Structured Review

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TRIM56 knockdown inactivated <t>TLR3/TRIF</t> signaling pathway in MM cells. (A) Western blot analysis of TLR3, TRIF, and RIP1 protein in RPMI8226 cells transfected with T56si-2 or si-control. (B) Quantitative analysis of TLR3, TRIF, and RIP1 protein levels. * p
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Images

1) Product Images from "TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling"

Article Title: TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling

Journal: Yonsei Medical Journal

doi: 10.3349/ymj.2018.59.1.43

TRIM56 knockdown inactivated TLR3/TRIF signaling pathway in MM cells. (A) Western blot analysis of TLR3, TRIF, and RIP1 protein in RPMI8226 cells transfected with T56si-2 or si-control. (B) Quantitative analysis of TLR3, TRIF, and RIP1 protein levels. * p
Figure Legend Snippet: TRIM56 knockdown inactivated TLR3/TRIF signaling pathway in MM cells. (A) Western blot analysis of TLR3, TRIF, and RIP1 protein in RPMI8226 cells transfected with T56si-2 or si-control. (B) Quantitative analysis of TLR3, TRIF, and RIP1 protein levels. * p

Techniques Used: Western Blot, Transfection

TRIM56 deficiency promoted the progression of MM via interrupting TLR3/TRIF signaling pathway. RPMI8226 cells were transfected with T56si-2 or along with poly (I:C) for further analysis at 48 h post-transfection. (A) Cell viability of transfected RPMI8226 cells was assessed by MTT assay. (B) Apoptotic rate of transfected RPMI8226 cells was detected by flow cytometry. (C) The relative caspase3 activity of transfected RPMI8226 cells was measured by the Caspase3 Colorimetric Assay Kit. The concentrations of secreted cytokines IFN-β (D), IL-6 (E), and TNF-α (F) in transfected RPMI8226 cells were measured by ELISA. (G) Western blot analysis of TLR3, TRIF, and RIP1 protein expression in transfected RPMI8226 cells. * p
Figure Legend Snippet: TRIM56 deficiency promoted the progression of MM via interrupting TLR3/TRIF signaling pathway. RPMI8226 cells were transfected with T56si-2 or along with poly (I:C) for further analysis at 48 h post-transfection. (A) Cell viability of transfected RPMI8226 cells was assessed by MTT assay. (B) Apoptotic rate of transfected RPMI8226 cells was detected by flow cytometry. (C) The relative caspase3 activity of transfected RPMI8226 cells was measured by the Caspase3 Colorimetric Assay Kit. The concentrations of secreted cytokines IFN-β (D), IL-6 (E), and TNF-α (F) in transfected RPMI8226 cells were measured by ELISA. (G) Western blot analysis of TLR3, TRIF, and RIP1 protein expression in transfected RPMI8226 cells. * p

Techniques Used: Transfection, MTT Assay, Flow Cytometry, Cytometry, Activity Assay, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Western Blot, 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 regulates poly(I:C)-triggered signaling through TLR3. (A) Mex3B(G83/177D) inhibits poly(I:C)- but not TRIF-induced signaling. The 293 cells (1 × 10 5 ) were transfected with the indicated plasmids and increased amounts of a Mex3B plasmid. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays were performed. (B) TLR3(C95/122A) inhibits poly(I:C)-induced, TLR3/Mex3B-mediated signaling. The experiments were similarly performed as in A . (C) Knockdown of Mex3B inhibits poly(I:C)- but not TRIF- or TBK1-mediated activation of the IFN-β promoter. The 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and either a control or Mex3B-RNAi plasmid (0.5 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h followed by either treatment with poly(I:C) (20 μg/ml) or retransfection with TRIF or TBK-1 plasmids (0.1 μg). Luciferase assays were performed 6 h after treatment or 18 h after retransfection. Data are represented as mean ± SEM. * P
Figure Legend Snippet: Mex3B regulates poly(I:C)-triggered signaling through TLR3. (A) Mex3B(G83/177D) inhibits poly(I:C)- but not TRIF-induced signaling. The 293 cells (1 × 10 5 ) were transfected with the indicated plasmids and increased amounts of a Mex3B plasmid. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays were performed. (B) TLR3(C95/122A) inhibits poly(I:C)-induced, TLR3/Mex3B-mediated signaling. The experiments were similarly performed as in A . (C) Knockdown of Mex3B inhibits poly(I:C)- but not TRIF- or TBK1-mediated activation of the IFN-β promoter. The 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and either a control or Mex3B-RNAi plasmid (0.5 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h followed by either treatment with poly(I:C) (20 μg/ml) or retransfection with TRIF or TBK-1 plasmids (0.1 μg). Luciferase assays were performed 6 h after treatment or 18 h after retransfection. Data are represented as mean ± SEM. * P

Techniques Used: Transfection, Plasmid Preparation, Luciferase, Activation Assay

Related Articles

Transduction:

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA). .. Blots were probed with anti-actin (Sigma-Aldrich, Dorset, UK) as a loading control.

Centrifugation:

Article Title: Spliceosome SNRNP200 Promotes Viral RNA Sensing and IRF3 Activation of Antiviral Response
Article Snippet: Cell lysates were clarified by centrifugation at 13,000 g for 20 min at 4°C and subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). .. Western blot analysis was performed using mouse anti-PHF5A (Abnova), anti-IRF3 (Santa Cruz), anti-TRAF3 (Santa Cruz), anti-RIG-I (Alexis Biochemicals), anti-ACTIN (Chemicon International), anti-TBK1 (Imgenex and Santa Cruz), anti-IKBKE (Santa Cruz), anti-TUBULIN (ICN), anti-GAPDH (RDI) and rabbit anti-SNRNP200 (Sigma-Aldrich), anti-SF3A1 (Santa Cruz), anti-RELA (Santa Cruz), anti-NHP2L1 (Abcam), anti-DDX3X (Bethyl), anti-DDX60 (Abcam), TRIF (Cell Signaling), anti-ISG56 (Novus Biologicals), anti-MDA5 (Alexis Biochemicals), anti-MAVS (Alexis Biochemicals), anti-IKBKE (eBioscience), STAT1 (ABCAM), STAT1 tyr701 (ABCAM), IFNAR1 (Santa Cruz) and anti-IRF3-P-ser386 (Abcam).

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.

Filtration:

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: Lentiviruses were concentrated and purified using tangential flow filtration (Minimate™; PAL Corp., Hampshire, UK), and titred in HT1080 cells or by p24 enzyme-linked immunosorbent assay (ELISA) (Cell Biolabs, San Diego, CA). .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA).

Cytometry:

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: GFP expression in transduced cells was determined by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ), and data were analysed using flowjo software (Tree Star Inc., Ashland, OR). .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA).

Blocking Assay:

Article Title: ENDOTHELIAL CELL TOLERANCE TO LIPOPOLYSACCHARIDE CHALLENGE IS INDUCED BY MONOPHOSPHORYL LIPID A
Article Snippet: Membranes were blocked with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE) for 1 hour at room-temperature. .. Antibodies were as follows: IKKαβ, phospho-IKKβ, IRAK-M, p-p38, p38, p-ERK, ERK, p-JNK, JNK, MyD88, TRIF (Cell Signaling Technology, Danvers, MA), IRF3 (Santa Cruz Biotechnology, Inc., Dallas, TX), p-IRF3 (EMD Milipore, Billerica, MA), Tubulin (Vanderbilt Antibody Core, Nashville, TN).

Electrophoresis:

Article Title: ENDOTHELIAL CELL TOLERANCE TO LIPOPOLYSACCHARIDE CHALLENGE IS INDUCED BY MONOPHOSPHORYL LIPID A
Article Snippet: Paragraph title: Western Blot and Electrophoresis ... Antibodies were as follows: IKKαβ, phospho-IKKβ, IRAK-M, p-p38, p38, p-ERK, ERK, p-JNK, JNK, MyD88, TRIF (Cell Signaling Technology, Danvers, MA), IRF3 (Santa Cruz Biotechnology, Inc., Dallas, TX), p-IRF3 (EMD Milipore, Billerica, MA), Tubulin (Vanderbilt Antibody Core, Nashville, TN).

Incubation:

Article Title: TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling
Article Snippet: .. After being blocked with 5% non-fat dry milk in tris-buffered saline with Tween 20, the membranes were blotted overnight at 4℃ with the following primary antibodies: TRIM56 (1:1000; Cell Signaling Technology, Danvers, MA, USA), TLR3 (1:500; Cell Signaling Technology), TRIF (1:1500; Cell Signaling Technology), RIP1 (1:500; Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibody anti-mouse IgG (1:2000; Millipore) for 1 h. Protein signals were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA). .. Cell viability assay Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer's protocol.

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: In experiments investigating cell survival, neutrophils were incubated with lentiviruses for 4 hr to allow novel protein expression, and then stimulated for a further 4 hr with 0·1 ng/ml purified Escherichia coli R515 LPS (Alexis, San Diego, CA) or 50 U/ml granulocyte–macrophage colony-stimulating factor (GM-CSF; Peprotech, Rocky Hill, NJ). .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA).

Article Title: ENDOTHELIAL CELL TOLERANCE TO LIPOPOLYSACCHARIDE CHALLENGE IS INDUCED BY MONOPHOSPHORYL LIPID A
Article Snippet: Antibodies were as follows: IKKαβ, phospho-IKKβ, IRAK-M, p-p38, p38, p-ERK, ERK, p-JNK, JNK, MyD88, TRIF (Cell Signaling Technology, Danvers, MA), IRF3 (Santa Cruz Biotechnology, Inc., Dallas, TX), p-IRF3 (EMD Milipore, Billerica, MA), Tubulin (Vanderbilt Antibody Core, Nashville, TN). .. Afterwards, membranes were incubated with fluorescent secondary antibodies and analyzed using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE).

Expressing:

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA). .. Blots were probed with anti-actin (Sigma-Aldrich, Dorset, UK) as a loading control.

BIA-KA:

Article Title: TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling
Article Snippet: Western blot analysis Total protein lysates were extracted from MM cells by RIPA lysis buffer (Beyotime, Jiangsu, China) with EDTA-free Protease Inhibitor Cocktail (Roche, Mannheim, Germany) and quantified using an Enhanced BCA protein assay kit (Beyotime). .. After being blocked with 5% non-fat dry milk in tris-buffered saline with Tween 20, the membranes were blotted overnight at 4℃ with the following primary antibodies: TRIM56 (1:1000; Cell Signaling Technology, Danvers, MA, USA), TLR3 (1:500; Cell Signaling Technology), TRIF (1:1500; Cell Signaling Technology), RIP1 (1:500; Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibody anti-mouse IgG (1:2000; Millipore) for 1 h. Protein signals were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA).

Modification:

Article Title: Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells
Article Snippet: Cells were then lysed with a Modified RIPA buffer (Tris-HCl 50 mM, 1% NP-40, 0.25% Na-deoxycholate, NaCl 150 mM, EDTA 1 mM pH 7.4) with 1X protease inhibitors (Roche 11 836 170 001) and 1X phophatase inhibitors (Fisher Scientific, PI-78420). .. Membranes were blocked for 1 hour with 5% milk in TBST or PBST, washed, and proteins were analyzed by immunoblotting with standard methods using antibodies specific to RIG-I (Enzo Lifesciences), MDA5 (Cell Signaling Technology), MyD88 (Cell Signaling Technology), IL-28B/IFNλ3 (R & D Systems), RNase L (Thermo Scientific), TRIF (Cell Signaling Technology), tubulin (Sigma), and GAPDH (Abcam).

Western Blot:

Article Title: TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling
Article Snippet: Paragraph title: Western blot analysis ... After being blocked with 5% non-fat dry milk in tris-buffered saline with Tween 20, the membranes were blotted overnight at 4℃ with the following primary antibodies: TRIM56 (1:1000; Cell Signaling Technology, Danvers, MA, USA), TLR3 (1:500; Cell Signaling Technology), TRIF (1:1500; Cell Signaling Technology), RIP1 (1:500; Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibody anti-mouse IgG (1:2000; Millipore) for 1 h. Protein signals were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA).

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA). .. Blots were probed with anti-actin (Sigma-Aldrich, Dorset, UK) as a loading control.

Article Title: ENDOTHELIAL CELL TOLERANCE TO LIPOPOLYSACCHARIDE CHALLENGE IS INDUCED BY MONOPHOSPHORYL LIPID A
Article Snippet: Paragraph title: Western Blot and Electrophoresis ... Antibodies were as follows: IKKαβ, phospho-IKKβ, IRAK-M, p-p38, p38, p-ERK, ERK, p-JNK, JNK, MyD88, TRIF (Cell Signaling Technology, Danvers, MA), IRF3 (Santa Cruz Biotechnology, Inc., Dallas, TX), p-IRF3 (EMD Milipore, Billerica, MA), Tubulin (Vanderbilt Antibody Core, Nashville, TN).

Article Title: Spliceosome SNRNP200 Promotes Viral RNA Sensing and IRF3 Activation of Antiviral Response
Article Snippet: .. Western blot analysis was performed using mouse anti-PHF5A (Abnova), anti-IRF3 (Santa Cruz), anti-TRAF3 (Santa Cruz), anti-RIG-I (Alexis Biochemicals), anti-ACTIN (Chemicon International), anti-TBK1 (Imgenex and Santa Cruz), anti-IKBKE (Santa Cruz), anti-TUBULIN (ICN), anti-GAPDH (RDI) and rabbit anti-SNRNP200 (Sigma-Aldrich), anti-SF3A1 (Santa Cruz), anti-RELA (Santa Cruz), anti-NHP2L1 (Abcam), anti-DDX3X (Bethyl), anti-DDX60 (Abcam), TRIF (Cell Signaling), anti-ISG56 (Novus Biologicals), anti-MDA5 (Alexis Biochemicals), anti-MAVS (Alexis Biochemicals), anti-IKBKE (eBioscience), STAT1 (ABCAM), STAT1 tyr701 (ABCAM), IFNAR1 (Santa Cruz) and anti-IRF3-P-ser386 (Abcam). .. HRP-conjugated secondary antibodies were from Bio-Rad.

Article Title: Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells
Article Snippet: Paragraph title: Western blots ... Membranes were blocked for 1 hour with 5% milk in TBST or PBST, washed, and proteins were analyzed by immunoblotting with standard methods using antibodies specific to RIG-I (Enzo Lifesciences), MDA5 (Cell Signaling Technology), MyD88 (Cell Signaling Technology), IL-28B/IFNλ3 (R & D Systems), RNase L (Thermo Scientific), TRIF (Cell Signaling Technology), tubulin (Sigma), and GAPDH (Abcam).

Flow Cytometry:

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: GFP expression in transduced cells was determined by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ), and data were analysed using flowjo software (Tree Star Inc., Ashland, OR). .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA).

Protease Inhibitor:

Article Title: TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling
Article Snippet: Western blot analysis Total protein lysates were extracted from MM cells by RIPA lysis buffer (Beyotime, Jiangsu, China) with EDTA-free Protease Inhibitor Cocktail (Roche, Mannheim, Germany) and quantified using an Enhanced BCA protein assay kit (Beyotime). .. After being blocked with 5% non-fat dry milk in tris-buffered saline with Tween 20, the membranes were blotted overnight at 4℃ with the following primary antibodies: TRIM56 (1:1000; Cell Signaling Technology, Danvers, MA, USA), TLR3 (1:500; Cell Signaling Technology), TRIF (1:1500; Cell Signaling Technology), RIP1 (1:500; Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibody anti-mouse IgG (1:2000; Millipore) for 1 h. Protein signals were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA).

Article Title: Spliceosome SNRNP200 Promotes Viral RNA Sensing and IRF3 Activation of Antiviral Response
Article Snippet: Western immunoblot analysis Cells were washed twice with ice-cold phosphate-buffered saline (PBS; Wisent), harvested and lysed in 10mM Tris-HCl, 100mM NaCl, 0.5% Triton X-100, pH7.6 with EDTA-free Protease Inhibitor Cocktail (Roche). .. Western blot analysis was performed using mouse anti-PHF5A (Abnova), anti-IRF3 (Santa Cruz), anti-TRAF3 (Santa Cruz), anti-RIG-I (Alexis Biochemicals), anti-ACTIN (Chemicon International), anti-TBK1 (Imgenex and Santa Cruz), anti-IKBKE (Santa Cruz), anti-TUBULIN (ICN), anti-GAPDH (RDI) and rabbit anti-SNRNP200 (Sigma-Aldrich), anti-SF3A1 (Santa Cruz), anti-RELA (Santa Cruz), anti-NHP2L1 (Abcam), anti-DDX3X (Bethyl), anti-DDX60 (Abcam), TRIF (Cell Signaling), anti-ISG56 (Novus Biologicals), anti-MDA5 (Alexis Biochemicals), anti-MAVS (Alexis Biochemicals), anti-IKBKE (eBioscience), STAT1 (ABCAM), STAT1 tyr701 (ABCAM), IFNAR1 (Santa Cruz) and anti-IRF3-P-ser386 (Abcam).

Imaging:

Article Title: ENDOTHELIAL CELL TOLERANCE TO LIPOPOLYSACCHARIDE CHALLENGE IS INDUCED BY MONOPHOSPHORYL LIPID A
Article Snippet: Antibodies were as follows: IKKαβ, phospho-IKKβ, IRAK-M, p-p38, p38, p-ERK, ERK, p-JNK, JNK, MyD88, TRIF (Cell Signaling Technology, Danvers, MA), IRF3 (Santa Cruz Biotechnology, Inc., Dallas, TX), p-IRF3 (EMD Milipore, Billerica, MA), Tubulin (Vanderbilt Antibody Core, Nashville, TN). .. Afterwards, membranes were incubated with fluorescent secondary antibodies and analyzed using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE).

Recombinant:

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.

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.

Purification:

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: In experiments investigating cell survival, neutrophils were incubated with lentiviruses for 4 hr to allow novel protein expression, and then stimulated for a further 4 hr with 0·1 ng/ml purified Escherichia coli R515 LPS (Alexis, San Diego, CA) or 50 U/ml granulocyte–macrophage colony-stimulating factor (GM-CSF; Peprotech, Rocky Hill, NJ). .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA).

Polyacrylamide Gel Electrophoresis:

Article Title: TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling
Article Snippet: Equal amounts of total protein (10 µg) were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA) overnight. .. After being blocked with 5% non-fat dry milk in tris-buffered saline with Tween 20, the membranes were blotted overnight at 4℃ with the following primary antibodies: TRIM56 (1:1000; Cell Signaling Technology, Danvers, MA, USA), TLR3 (1:500; Cell Signaling Technology), TRIF (1:1500; Cell Signaling Technology), RIP1 (1:500; Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibody anti-mouse IgG (1:2000; Millipore) for 1 h. Protein signals were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA).

SDS Page:

Article Title: TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling
Article Snippet: Equal amounts of total protein (10 µg) were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA) overnight. .. After being blocked with 5% non-fat dry milk in tris-buffered saline with Tween 20, the membranes were blotted overnight at 4℃ with the following primary antibodies: TRIM56 (1:1000; Cell Signaling Technology, Danvers, MA, USA), TLR3 (1:500; Cell Signaling Technology), TRIF (1:1500; Cell Signaling Technology), RIP1 (1:500; Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibody anti-mouse IgG (1:2000; Millipore) for 1 h. Protein signals were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA).

Article Title: Spliceosome SNRNP200 Promotes Viral RNA Sensing and IRF3 Activation of Antiviral Response
Article Snippet: Cell lysates were clarified by centrifugation at 13,000 g for 20 min at 4°C and subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). .. Western blot analysis was performed using mouse anti-PHF5A (Abnova), anti-IRF3 (Santa Cruz), anti-TRAF3 (Santa Cruz), anti-RIG-I (Alexis Biochemicals), anti-ACTIN (Chemicon International), anti-TBK1 (Imgenex and Santa Cruz), anti-IKBKE (Santa Cruz), anti-TUBULIN (ICN), anti-GAPDH (RDI) and rabbit anti-SNRNP200 (Sigma-Aldrich), anti-SF3A1 (Santa Cruz), anti-RELA (Santa Cruz), anti-NHP2L1 (Abcam), anti-DDX3X (Bethyl), anti-DDX60 (Abcam), TRIF (Cell Signaling), anti-ISG56 (Novus Biologicals), anti-MDA5 (Alexis Biochemicals), anti-MAVS (Alexis Biochemicals), anti-IKBKE (eBioscience), STAT1 (ABCAM), STAT1 tyr701 (ABCAM), IFNAR1 (Santa Cruz) and anti-IRF3-P-ser386 (Abcam).

Article Title: Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells
Article Snippet: Samples were separated using SDS-PAGE on Mini-protean TGX Any kD gels (Bio-rad, 456-9033) and transferred onto a nitrocellulose membrane using a wet transfer system. .. Membranes were blocked for 1 hour with 5% milk in TBST or PBST, washed, and proteins were analyzed by immunoblotting with standard methods using antibodies specific to RIG-I (Enzo Lifesciences), MDA5 (Cell Signaling Technology), MyD88 (Cell Signaling Technology), IL-28B/IFNλ3 (R & D Systems), RNase L (Thermo Scientific), TRIF (Cell Signaling Technology), tubulin (Sigma), and GAPDH (Abcam).

Plasmid Preparation:

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA). .. Blots were probed with anti-actin (Sigma-Aldrich, Dorset, UK) as a loading control.

Software:

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: GFP expression in transduced cells was determined by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ), and data were analysed using flowjo software (Tree Star Inc., Ashland, OR). .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA).

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: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: Lentiviruses were concentrated and purified using tangential flow filtration (Minimate™; PAL Corp., Hampshire, UK), and titred in HT1080 cells or by p24 enzyme-linked immunosorbent assay (ELISA) (Cell Biolabs, San Diego, CA). .. Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA).

Activation Assay:

Article Title: Pathways regulating lipopolysaccharide-induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils
Article Snippet: Protein expression was also determined by western blotting of lysates from neutrophils transduced with empty vector, eGFP, GFP-Bid, kmIRAK-1 or dnTRIF encoding lentiviruses for 4 hr, using antibodies to GFP (Roche Diagnostics, Burgess Hill, UK), Bid, IRAK-1 or TRIF (Cell Signalling, Boston, MA). .. Activation of nuclear factor (NF)-κB was determined by detection of phosphorylated IκBα.

Lysis:

Article Title: TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling
Article Snippet: Western blot analysis Total protein lysates were extracted from MM cells by RIPA lysis buffer (Beyotime, Jiangsu, China) with EDTA-free Protease Inhibitor Cocktail (Roche, Mannheim, Germany) and quantified using an Enhanced BCA protein assay kit (Beyotime). .. After being blocked with 5% non-fat dry milk in tris-buffered saline with Tween 20, the membranes were blotted overnight at 4℃ with the following primary antibodies: TRIM56 (1:1000; Cell Signaling Technology, Danvers, MA, USA), TLR3 (1:500; Cell Signaling Technology), TRIF (1:1500; Cell Signaling Technology), RIP1 (1:500; Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibody anti-mouse IgG (1:2000; Millipore) for 1 h. Protein signals were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA).

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    Cell Signaling Technology Inc trif antibody
    Trif Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    trif antibody - by Bioz Stars, 2020-01
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