cy5-hsv60  (Sangon Biotech)

Journal: iScience

Article Title: Porcine NLRC3 specially binds short dsDNA to regulate cGAS activation

doi: 10.1016/j.isci.2024.111145

Figure Lengend Snippet: Porcine NLRC3 binds to viral DNA via NBD and LRR domain (A) Schematics view of the domain architecture of porcine and human NLRC3. (B) Electrostatic potential surface maps of pNLRC3 FL by AlphaFold (blue represents positive charge enriched area, red represents negative charge enriched area). (C) Streptavidin pull-down assay of HA-pNLRC3 FL , HA-hNLRC3 FL , HA-hcGAS FL and HA-hRIG-I FL in HEK-293T cells. The whole cell lysate for streptavidin pull-down was incubated with biotin-HSV60. HA-hNLRC3 FL and HA-hcGAS FL serve as positive controls, and HA-hRIG-I FL is a negative control. (D) Streptavidin pull-down assay for the binding of biotin-HSV60 dsDNA to HA-pNLRC3 FL or indicated domains of pNLRC3 in HEK-293T cells. (E and F) Streptavidin pull-down assay of HA-pNLRC3 FL (E) and HA-hNLRC3 FL (F) binding to biotin-HSV60 dsDNA, biotin-HSV60 DNA-RNA hybrids and biotin-HSV60 dsRNA. (G–I) Streptavidin pull-down assay for the binding of biotin-HSV60 dsDNA to Strep-II-pNLRC3 FL (G), GST-pNLRC3 NBD (H) and His-pNLRC3 LRR (I) recombinant proteins.

Article Snippet: The full-length pcGAS and the Salmon Sperm DNA (Thermo Fisher, #15632011) or HSV60 (synthesized by Sangon Biotech) were incubated in reaction buffer containing 2 mM GTP, 2 mM ATP, 5 mM MgCl 2 and 20 mM HEPES (pH 7.5) for 2 h at 37°C.

Techniques: Pull Down Assay, Incubation, Negative Control, Binding Assay, Recombinant

Journal: iScience

Article Title: Porcine NLRC3 specially binds short dsDNA to regulate cGAS activation

doi: 10.1016/j.isci.2024.111145

Figure Lengend Snippet: Human and porcine NLRC3 prefer different lengths of dsDNA (A) Computationally derived binding model of hNLRC3 NBD-LRR with HSV15 (upper) and HSV60 (bottom) dsDNA. hNLRC3, NBD is highlighted in blue, and LRR is highlighted in purple. (B and C) Computationally derived binding model of pNLRC3 NBD-LRR with HSV15 (B) and HSV60 (C) dsDNA. The binding conformations between pNLRC3 and dsDNA were derived from all-atom MD simulations at the equilibrium states. HSV15 associated with either NBD or LRR domains, while HSV60 dissociated from pNLRC3 (distance >50 Å) in representative conformations suggesting weaker affinities. pNLRC3, NBD is highlighted in yellow, and LRR is highlighted in pink. (D and E) The distance between 15 bp dsDNA (D) or 60 bp dsDNA (E) and pNLRC3 was estimated by measuring the distance between pNLRC3 and DNA backbone atom. (F) MST assays showing pNLRC3 binding affinities with HSV30, HSV60 and HSV120. Each experiment was independently repeated three times. (G and H) Streptavidin pull-down assay for the binding of biotin-HSV30, biotin-HSV60 and biotin-HSV120 dsDNA to HA-pNLRC3 FL (G) or HA-hNLRC3 FL (H) transfected in HEK-293T cells. (I and J) Streptavidin pull-down assay of HA-pNLRC3 FL (I) and HA-hNLRC3 FL (J) binding to biotin-HSV15 DNA/RNA hybrids and biotin-HSV60 DNA/RNA hybrids.

Article Snippet: The full-length pcGAS and the Salmon Sperm DNA (Thermo Fisher, #15632011) or HSV60 (synthesized by Sangon Biotech) were incubated in reaction buffer containing 2 mM GTP, 2 mM ATP, 5 mM MgCl 2 and 20 mM HEPES (pH 7.5) for 2 h at 37°C.

Techniques: Derivative Assay, Binding Assay, Pull Down Assay, Transfection

Journal: iScience

Article Title: Porcine NLRC3 specially binds short dsDNA to regulate cGAS activation

doi: 10.1016/j.isci.2024.111145

Figure Lengend Snippet: The replacement of a single residue within LRR 13-16 determines the preference of different dsDNA by NLRC3 (A) The three dimensional structure (upper) and electrostatic potential surface maps(bottom) alignment of the NBD and LRR domain of pNLRC3 and hNLRC3. (B) Phylogenetic analysis of NLRC3 proteins in different species including invertebrate and vertebrate. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method. Evolutionary analyses were conducted in MEGA7. (C) The amino acid sequence alignment of pNLRC3 G1032 site in 14 different species. (D and E) Streptavidin pull-down assay of HA-pNLRC3 FL WT and G1032R mutant (D) or HA-hNLRC3 FL WT and R1031G mutant (E) binding to biotin-HSV60. (F and G) Streptavidin pull-down assay for the binding of different lengths biotin-dsDNA to HA-pNLRC3 G1032R mutant (F) and HA-hNLRC3 R1031G mutant (G). (H and I) Streptavidin pull-down assay for the binding of different lengths biotin-DNA/RNA hybrids to HA-pNLRC3 G1032R mutant (H) and HA-hNLRC3 R1031G mutant (I).

Article Snippet: The full-length pcGAS and the Salmon Sperm DNA (Thermo Fisher, #15632011) or HSV60 (synthesized by Sangon Biotech) were incubated in reaction buffer containing 2 mM GTP, 2 mM ATP, 5 mM MgCl 2 and 20 mM HEPES (pH 7.5) for 2 h at 37°C.

Techniques: Residue, Sequencing, Pull Down Assay, Mutagenesis, Binding Assay

Journal: iScience

Article Title: Porcine NLRC3 specially binds short dsDNA to regulate cGAS activation

doi: 10.1016/j.isci.2024.111145

Figure Lengend Snippet: Porcine NLRC3 interferes with DNA binding and attenuates enzymatic function of cGAS (A) Schematic diagram of pNLRC3 and pcGAS domains. (B and C) Anti-Flag immunoprecipitates of HA-pcGAS (B) or HA-pSTING (C) with Flag-pNLRC3 mutants in HEK-293T cells. The cell lysates and immunoprecipitates were analyzed by immunoblotting. (D and E) Anti-Flag immunoprecipitates of HA-pcGAS CT (D) and V5-pcGAS NT (E) with Flag-pNLRC3 in HEK-293T cells. (F) Anti-Flag immunoprecipitates of HA-pcGAS CT with Flag-pNLRC3 NBD in HEK-293T cells. (G) Streptavidin pull-down assay for the binding of biotin-HSV60 dsDNA to HA-pcGAS in HEK-293T cells. Flag-pNLRC3 was added in a dose as a competitor. (H) cGAS activity assay by ion-exchange chromatography. 10 μM pcGAS FL was incubated with the Salmon Sperm DNA in reaction buffer at 37°C for 2 h. Analyzing the effects of pNLRC3 FL or pNLRC3 LRR on cGAMP production using a Mono Q ion-exchange column.

Article Snippet: The full-length pcGAS and the Salmon Sperm DNA (Thermo Fisher, #15632011) or HSV60 (synthesized by Sangon Biotech) were incubated in reaction buffer containing 2 mM GTP, 2 mM ATP, 5 mM MgCl 2 and 20 mM HEPES (pH 7.5) for 2 h at 37°C.

Techniques: Binding Assay, Western Blot, Pull Down Assay, Activity Assay, Ion Exchange Chromatography, Incubation

Journal: iScience

Article Title: Porcine NLRC3 specially binds short dsDNA to regulate cGAS activation

doi: 10.1016/j.isci.2024.111145

Figure Lengend Snippet: Porcine NLRC3 facilitates the activation of cGAS by binding short dsDNA (A and B) Anti-Flag immunoprecipitates from HEK-293T cells transfected with Flag-pNLRC3 and HA-pcGAS and increasing HSV60 (A) or HSV30 (B) dsDNA in a dose. (C and D) Anti-Flag immunoprecipitates from HEK-293T cells transfected with Flag-pNLRC3 and HA-pSTING and increasing HSV60 (C) or HSV30 (D) dsDNA in a dose. (E) cGAS activity assay was performed with HSV60 to detect the cGAMP level when adding equal pNLRC3 WT or pNLRC3 G1032R lysate from HEK-293T cells. The total proteins were determined by BCA quantitative kit. (F) cGAS activity assay was performed with HSV60 to detect the cGAMP level when adding equal hNLRC3 WT or hNLRC3 R1031G lysate from HEK-293T cells. The total proteins were determined by BCA quantitative kit.

Article Snippet: The full-length pcGAS and the Salmon Sperm DNA (Thermo Fisher, #15632011) or HSV60 (synthesized by Sangon Biotech) were incubated in reaction buffer containing 2 mM GTP, 2 mM ATP, 5 mM MgCl 2 and 20 mM HEPES (pH 7.5) for 2 h at 37°C.

Techniques: Activation Assay, Binding Assay, Transfection, Activity Assay

Journal: iScience

Article Title: Porcine NLRC3 specially binds short dsDNA to regulate cGAS activation

doi: 10.1016/j.isci.2024.111145

Figure Lengend Snippet:

Article Snippet: The full-length pcGAS and the Salmon Sperm DNA (Thermo Fisher, #15632011) or HSV60 (synthesized by Sangon Biotech) were incubated in reaction buffer containing 2 mM GTP, 2 mM ATP, 5 mM MgCl 2 and 20 mM HEPES (pH 7.5) for 2 h at 37°C.

Techniques: Lysis, Western Blot, Protease Inhibitor, Stripping Membranes, Marker, Bicinchoninic Acid Protein Assay, Cloning, Mutagenesis, Purification, RNA Extraction, Reporter Assay, Software