Techniques: Reverse Transcription, Negative Control, Immunolabeling, Expressing, Cytometry, Control, Staining

Journal: eLife

Article Title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

doi: 10.7554/elife.78044

Figure Lengend Snippet: Figure 1. Construction of fibroblast cell line expressing Toll-like receptor 2-4 (TLR2-4). (A) Structure of fusion protein TLR2-4. Total RNA was extracted from goat peripheral blood and was reverse- transcribed into cDNA. TLR2 extracellular domain and TLR4 transmembrane and intracellular domain were amplified from cDNA. (B) Schematic representation of the small guide RNA2 (sgRNA2) and its target genome DNA. The sgRNA target 2 was located on the first intron of SETD5 and the PAM site is colored blue. (C) The expression vector of sgRNA2-CRISPR/Cas9 was constructed by joining the crRNA-oligo into PX458 vector containing GFP. TLR2-4 was inserted into pRosa26-promoter vector, and the expression construction of CMV-tdTomato was cloned into reconstructed pRosa26-TLR2-4 vector. Both PX458-sgRNA and pRosa26-TLR2-4-tdTomato were co-transfected into goat fibroblasts.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Capra hircus) TLR2 GenBank NC_030824.1 Gene (Capra hircus) TLR4 GenBank NC_030815.1 Strain, strain background (Staphylococcus aureus) ATCC29213 GenBank U77328 Bacteria cells Transfected construct PX458 (pSpCas9(BB)–2A- GFP) Addgene RRID: Addgene_48138 PMID:24157548 Transfected construct pRosa26- promoter Addgene RRID: Addgene_21710 PMID:9108056 Transfected construct (Capra hircus) siRNA to endogenous TLR2 GenePharma Sense: 5’- GCAC UUCA ACCC UCCC UUUTT- 3’ Antisense: 5’- AAAG GGAG GGUU GAAG UGCTT- 3’ Sequence- based reagent siRNA: nontargetin control GenePharma Silencer Select Antibody nti- MyC (Mouse monoclonal) Proteintech Cat# 60003–2- lg RRID: AB_2883088 IF (1:100), WB (1:1000) Wang, Qi et al. eLife 2022;11:e78044.

Techniques: Expressing, Reverse Transcription, Amplification, Plasmid Preparation, CRISPR, Construct, Clone Assay, Transfection

Journal: eLife

Article Title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

doi: 10.7554/elife.78044

Figure Lengend Snippet: Figure 2. Generation of clone goat expressing Toll-like receptor 2-4 (TLR2-4). (A) The clone goat expressing TLR2-4 was prepared by somatic cell nuclear transfer. (B) Schematic representation of the site-specific integration of primers S-M and T-R. (C) Genomic DNA of macrophages from transgenic and wild-type (WT) goat was extracted. The PCR products were amplified using primers S-M and T-R. The sequences of primers are listed in Supplementary file 2D. (D) Schematic representation of the primers U-L1 and U-L. The sequences of primers are listed in Supplementary file 2D. (E) Genomic DNA of macrophages from transgenic and WT goat was extracted. The products of PCR were amplified using primers U-L1 and U-L. (F) The mRNA expression of TLR2-4 in macrophages was detected by RT-PCR. 331 bp length fragments were amplified by Primer P1, and 188 bp

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Capra hircus) TLR2 GenBank NC_030824.1 Gene (Capra hircus) TLR4 GenBank NC_030815.1 Strain, strain background (Staphylococcus aureus) ATCC29213 GenBank U77328 Bacteria cells Transfected construct PX458 (pSpCas9(BB)–2A- GFP) Addgene RRID: Addgene_48138 PMID:24157548 Transfected construct pRosa26- promoter Addgene RRID: Addgene_21710 PMID:9108056 Transfected construct (Capra hircus) siRNA to endogenous TLR2 GenePharma Sense: 5’- GCAC UUCA ACCC UCCC UUUTT- 3’ Antisense: 5’- AAAG GGAG GGUU GAAG UGCTT- 3’ Sequence- based reagent siRNA: nontargetin control GenePharma Silencer Select Antibody nti- MyC (Mouse monoclonal) Proteintech Cat# 60003–2- lg RRID: AB_2883088 IF (1:100), WB (1:1000) Wang, Qi et al. eLife 2022;11:e78044.

Techniques: Expressing, Transgenic Assay, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

doi: 10.7554/elife.78044

Figure Lengend Snippet: Figure 3. Toll-like receptor 2-4 (TLR2-4) promoted autophagy-dependent elimination of Staphylococcus aureus in macrophages. (A) S. aureus burden in macrophages was estimated by the mean fluorescence intensity (MFI) of macrophages infected with fluorescein isothiocyanate (FITC)-labeled S. aureus using flow cytometry (multiplicity of infection [MOI] = 10). MFI of FITC in macrophages was calculated by FlowJo software. (B) S. aureus infection index in macrophages was compared according to the results of MFI (n=3 biologically independent samples). (C) The clearance of S. aureus in macrophages was assessed by colony-forming unit (CFU) method. Macrophages were treated with S. aureus (MOI = 10) for 1 hr. After 4, 8, 12, 24 hr, survived S.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Capra hircus) TLR2 GenBank NC_030824.1 Gene (Capra hircus) TLR4 GenBank NC_030815.1 Strain, strain background (Staphylococcus aureus) ATCC29213 GenBank U77328 Bacteria cells Transfected construct PX458 (pSpCas9(BB)–2A- GFP) Addgene RRID: Addgene_48138 PMID:24157548 Transfected construct pRosa26- promoter Addgene RRID: Addgene_21710 PMID:9108056 Transfected construct (Capra hircus) siRNA to endogenous TLR2 GenePharma Sense: 5’- GCAC UUCA ACCC UCCC UUUTT- 3’ Antisense: 5’- AAAG GGAG GGUU GAAG UGCTT- 3’ Sequence- based reagent siRNA: nontargetin control GenePharma Silencer Select Antibody nti- MyC (Mouse monoclonal) Proteintech Cat# 60003–2- lg RRID: AB_2883088 IF (1:100), WB (1:1000) Wang, Qi et al. eLife 2022;11:e78044.

Techniques: Fluorescence, Infection, Labeling, Flow Cytometry, Software

Journal: eLife

Article Title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

doi: 10.7554/elife.78044

Figure Lengend Snippet: Figure 4. Identification of autophagy-related genes involved in clearance of Staphylococcus aureus in transgenic macrophages. Macrophages from the Toll-like receptor 2-4 (TLR2-4) and two wild-type goats (WT1, WT2) were infected with S. aureus for 1 hr (multiplicity of infection [MOI] = 10), and then after washing three times with PBS, were cultured in DMEM containing gentamicin (200 μg/ml) for 1 hr. Subsequently, total RNA of macrophages was extracted to perform RNA-seq when macrophages were cultured with DMEM containing gentamicin (25 μg/ml) for 8 hr. (A) Spearsman’s correlation coefficient were calculated between the two wild-type macrophages on the basis of total transcripts. (B) Gene Ontology (GO) enrichment terms of

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Capra hircus) TLR2 GenBank NC_030824.1 Gene (Capra hircus) TLR4 GenBank NC_030815.1 Strain, strain background (Staphylococcus aureus) ATCC29213 GenBank U77328 Bacteria cells Transfected construct PX458 (pSpCas9(BB)–2A- GFP) Addgene RRID: Addgene_48138 PMID:24157548 Transfected construct pRosa26- promoter Addgene RRID: Addgene_21710 PMID:9108056 Transfected construct (Capra hircus) siRNA to endogenous TLR2 GenePharma Sense: 5’- GCAC UUCA ACCC UCCC UUUTT- 3’ Antisense: 5’- AAAG GGAG GGUU GAAG UGCTT- 3’ Sequence- based reagent siRNA: nontargetin control GenePharma Silencer Select Antibody nti- MyC (Mouse monoclonal) Proteintech Cat# 60003–2- lg RRID: AB_2883088 IF (1:100), WB (1:1000) Wang, Qi et al. eLife 2022;11:e78044.

Techniques: Transgenic Assay, Infection, Cell Culture, RNA Sequencing

Journal: eLife

Article Title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

doi: 10.7554/elife.78044

Figure Lengend Snippet: Figure 5. Toll-like receptor (TLR2-4) enhanced Staphylococcus aureus-induced autophagy via activating JNK/ERK signaling. (A) The change in phosphorylation of JNK, ERK, and p38 in S. aureus-infected macrophages. Macrophages were pretreated with DMSO (carrier), Takinib (200 μM), or Amlexanox (100 μM) for 12 hr, then stimulated with S. aureus for 4 hr. Phosphorylation of JNK, ERK, and p38 was observed by Western blot. (B) The level of LC3-II was detected in macrophages after S. aureus challenge. Macrophages were pretreated with DMSO, Dynasore (50 μM), SP600125 (200 μM), PD98059 (200 μM), SB203580 (200 μM), or Amlexanox for 12 hr, respectively, then treated with S. aureus (multiplicity of infection [MOI] = 10) for 4 hr.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Capra hircus) TLR2 GenBank NC_030824.1 Gene (Capra hircus) TLR4 GenBank NC_030815.1 Strain, strain background (Staphylococcus aureus) ATCC29213 GenBank U77328 Bacteria cells Transfected construct PX458 (pSpCas9(BB)–2A- GFP) Addgene RRID: Addgene_48138 PMID:24157548 Transfected construct pRosa26- promoter Addgene RRID: Addgene_21710 PMID:9108056 Transfected construct (Capra hircus) siRNA to endogenous TLR2 GenePharma Sense: 5’- GCAC UUCA ACCC UCCC UUUTT- 3’ Antisense: 5’- AAAG GGAG GGUU GAAG UGCTT- 3’ Sequence- based reagent siRNA: nontargetin control GenePharma Silencer Select Antibody nti- MyC (Mouse monoclonal) Proteintech Cat# 60003–2- lg RRID: AB_2883088 IF (1:100), WB (1:1000) Wang, Qi et al. eLife 2022;11:e78044.

Techniques: Phospho-proteomics, Infection, Western Blot

Journal: eLife

Article Title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

doi: 10.7554/elife.78044

Figure Lengend Snippet: Figure 6. Toll-like receptor 2-4 (TLR2-4) induced autophagy flux through the cyclic adenosine phosphate (cAMP) pathway in Staphylococcus aureus- infected macrophages. (A) TLR2-4 promoted the expression of ATG5 and ATG12 in macrophages after S. aureus infection. The mRNA relative expression of ATG5 and ATG12 was monitored by real-time reverse transcription PCR (qRT-PCR) when macrophages were stimulated with S. aureus (multiplicity of infection [MOI] = 10) for 4 hr (n=3 biologically independent samples). (B) Inhibiting JNK or ERK1/2 increased the mRNA level of ATG5 and

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Capra hircus) TLR2 GenBank NC_030824.1 Gene (Capra hircus) TLR4 GenBank NC_030815.1 Strain, strain background (Staphylococcus aureus) ATCC29213 GenBank U77328 Bacteria cells Transfected construct PX458 (pSpCas9(BB)–2A- GFP) Addgene RRID: Addgene_48138 PMID:24157548 Transfected construct pRosa26- promoter Addgene RRID: Addgene_21710 PMID:9108056 Transfected construct (Capra hircus) siRNA to endogenous TLR2 GenePharma Sense: 5’- GCAC UUCA ACCC UCCC UUUTT- 3’ Antisense: 5’- AAAG GGAG GGUU GAAG UGCTT- 3’ Sequence- based reagent siRNA: nontargetin control GenePharma Silencer Select Antibody nti- MyC (Mouse monoclonal) Proteintech Cat# 60003–2- lg RRID: AB_2883088 IF (1:100), WB (1:1000) Wang, Qi et al. eLife 2022;11:e78044.

Techniques: Infection, Expressing, Reverse Transcription, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: GroEL1, a heat shock protein 60 of Chlamydia pneumoniae, induces lectin-like oxidized low-density lipoprotein receptor 1 expression in endothelial cells and enhances atherogenesis in hypercholesterolemic rabbits.

doi: 10.4049/jimmunol.1003116

Figure Lengend Snippet: FIGURE 3. GroEL1-induced LOX-1 expression in HCAECs is medi- ated by TLR4. A, TLR4 and TLR2 protein expression was analyzed. B, HCAECs were pretreated with anti-hTLR4 Ab or transfected with TLR4 siRNA prior to GroEL1 treatment. LOX-1 mRNA expression was analyzed by real-time PCR. C, HCAECs were pretreated with anti-hTLR2 Ab or transfected with TLR2 siRNA prior to GroEL1 treatment. LOX-1 mRNA expression was analyzed by real-time PCR. Data represent the results of three independent experiments (mean 6 SEM). *p , 0.05.

Article Snippet: The membranes were probedwith mouse anti–LOX-1 (Santa Cruz Biotechnology), anti-SREC (Santa Cruz Biotechnology), anti– SR-B1 (Santa Cruz Biotechnology), anti-endothelial NO synthase ([eNOS], Millipore). anti–phospho-eNOS (Millopore), anti-Akt (Anaspec), anti–phospho-Akt (Merck, Darmstadt, Geremany), goat anti-TLR4 (R&D Systems), goat anti-TLR2 Ab (R&D Systems), rabbit anti-p38, rabbit anti–phosphop38, rabbit anti–stress-activated protein kinase (SAPK)/JNK, rabbit anti– phospho-SAPK/JNK, rabbit anti-p44/p42 MAPK, or mouse anti–phosphop44/p42 MAPK Ab (all MAPK Abs were purchased from Cell Signaling Technology).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction