immunoblotting with anti tlr4  (R&D Systems)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Open reading frame mining identifies a TLR4 binding domain in the primary sequence of ECRG4

doi: 10.1007/s00018-019-03159-5

Figure Lengend Snippet: Internalization of the ECRG437–63-targeted phage into reporter HEK293 cells engineered to express TLR4-MD2-CD14 innate immunity complex. Panel a–e Targeting control HEK293 cells Wild type HEK cells were incubated with 1011 PFU/ml of wild type pUC198 (empty), EGF1–53-targeted pUC198, ECRG437–63-targeted pUC198, ECRG471–148-targeted pUC198 or ECRG4133–148-targeted pUC198 phage, respectively. Cells were fixed and phage internalization was visualized by immunofluorescence using an anti-phage antibody. Nuclei (blue) stained with DAPI. Panel f–j Targeting TLR4 expressing HEK cells. HEKblue™ cells with the innate immunity receptor complex were incubated with 1011 PFU/ml of wild-type pUC198 (empty), EGF1–53-targeted pUC198, ECRG437–63-targeted pUC198, Ecrg471–148-targeted pUC198 or ECRG4133–148-targeted pUC198 phage, respectively. Cells were fixed and phage internalization was visualized by immunofluorescence using an anti-phage antibody. The internalization into parental HEK293 cells was minimal for all phages. However, strong perinuclear staining (red) was observed in HEKblue™ cells incubated with ECRG437–63-targeted pUC198 (panel h) and ECRG4133–148-targeted pUC198 phage (panel j). Nuclei (blue) stained with DAPI. Panel k Quantification of phage internalization into HEKblue cells by real-time PCR. HEKblue™ cells were incubated with 1011 PFU/ml of the pUC198 phages indicated and DNA of internalized phage DNA was quantified by real-time PCR. The internalization was normalized to that of parental HEK293 cells. Data were presented as mean ± standard deviation, and experiments were repeated at least three times in triplicate. Panel l: Activation of NFkB in HEKblue™ cells. HEKblue™ cells were incubated with the indicated concentrations (ng/ml) of lipopolysaccharide (circles), recombinant ECRG431–70 (squares) or ECRG4133–148 (triangles) and the production of SEAP measured by optical density

Article Snippet: The beads were the centrifuged and bound proteins recovered with SDS loading buffer and resolved on a 10% SDS-PAGE before transfer to a PVDF membrane and immunoblotting with anti-TLR4 (CAT#119079, Abcam) at a dilution of 1:5000 [ 44 ].

Techniques: Incubation, Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Activation Assay, Recombinant

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Open reading frame mining identifies a TLR4 binding domain in the primary sequence of ECRG4

doi: 10.1007/s00018-019-03159-5

Figure Lengend Snippet: ECRG437–63 interacts with TLR4 in the TLR4-CD14-MD2 innate immune complex. a Anti ECRG4 immunoprecipitation of TLR4: HEK293 cells were transiently transfected for 36 h with either ECRG4 or the ECRG4∆31–70 lacking the active domain, along with a TLR4-FLAG plasmid. Cells were then lysed and ECRG4 immunoprecipitated with anti-ECRG4 for immunoblotting with an anti-TLR4 antibody. Input was loaded 1/30 of the amount used in immunoprecipitation and used to demonstrate equivalent starting material with each sample. b Anti-TLR4 immunoprecipitation of ECRG4: HEK293 cells were transiently transfected for 36 h with either ECRG4 or ECRG4∆31–70 and TLR4-FLAG. Cells were lysed and protein immunoprecipitated with anti-FLAG antibodies that precipitate TLR4 which was then immunoblotted with anti-ECRG4 antibodies. Input was loaded 1/30 of the amount used in immunoprecipitation and used to demonstrate equivalent starting material with each sample

Article Snippet: The beads were the centrifuged and bound proteins recovered with SDS loading buffer and resolved on a 10% SDS-PAGE before transfer to a PVDF membrane and immunoblotting with anti-TLR4 (CAT#119079, Abcam) at a dilution of 1:5000 [ 44 ].

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot