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ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and <t>streptavidin-Sepharose</t> beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
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1) Product Images from "ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response"

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

Journal: Nature Communications

doi: 10.1038/s41467-018-05559-w

ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
Figure Legend Snippet: ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

Techniques Used: Transfection, Incubation, In Vitro, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction

ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P
Figure Legend Snippet: ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P

Techniques Used: Binding Assay, Transfection, Incubation, In Vitro, Western Blot, Clone Assay, Generated, CRISPR, Microscale Thermophoresis, Recombinant, Purification, Staining, Infection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing

2) Product Images from "The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response"

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Journal: Cell Research

doi: 10.1038/cr.2016.16

Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.
Figure Legend Snippet: Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.

Techniques Used: Ligand Binding Assay, Marker, Transfection, Staining, Confocal Microscopy, Isolation, Over Expression, Plasmid Preparation, Binding Assay, Incubation

3) Product Images from "ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3"

Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3

Journal: Journal of Molecular Cell Biology

doi: 10.1093/jmcb/mjaa004

ZCCHC3 promotes the recruitment of TRIF to TLR3. ( A ) Overexpression of ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin-sepharose beads. Precipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. ( B ) Endogenous ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). Lysate of 293-TLR3 cells (1 × 10 7 ) was incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays and immunoblotting. ( C ) ZCCHC3 localizes in the cytosolic but not the lumen of endosomes. 293-TLR3 cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosomal and lysosomal fractions were isolated and left untreated or treated with proteinase K (2 μg/ml) or proteinase K plus 0.2% Triton X-100 for 15 min at room temperature. The samples were then analyzed by immunoblotting with the indicates antibodies. ( D ) ZCCHC3 promotes TLR3–TRIF but not TLR4–TRIF interaction in mammalian overexpression system. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cells were lysed for co-immunoprecipitation and immunoblotting with the indicated antibodies. ( E ) Knockdown of ZCCHC3 disrupts TLR3–TRIF but not TLR4–TRIF interaction. ZCCHC3-knockdown or control 293-TLR3 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were harvested and lysed for co-immunoprecipitation before immunoblotting with the indicated antibodies. ( F ) Knockdown of ZCCHC3 disrupts the recruitment of TRIF to TLR3. ZCCHC3-knockdown or control 293-TLR3 cells (1 × 10 7 ) were treated with poly(I:C) (50 μg/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-TLR3 and analyzed by immunoblotting with the indicated antibodies. ( G ) Stimulation of poly(I:C) but not LPS induces the interaction of ZCCHC3 with TRIF. The indicated cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) or LPS (200 ng/ml) for the indicated times before co-immunoprecipitation and immunoblotting with the indicated antibodies.
Figure Legend Snippet: ZCCHC3 promotes the recruitment of TRIF to TLR3. ( A ) Overexpression of ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin-sepharose beads. Precipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. ( B ) Endogenous ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). Lysate of 293-TLR3 cells (1 × 10 7 ) was incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays and immunoblotting. ( C ) ZCCHC3 localizes in the cytosolic but not the lumen of endosomes. 293-TLR3 cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosomal and lysosomal fractions were isolated and left untreated or treated with proteinase K (2 μg/ml) or proteinase K plus 0.2% Triton X-100 for 15 min at room temperature. The samples were then analyzed by immunoblotting with the indicates antibodies. ( D ) ZCCHC3 promotes TLR3–TRIF but not TLR4–TRIF interaction in mammalian overexpression system. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cells were lysed for co-immunoprecipitation and immunoblotting with the indicated antibodies. ( E ) Knockdown of ZCCHC3 disrupts TLR3–TRIF but not TLR4–TRIF interaction. ZCCHC3-knockdown or control 293-TLR3 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were harvested and lysed for co-immunoprecipitation before immunoblotting with the indicated antibodies. ( F ) Knockdown of ZCCHC3 disrupts the recruitment of TRIF to TLR3. ZCCHC3-knockdown or control 293-TLR3 cells (1 × 10 7 ) were treated with poly(I:C) (50 μg/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-TLR3 and analyzed by immunoblotting with the indicated antibodies. ( G ) Stimulation of poly(I:C) but not LPS induces the interaction of ZCCHC3 with TRIF. The indicated cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) or LPS (200 ng/ml) for the indicated times before co-immunoprecipitation and immunoblotting with the indicated antibodies.

Techniques Used: Over Expression, Binding Assay, Transfection, Incubation, Isolation, Immunoprecipitation

4) Product Images from "New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase"

Article Title: New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase

Journal: Bioconjugate chemistry

doi: 10.1021/acs.bioconjchem.6b00164

Validation of solid-phase, fluoride triggered mAb- 2 elution. (a) Brightfield (i) and fluorescent (ii) images of the solid-phase generator. A spin column loaded with a streptavidin-agarose that binds biotin on mAb- 1 . (b) SDS-PAGE gel of eluent generated
Figure Legend Snippet: Validation of solid-phase, fluoride triggered mAb- 2 elution. (a) Brightfield (i) and fluorescent (ii) images of the solid-phase generator. A spin column loaded with a streptavidin-agarose that binds biotin on mAb- 1 . (b) SDS-PAGE gel of eluent generated

Techniques Used: SDS Page, Generated

Radiolabeling of [ 18 F]-mAb- 2 . (a) Radioactive, SEC HPLC of [ 18 F]-mAb- 2 generated by solution fluoridation of mAb- 1 (1 h, [ 18 F]-hydrogen fluoride, pH 3). No streptavidin-agarose was utilized in this synthesis. (b) Radioactive, SEC HPLC [ 18 F]-mAb- 2 fluoride
Figure Legend Snippet: Radiolabeling of [ 18 F]-mAb- 2 . (a) Radioactive, SEC HPLC of [ 18 F]-mAb- 2 generated by solution fluoridation of mAb- 1 (1 h, [ 18 F]-hydrogen fluoride, pH 3). No streptavidin-agarose was utilized in this synthesis. (b) Radioactive, SEC HPLC [ 18 F]-mAb- 2 fluoride

Techniques Used: Radioactivity, Size-exclusion Chromatography, High Performance Liquid Chromatography, Generated

5) Product Images from "The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response"

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Journal: Cell Research

doi: 10.1038/cr.2016.16

Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.
Figure Legend Snippet: Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.

Techniques Used: Ligand Binding Assay, Marker, Transfection, Staining, Confocal Microscopy, Isolation, Over Expression, Plasmid Preparation, Binding Assay, Incubation

6) Product Images from "ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response"

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

Journal: Nature Communications

doi: 10.1038/s41467-018-05559-w

ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
Figure Legend Snippet: ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

Techniques Used: Transfection, Incubation, In Vitro, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction

ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P
Figure Legend Snippet: ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P

Techniques Used: Binding Assay, Transfection, Incubation, In Vitro, Western Blot, Clone Assay, Generated, CRISPR, Microscale Thermophoresis, Recombinant, Purification, Staining, Infection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing

7) Product Images from "New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase"

Article Title: New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase

Journal: Bioconjugate chemistry

doi: 10.1021/acs.bioconjchem.6b00164

Validation of solid-phase, fluoride triggered mAb- 2 elution. (a) Brightfield (i) and fluorescent (ii) images of the solid-phase generator. A spin column loaded with a streptavidin-agarose that binds biotin on mAb- 1 . (b) SDS-PAGE gel of eluent generated
Figure Legend Snippet: Validation of solid-phase, fluoride triggered mAb- 2 elution. (a) Brightfield (i) and fluorescent (ii) images of the solid-phase generator. A spin column loaded with a streptavidin-agarose that binds biotin on mAb- 1 . (b) SDS-PAGE gel of eluent generated

Techniques Used: SDS Page, Generated

Radiolabeling of [ 18 F]-mAb- 2 . (a) Radioactive, SEC HPLC of [ 18 F]-mAb- 2 generated by solution fluoridation of mAb- 1 (1 h, [ 18 F]-hydrogen fluoride, pH 3). No streptavidin-agarose was utilized in this synthesis. (b) Radioactive, SEC HPLC [ 18 F]-mAb- 2 fluoride
Figure Legend Snippet: Radiolabeling of [ 18 F]-mAb- 2 . (a) Radioactive, SEC HPLC of [ 18 F]-mAb- 2 generated by solution fluoridation of mAb- 1 (1 h, [ 18 F]-hydrogen fluoride, pH 3). No streptavidin-agarose was utilized in this synthesis. (b) Radioactive, SEC HPLC [ 18 F]-mAb- 2 fluoride

Techniques Used: Radioactivity, Size-exclusion Chromatography, High Performance Liquid Chromatography, Generated

8) Product Images from "New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase"

Article Title: New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase

Journal: Bioconjugate chemistry

doi: 10.1021/acs.bioconjchem.6b00164

Validation of solid-phase, fluoride triggered mAb- 2 elution. (a) Brightfield (i) and fluorescent (ii) images of the solid-phase generator. A spin column loaded with a streptavidin-agarose that binds biotin on mAb- 1 . (b) SDS-PAGE gel of eluent generated
Figure Legend Snippet: Validation of solid-phase, fluoride triggered mAb- 2 elution. (a) Brightfield (i) and fluorescent (ii) images of the solid-phase generator. A spin column loaded with a streptavidin-agarose that binds biotin on mAb- 1 . (b) SDS-PAGE gel of eluent generated

Techniques Used: SDS Page, Generated

Radiolabeling of [ 18 F]-mAb- 2 . (a) Radioactive, SEC HPLC of [ 18 F]-mAb- 2 generated by solution fluoridation of mAb- 1 (1 h, [ 18 F]-hydrogen fluoride, pH 3). No streptavidin-agarose was utilized in this synthesis. (b) Radioactive, SEC HPLC [ 18 F]-mAb- 2 fluoride
Figure Legend Snippet: Radiolabeling of [ 18 F]-mAb- 2 . (a) Radioactive, SEC HPLC of [ 18 F]-mAb- 2 generated by solution fluoridation of mAb- 1 (1 h, [ 18 F]-hydrogen fluoride, pH 3). No streptavidin-agarose was utilized in this synthesis. (b) Radioactive, SEC HPLC [ 18 F]-mAb- 2 fluoride

Techniques Used: Radioactivity, Size-exclusion Chromatography, High Performance Liquid Chromatography, Generated

9) Product Images from "New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase"

Article Title: New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase

Journal: Bioconjugate chemistry

doi: 10.1021/acs.bioconjchem.6b00164

Validation of solid-phase, fluoride triggered mAb- 2 elution. (a) Brightfield (i) and fluorescent (ii) images of the solid-phase generator. A spin column loaded with a streptavidin-agarose that binds biotin on mAb- 1 . (b) SDS-PAGE gel of eluent generated
Figure Legend Snippet: Validation of solid-phase, fluoride triggered mAb- 2 elution. (a) Brightfield (i) and fluorescent (ii) images of the solid-phase generator. A spin column loaded with a streptavidin-agarose that binds biotin on mAb- 1 . (b) SDS-PAGE gel of eluent generated

Techniques Used: SDS Page, Generated

Radiolabeling of [ 18 F]-mAb- 2 . (a) Radioactive, SEC HPLC of [ 18 F]-mAb- 2 generated by solution fluoridation of mAb- 1 (1 h, [ 18 F]-hydrogen fluoride, pH 3). No streptavidin-agarose was utilized in this synthesis. (b) Radioactive, SEC HPLC [ 18 F]-mAb- 2 fluoride
Figure Legend Snippet: Radiolabeling of [ 18 F]-mAb- 2 . (a) Radioactive, SEC HPLC of [ 18 F]-mAb- 2 generated by solution fluoridation of mAb- 1 (1 h, [ 18 F]-hydrogen fluoride, pH 3). No streptavidin-agarose was utilized in this synthesis. (b) Radioactive, SEC HPLC [ 18 F]-mAb- 2 fluoride

Techniques Used: Radioactivity, Size-exclusion Chromatography, High Performance Liquid Chromatography, Generated

Related Articles

Flow Cytometry:

Article Title: New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase
Article Snippet: .. Room temperature nitrogen flow was passed over the streptavidin-agarose as fluoridation proceeded for 69 min (TOR = 69 min) before 50 μL of 250 mM PBS (pH 6.5) was added to neutralize. .. The microspin column containing [18 F]-mAb- 2 /Cetuximab- 2 was centrifuged to elute radiolabeled [18 F]-mAb- 2 /Cetuximab- 2 , which was chromatographed by SEC HPLC using an isocratic 25 mM PBS (pH 6.5) and 200 mM NaCl solution as an eluent ([18 F]-mAb- 2 /Cetuximab- 2 was purified with 250 mM PBS solution 0 mM NaCl (pH 6.5)).

Luciferase:

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response
Article Snippet: .. Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers. .. Mouse antisera against human Mex3B were raised against recombinant human Mex3B fragments containing aa236-517 and aa1-517, respectively.

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response
Article Snippet: .. Reagents, antibodies, cells, and viruses Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers. .. Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response
Article Snippet: .. Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers. .. Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3
Article Snippet: .. Reagents, antibodies, and cells poly(I:C), R848, PGN, and Lipofectamine 2000 (InvivoGen); MPLA and LPS (Sigma); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); puromycin and EZ-link Psoralen–PEG3 –Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6, and TNFα (BioLegend). .. Mouse monoclonal antibodies against HA (Origene), FLAG, and β-actin, anti-HA-Peroxidase, and anti-FLAG-M2-Peroxidase (Sigma) and rabbit monoclonal antibodies against TLR3, phosphor-IRF3, p65 (Cell Signaling Technology), phosphor-TBK1, TBK1, TRIF (Abcam), and IRF3 (Santa Cruz Biotechnology) were purchased from the indicated manufacturers.

Isolation:

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response
Article Snippet: .. Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers. .. Mouse antisera against human Mex3B were raised against recombinant human Mex3B fragments containing aa236-517 and aa1-517, respectively.

Enzyme-linked Immunosorbent Assay:

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response
Article Snippet: .. Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers. .. Mouse antisera against human Mex3B were raised against recombinant human Mex3B fragments containing aa236-517 and aa1-517, respectively.

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response
Article Snippet: .. Reagents, antibodies, cells, and viruses Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers. .. Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response
Article Snippet: .. Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers. .. Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3
Article Snippet: .. Reagents, antibodies, and cells poly(I:C), R848, PGN, and Lipofectamine 2000 (InvivoGen); MPLA and LPS (Sigma); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); puromycin and EZ-link Psoralen–PEG3 –Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6, and TNFα (BioLegend). .. Mouse monoclonal antibodies against HA (Origene), FLAG, and β-actin, anti-HA-Peroxidase, and anti-FLAG-M2-Peroxidase (Sigma) and rabbit monoclonal antibodies against TLR3, phosphor-IRF3, p65 (Cell Signaling Technology), phosphor-TBK1, TBK1, TRIF (Abcam), and IRF3 (Santa Cruz Biotechnology) were purchased from the indicated manufacturers.

Incubation:

Article Title: New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase
Article Snippet: .. 19.5 μL of mAb- 1 /Cetuximab- 1 (372 pmol) was added to 30 μL streptavidin-agarose (Solulink, N-1000-005) and incubated for 5 min. .. The mixture was transferred to a Spin-X 0.22 μm cellulose acetate filter (Costar, 8161) or a microspin column (30 μm polyethylene filter, Thermo Scientific, 89879) and was centrifuged at 4000 rcf for 1 min.

Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response
Article Snippet: .. Lysates were incubated with biotinylated-poly(I:C) for 1 h at 4 °C and then incubated with streptavidin agarose for another 2 h at 4 °C. .. The beads were washed three times with lysis buffer and analyzed by immunoblot.

other:

Article Title: New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase
Article Snippet: The filtrate was discarded and streptavidin-agarose was resuspended in 500 μL of diH2 O.

Recombinant:

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response
Article Snippet: .. Reagents, antibodies, cells, and viruses Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers. .. Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response
Article Snippet: .. Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers. .. Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

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    Solulink streptavidin agarose
    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and <t>streptavidin-Sepharose</t> beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
    Streptavidin Agarose, supplied by Solulink, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin agarose/product/Solulink
    Average 90 stars, based on 3 article reviews
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    Solulink streptavidin agarose beads
    Cep170 is an FHDC1-interacting protein. BirA*-tagged FHDC1 was expressed in NIH 3T3 cells by transient transfection. Ciliogenesis was induced as before in low-serum media. BirA*-FHDC1 (red) was detected by virtue of the encoded myc-tag. (A) BirA*-FHDC1 is recruited to the elongated cilia and, following exogenous biotin treatment, induces biotinylation of cytoplasmic microtubules (green) and the cilia (acetylated tubulin, white). Specific punctate structures within the cytoplasmic microtubule network were also labeled (arrows). Biotinylated proteins were detected with <t>Alexa488-streptavidin.</t> (B) BirA*-FHDC1 (red) induces biotinylation of puncta (green) that colocalize with γ-tubulin puncta detected with anti–γ-tubulin (white). (C) BirA*-FHDC1 induces biotinylation of endogenous Cep170. As in B, BirA*-FHDC1 was expressed by transient transfection and its ability to induce biotinylation of the endogenous Cep170 was assessed by IB. myc-FHDC1 and FMNL2-BirA* were included as negative controls. Total biotinylated proteins were isolated from each sample using streptavidin–agarose beads. The eluted proteins were immunoblotted using the indicated antibodies. Endogenous Cep170 is only present in the pool of biotinylated proteins induced by BirA*-FHDC1 (arrow).
    Streptavidin Agarose Beads, supplied by Solulink, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

    Journal: Nature Communications

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    doi: 10.1038/s41467-018-05559-w

    Figure Lengend Snippet: ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

    Article Snippet: Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers.

    Techniques: Transfection, Incubation, In Vitro, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction

    ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P

    Journal: Nature Communications

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    doi: 10.1038/s41467-018-05559-w

    Figure Lengend Snippet: ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P

    Article Snippet: Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers.

    Techniques: Binding Assay, Transfection, Incubation, In Vitro, Western Blot, Clone Assay, Generated, CRISPR, Microscale Thermophoresis, Recombinant, Purification, Staining, Infection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing

    Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.

    Journal: Cell Research

    Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.

    Article Snippet: Lysates were incubated with biotinylated-poly(I:C) for 1 h at 4 °C and then incubated with streptavidin agarose for another 2 h at 4 °C.

    Techniques: Ligand Binding Assay, Marker, Transfection, Staining, Confocal Microscopy, Isolation, Over Expression, Plasmid Preparation, Binding Assay, Incubation

    ZCCHC3 promotes the recruitment of TRIF to TLR3. ( A ) Overexpression of ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin-sepharose beads. Precipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. ( B ) Endogenous ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). Lysate of 293-TLR3 cells (1 × 10 7 ) was incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays and immunoblotting. ( C ) ZCCHC3 localizes in the cytosolic but not the lumen of endosomes. 293-TLR3 cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosomal and lysosomal fractions were isolated and left untreated or treated with proteinase K (2 μg/ml) or proteinase K plus 0.2% Triton X-100 for 15 min at room temperature. The samples were then analyzed by immunoblotting with the indicates antibodies. ( D ) ZCCHC3 promotes TLR3–TRIF but not TLR4–TRIF interaction in mammalian overexpression system. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cells were lysed for co-immunoprecipitation and immunoblotting with the indicated antibodies. ( E ) Knockdown of ZCCHC3 disrupts TLR3–TRIF but not TLR4–TRIF interaction. ZCCHC3-knockdown or control 293-TLR3 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were harvested and lysed for co-immunoprecipitation before immunoblotting with the indicated antibodies. ( F ) Knockdown of ZCCHC3 disrupts the recruitment of TRIF to TLR3. ZCCHC3-knockdown or control 293-TLR3 cells (1 × 10 7 ) were treated with poly(I:C) (50 μg/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-TLR3 and analyzed by immunoblotting with the indicated antibodies. ( G ) Stimulation of poly(I:C) but not LPS induces the interaction of ZCCHC3 with TRIF. The indicated cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) or LPS (200 ng/ml) for the indicated times before co-immunoprecipitation and immunoblotting with the indicated antibodies.

    Journal: Journal of Molecular Cell Biology

    Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3

    doi: 10.1093/jmcb/mjaa004

    Figure Lengend Snippet: ZCCHC3 promotes the recruitment of TRIF to TLR3. ( A ) Overexpression of ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin-sepharose beads. Precipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. ( B ) Endogenous ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). Lysate of 293-TLR3 cells (1 × 10 7 ) was incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays and immunoblotting. ( C ) ZCCHC3 localizes in the cytosolic but not the lumen of endosomes. 293-TLR3 cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosomal and lysosomal fractions were isolated and left untreated or treated with proteinase K (2 μg/ml) or proteinase K plus 0.2% Triton X-100 for 15 min at room temperature. The samples were then analyzed by immunoblotting with the indicates antibodies. ( D ) ZCCHC3 promotes TLR3–TRIF but not TLR4–TRIF interaction in mammalian overexpression system. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cells were lysed for co-immunoprecipitation and immunoblotting with the indicated antibodies. ( E ) Knockdown of ZCCHC3 disrupts TLR3–TRIF but not TLR4–TRIF interaction. ZCCHC3-knockdown or control 293-TLR3 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were harvested and lysed for co-immunoprecipitation before immunoblotting with the indicated antibodies. ( F ) Knockdown of ZCCHC3 disrupts the recruitment of TRIF to TLR3. ZCCHC3-knockdown or control 293-TLR3 cells (1 × 10 7 ) were treated with poly(I:C) (50 μg/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-TLR3 and analyzed by immunoblotting with the indicated antibodies. ( G ) Stimulation of poly(I:C) but not LPS induces the interaction of ZCCHC3 with TRIF. The indicated cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) or LPS (200 ng/ml) for the indicated times before co-immunoprecipitation and immunoblotting with the indicated antibodies.

    Article Snippet: Reagents, antibodies, and cells poly(I:C), R848, PGN, and Lipofectamine 2000 (InvivoGen); MPLA and LPS (Sigma); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); puromycin and EZ-link Psoralen–PEG3 –Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6, and TNFα (BioLegend).

    Techniques: Over Expression, Binding Assay, Transfection, Incubation, Isolation, Immunoprecipitation

    Cep170 is an FHDC1-interacting protein. BirA*-tagged FHDC1 was expressed in NIH 3T3 cells by transient transfection. Ciliogenesis was induced as before in low-serum media. BirA*-FHDC1 (red) was detected by virtue of the encoded myc-tag. (A) BirA*-FHDC1 is recruited to the elongated cilia and, following exogenous biotin treatment, induces biotinylation of cytoplasmic microtubules (green) and the cilia (acetylated tubulin, white). Specific punctate structures within the cytoplasmic microtubule network were also labeled (arrows). Biotinylated proteins were detected with Alexa488-streptavidin. (B) BirA*-FHDC1 (red) induces biotinylation of puncta (green) that colocalize with γ-tubulin puncta detected with anti–γ-tubulin (white). (C) BirA*-FHDC1 induces biotinylation of endogenous Cep170. As in B, BirA*-FHDC1 was expressed by transient transfection and its ability to induce biotinylation of the endogenous Cep170 was assessed by IB. myc-FHDC1 and FMNL2-BirA* were included as negative controls. Total biotinylated proteins were isolated from each sample using streptavidin–agarose beads. The eluted proteins were immunoblotted using the indicated antibodies. Endogenous Cep170 is only present in the pool of biotinylated proteins induced by BirA*-FHDC1 (arrow).

    Journal: Molecular Biology of the Cell

    Article Title: Actin-dependent regulation of cilia length by the inverted formin FHDC1

    doi: 10.1091/mbc.E18-02-0088

    Figure Lengend Snippet: Cep170 is an FHDC1-interacting protein. BirA*-tagged FHDC1 was expressed in NIH 3T3 cells by transient transfection. Ciliogenesis was induced as before in low-serum media. BirA*-FHDC1 (red) was detected by virtue of the encoded myc-tag. (A) BirA*-FHDC1 is recruited to the elongated cilia and, following exogenous biotin treatment, induces biotinylation of cytoplasmic microtubules (green) and the cilia (acetylated tubulin, white). Specific punctate structures within the cytoplasmic microtubule network were also labeled (arrows). Biotinylated proteins were detected with Alexa488-streptavidin. (B) BirA*-FHDC1 (red) induces biotinylation of puncta (green) that colocalize with γ-tubulin puncta detected with anti–γ-tubulin (white). (C) BirA*-FHDC1 induces biotinylation of endogenous Cep170. As in B, BirA*-FHDC1 was expressed by transient transfection and its ability to induce biotinylation of the endogenous Cep170 was assessed by IB. myc-FHDC1 and FMNL2-BirA* were included as negative controls. Total biotinylated proteins were isolated from each sample using streptavidin–agarose beads. The eluted proteins were immunoblotted using the indicated antibodies. Endogenous Cep170 is only present in the pool of biotinylated proteins induced by BirA*-FHDC1 (arrow).

    Article Snippet: Protein concentration was determined by BCA assay and equal amounts of each lysate were incubated separately with streptavidin agarose beads (Solulink) for 3 h at 4°C.

    Techniques: Transfection, Labeling, Isolation