tlr2 plasmid tlr2 plasmid  (Addgene inc)

Journal: PLOS ONE

Article Title: ECDD-S16 targets vacuolar ATPase: A potential inhibitor compound for pyroptosis-induced inflammation

doi: 10.1371/journal.pone.0292340

Figure Lengend Snippet: Raw264.7 macrophages were transfected with TLR2-YFP (green) for 48 h. After that, the transfected cells were stimulated with Pam2CSK4 (1 μg/ml) for 1 h. Acidification of endosomes/lysosomes was then determined by adding the Lysotracker Red (LTR) dye to these cells in the presence or absence of ECDD-S16 and incubation was continued for 2 h. The treated cells were then fixed and processed for CLSM image analysis. (A) Confocal microscopy showing the colocalization of TLR2-YFP and LTR. Scale bar is 10 μm. (B) The co-localization of TLR2-YFP and LTR was quantified. All data were determined by one-way ANOVA followed by Tukey’s multiple comparison test. **** p < 0.0001.

Article Snippet: TLR2 plasmid (pcDNA3-TLR2-YFP) was a gift from Doug Golenbock (Addgene plasmid # 13016; http://n2t.net/addgene:13016 ; RRID:Addgene_13016).

Techniques: Transfection, Incubation, Confocal Microscopy, Comparison

Journal: Computational and Structural Biotechnology Journal

Article Title: The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes

doi: 10.1016/j.csbj.2021.01.023

Figure Lengend Snippet: Luciferase reporter activation after stimulation of Toll-like receptors (TLRs) -expressing Flp-In™-293 NF-κB cells with spores of Lichtheimia corymbifera after different treatments for 5 h . Flp-In™-293 NF-κB cells stably expressing different Toll like receptors (TLR1,2,4,5,6) and cofactors (CD14, MD2) and additional knock out of TLR5 (TLR5-ko) were generated for TLR activation assays. All cell lines have a stably integrated promotor that is responding to the transcription factor NF-κB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) that translocates from cell plasma to the nucleus upon TLR activation. The responsive promotor regulates the expression of the luciferase reporter gene. The selected TLR combinations were: TLR2-CD14 with TLR5-ko, TLR2-TLR6-CD14, TLR2-TLR1 -CD14, CD14- MD2- TLR4 and TLR5. The basic cell line with luciferase reporter gene was used as comparison and has an additional TLR5-ko of the natural gene. Cell lines were seeded into 96-well-plates. The activity of the luciferase after 5 h of stimulation was normalized to the mean value of the pre run (2 h before stimulation). Data was analyzed by two-way ANOVA and Bonferroni posttest (*** indicates P < 0.001, * indicates P < 0.05).

Article Snippet: Plasmids coding for Toll-like receptors (TLR) were purchased from Addgene (www.addgene.org): pcDNA3-TLR1-YFP, pcDNA3-TLR2-YFP, pcDNA3-TLR4-YFP, pcDNA3-TLR5-CFP, pcDNA3-TLR6-YFP, pcDNA3-CD14 (Addgene plasmids # 13014, # 13016, # 13018, # 13019, # 13020, # 13645) were a gift from Douglas T. Golenbock (University of Massachusetts Medical School, division of Infectious Disease and Immunology, Worcester, MA, USA) to Addgene).

Techniques: Luciferase, Activation Assay, Expressing, Stable Transfection, Knock-Out, Generated, Clinical Proteomics, Comparison, Activity Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes

doi: 10.1016/j.csbj.2021.01.023

Figure Lengend Snippet: Luciferase reporter activation after stimulation of TLR 2-CD14 expressing Flp-In™-293 NF-κB cells with spores of Lichtheimia corymbifera after different treatments for 10 h . The capability of kitalase-treated spores, heat-killed first then kitalase –treated spores, vinotaste-treated spores, heat-killed spores, pronase E, heat-killed spores then pronase E, UV-treated spores, and resting spores of LCV and LCA strains to stimulate expression of TLR2-CD14 was evaluated. Treating the spores of LCV and LCA strains with kitalase and heat-killed spores following kitalase treatment stimulated the expression of TLR2-CD14. Other treatments were comparable to resting spores of both strains. Data were analyzed by two-way ANOVA and Bonferroni posttest (*** indicates P < 0.001, * indicates P < 0.05).

Article Snippet: Plasmids coding for Toll-like receptors (TLR) were purchased from Addgene (www.addgene.org): pcDNA3-TLR1-YFP, pcDNA3-TLR2-YFP, pcDNA3-TLR4-YFP, pcDNA3-TLR5-CFP, pcDNA3-TLR6-YFP, pcDNA3-CD14 (Addgene plasmids # 13014, # 13016, # 13018, # 13019, # 13020, # 13645) were a gift from Douglas T. Golenbock (University of Massachusetts Medical School, division of Infectious Disease and Immunology, Worcester, MA, USA) to Addgene).

Techniques: Luciferase, Activation Assay, Expressing