tlr7 primary antibody monoclonal  (Bioss)

Journal: Biology

Article Title: Imiquimod (R837), a TLR7-Specific Agonist, Regulates Boar Sperm Motility via PI3K/GSK3α/β/Hexokinase Pathway

doi: 10.3390/biology14091182

Figure Lengend Snippet: The expression of TLR7 in boar sperm. ( A ) Localization and quantification of TLR7 (red) and DAPI (blue) in boar sperm. Sperm fluorescence scale bar = 50 μm. ( B ) Detection of TLR7 proteins in boar sperm by Western blotting.

Article Snippet: Sperm samples were placed on special glass slides and washed with PBS, air-dried, and permeabilized using TritonX-100/PBS (0.1% v / v ) for 1 h. Subsequently, the sperm sample was blocked with 10% goat serum ( v / v ) at room temperature for 30 min, followed by incubation with TLR7 primary antibody monoclonal (#bs 6601R, Bioss, 1:1000, Beijing, China) at 4 °C overnight.

Techniques: Expressing, Fluorescence, Western Blot

Journal: Biology

Article Title: Imiquimod (R837), a TLR7-Specific Agonist, Regulates Boar Sperm Motility via PI3K/GSK3α/β/Hexokinase Pathway

doi: 10.3390/biology14091182

Figure Lengend Snippet: Detection of TLR7 content in the upper layer and lower layer of boar sperm by FCM and WB. ( A , B ) The histogram of TLR7 expression was drawn based on flow cytometer detection. ( C ) The quantitative expression of TLR7 proteins divided by α-tubulin (internal control). ( D ) Gray analysis. Values are specified as mean ± standard error of the mean (SEM) of 4 replicates. Different lowercase letters indicate significant differences ( p < 0.05).

Article Snippet: Sperm samples were placed on special glass slides and washed with PBS, air-dried, and permeabilized using TritonX-100/PBS (0.1% v / v ) for 1 h. Subsequently, the sperm sample was blocked with 10% goat serum ( v / v ) at room temperature for 30 min, followed by incubation with TLR7 primary antibody monoclonal (#bs 6601R, Bioss, 1:1000, Beijing, China) at 4 °C overnight.

Techniques: Expressing, Flow Cytometry, Control

Journal: Biology

Article Title: Imiquimod (R837), a TLR7-Specific Agonist, Regulates Boar Sperm Motility via PI3K/GSK3α/β/Hexokinase Pathway

doi: 10.3390/biology14091182

Figure Lengend Snippet: The TLR7 signal transduction mechanism affects ATP production in boar sperm. In the lower-layer sperm, the TLR7 agonist R837 inhibits hexokinase activity and reduces ATP content by phosphorylation of PI3K and GSK3 proteins, thus leading to lower-layer sperm exhibiting low motility.

Article Snippet: Sperm samples were placed on special glass slides and washed with PBS, air-dried, and permeabilized using TritonX-100/PBS (0.1% v / v ) for 1 h. Subsequently, the sperm sample was blocked with 10% goat serum ( v / v ) at room temperature for 30 min, followed by incubation with TLR7 primary antibody monoclonal (#bs 6601R, Bioss, 1:1000, Beijing, China) at 4 °C overnight.

Techniques: Transduction, Activity Assay, Phospho-proteomics

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A, B ) In situ hybridization in 3dpf WT larvae probing for tlr7 and trpa1b mRNA respectively. ( C, D ) Larval locomotor assay of 5dpf tlr7 +/+ and tlr7 -/- ( C ) or trpa1b +/+ and trpa1b -/- ( D ) larvae. ( E, F ) Adult lip-rubbing behavioral assay of tlr7 +/+ /tlr7 -/- ( E ) and trpa1b +/+ /trpa1b -/- ( F ) fish. ( C ), ( D ), ( E ), ( F ), 100 μM IMQ used. ***p<0.001, **p<0.01, Student’s t -test. Bars represent mean ± s.e.m. ( G, H ) Representative calcium imaging traces of 3dpf trpa1b +/+ ( G ) and trpa1b -/- ( H ) larvae in a transgenic elavl3 HuC:GCaMP5 background exposed to 100 μM IMQ, 50 μM AITC, and 1 mM 2-APB. B = blood, TG = trigeminal ganglion.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: In Situ Hybridization, Behavioral Assay, Imaging, Transgenic Assay

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A ) The 7 bp deletion in the trpa1b coding sequence generated a premature stop codon at 1412 bp. ( B ) Amino acid sequences of the WT and mutant Trpa1b proteins. ( C ) In situ hybridization probing for trpa1b mRNA in WT 3dpf larval zebrafish, demonstrating expression in RB neurons. ( D ) Schematic of the Trpa1b protein structure, demonstrating that a truncated protein (in the unlikely event that it was translated) would lack a critical cysteine residue required for agonist binding . ( E ) Trpa1b -/- nonsense mutants locomoted more in response to increasing temperatures at levels equivalent to their WT/heterozygous siblings. ( F ) Normal AITC behavioral responses (increased locomotion) are abolished in trpa1b -/- nonsense mutants. ( G ) The 1 bp deletion in the tlr7 coding sequence generated a premature stop codon at bp 665. ( H ) Amino acid sequences of WT and mutant Tlr7 proteins. ( I ) Schematic of the Tlr7 protein, demonstrating that a truncated protein (in the unlikely event of translation) would lack critical functional domains. ( J ) In situ hybridization probing for tlr7 mRNA in WT 3dpf larval zebrafish. No tlr7 expression was observed in RB somatosensory neurons. Premature stops are denoted with red highlighting. EC = extracellular, IC = intracellular, TM = transmembrane domain, LRR = leucine rich repeat, TIR = Toll Interleukin-1 Resistance domain. C and N denote c- and n-termini. ( E ), Student’s t -test.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: Sequencing, Generated, Mutagenesis, In Situ Hybridization, Expressing, Residue, Binding Assay, Functional Assay

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A–C ) Calcium imaging of HEK cells transfected with zebrafish ( A ), mouse ( B ), and human ( C ) Trpa1 or Trpa1 + Tlr7 . ( D, E, F ) Calcium imaging dose response curves of HEK cells transfected with zebrafish ( D ), mouse ( E ), and human ( F ) Trpa1 , exposed to IMQ or AITC. ( A–F ) Numbers represent total cell counts per condition. ( G ) Patch clamp of HEK cell transfected with zebrafish trpa1b exposed to 100 μM IMQ. ( H ) Patch clamp dose response curve for HEK cells transfected with zebrafish trpa1b , mouse Trpa1 , or mouse Trpa1 + mouse Tlr7 . ( I ) Current density values of HEK cells exposed transfected with zebrafish trpa1b or mouse Trpa1 and exposed to 100 μM AITC or 100 μM IMQ. ( G–I ) n = 5 cells per condition. **p<0.01, Student’s t -test. Bars represent mean ± s.e.m.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: Imaging, Transfection, Patch Clamp

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A–D ) Average traces from calcium imaging experiments with HEK cells transiently transfected with zebrafish trpa1b ( n = 85) ( A ), zebrafish trpa1b + zebrafish tlr7 ( n = 80) ( B ), mouse Trpa1 ( n = 134) ( C ), or mouse Trpa1 + mouse Tlr7 ( n = 89) ( D ). n = 22, 23, 10, and 19 untransfected control HEK cells per experiment, respectively. No gross differences were observed between the two conditions for each species. ( E ) Quantification of peak fluorescence intensity achieved during stimulation with 100 μM IMQ across all HEK cell transfection conditions. In all species examined, no significant difference was observed between cells transfected with only Trpa1 and cells transfected with Trpa1 plus the corresponding Tlr7 . ( F–H ), Immunohistochemistry performed on HEK cells transfected with pIRES-eGFP only ( F ), mouse Tlr7 ( G ), or human TLR7 ( H ). As shown, TLR7 labeling (red) was only observed HEK cells transfected with Tlr7 constructs. ( I–J ), I/V curves from voltage clamp experiments using cells transfected with mTRPA1 ( I ) and mouse Trpa1 + mouse Tlr7 ( J ). As shown in ( I ), 100 μM IMQ elicited greater current influx in transfected cells than under basal conditions, an effect that disappeared during washout. Cells transfected with both mouse Trpa1 and mouse Tlr7 also showed significant current flux during IMQ stimulus, but this was not different from cells transfected only with mTRPA1. n = 5 cells per condition. ( K ) a table of EC50 values for both IMQ and AITC stimuli in cells transfected with zebrafish trpa1b , mouse Trpa1 , and human TRPA1 in . As shown, the EC50 values for IMQ are much greater than those of AITC for all species of TRPA1. ΔF/F (fluorescence intensity change) is expressed as a normalized 340 nm/380 nm intensity ratio. (*p<0.05, **p<0.01, ***p<0.001, Student’s t -test. Bars are expressed as means ± s.e.m.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: Imaging, Transfection, Control, Fluorescence, Immunohistochemistry, Labeling, Construct

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A ) A comparison of average peak fluorescence values obtained from Trpa1 or Trpa1 + Tlr7 transfected HEK cells in calcium imaging experiments following stimulation by 100 μM IMQ or 100 μM loxoribine. ( B, C ) Average traces from calcium experiments in which HEK cells were transfected with zebrafish trpa1b + zebrafish tlr7 ( n = 102) ( B ) or mouse Trpa1 + mouse Tlr7 ( n = 45) ( C ) and treated with 100 μM loxoribine and 100 μM AITC. 15 and 21 untransfected control cells were present in each respective experiment. As shown, loxoribine did not elicit any calcium flux. ( D ) I/V curve from voltage clamp experiments in which HEK cells were transfected with mouse Trpa1 + mouse Tlr7 and stimulated with both loxoribine (100 μM) and AITC (100 μM). While AITC elicited remarkable current influx, current change associated with application of loxoribine did not change current flow above baseline levels. ( E ) A dual luciferase assay to verify the functionality of transfected TLR7 and loxoribine. Cells transfected with only the two luciferase constructs and pIRES-eGFP did not demonstrate any NF-kB induction following stimulation with 200 μM loxoribine, as expected. Notable NF-kB induction was observed in cells transfected with the two luciferase constructs and either mouse Tlr7 or human TLR7 following stimulation with loxoribine. Intriguingly, cells transfected with the zebrafish tlr7 did not exhibit any significant NF-kB induction following application of loxoribine. ( F ) A dual luciferase assay in which both pIRES-eGFP-transfected control HEK cells and zebrafish tlr7 -transfected cells were treated with TLR7 agonists loxoribine (500 μM and 1 mM) and IMQ (100 μM and 500 μM). There was no difference in NF-kB induction between pIRES-eGFP-only and zebrafish tlr7- transfected cells, potentially indicating that zebrafish Tlr7 is unresponsive to typical mammalian TLR7 agonists. ΔF/F (fluorescence intensity change) is expressed as a normalized 340 nm/380 nm intensity ratio. Luminosity values are expressed as the firefly/renilla ratio of relative light units in counts per second (CPS) following background subtraction. *p<0.05, **p<0.01, ***p<0.001, Student’s t -test ( A, E ) or one-way ANOVA ( F ). Bars are expressed as means ± s.e.m.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: Comparison, Fluorescence, Transfection, Imaging, Control, Luciferase, Construct