Large-scale, arrayed format RNAi screens to identify genes essential for proliferation/viability were performed as described3 (link),14 (link). The effect of introducing each of the 5002 shRNAs (targeting 957 genes) was determined in 19 cell lines, and normalized using the B-score metric4 (link). Feature selection of shRNA B-score data was performed using the Comparative Marker Application Suite in GenePattern5 (link) and was independently analyzed using RIGER analysis6 (link) to compute NES for each gene. Secondary screen viability data was normalized using a percent of control statistic, given the biased nature of the candidate shRNA plate. Expression profiling was used to generate a signature that correlates with KRAS activation and implicated NF-κB signaling in cell lines and tumors dependent on KRAS. Regulation of NF-κB by TBK1 was shown using biochemical and cell biological approaches. Details of the analytical methods are provided in the Full Methods.
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TBK1 protein, human
TBK1 protein, human
TBK1, also known as TANK-binding kinase 1, is a serine/threonine protein kinase that plays a crucial role in innate immunity and inflammatory signaling pathways.
It is involved in the activation of transcription factors such as interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), which regulate the expression of genes involved in the immune response.
TBK1 is an important regulator of antiviral and antibacterial defenses, and it has been implicated in the pathogenesis of various inflammatory and autoimmune disorders.
Researchers studyting TBK1 can optimize their work with PubCompare.ai, an AI-powered tool that enhances reproducibility and accuaracy by helping them easily locate protocols from literature, preprints, and patents, while using intelligent comparisons to identify the best methods and products.
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It is involved in the activation of transcription factors such as interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), which regulate the expression of genes involved in the immune response.
TBK1 is an important regulator of antiviral and antibacterial defenses, and it has been implicated in the pathogenesis of various inflammatory and autoimmune disorders.
Researchers studyting TBK1 can optimize their work with PubCompare.ai, an AI-powered tool that enhances reproducibility and accuaracy by helping them easily locate protocols from literature, preprints, and patents, while using intelligent comparisons to identify the best methods and products.
Experience seamless and efficient TBK1 research with PubCompare.ai.
Most cited protocols related to «TBK1 protein, human»
Biopharmaceuticals
Cell Lines
Cells
Genes
K-ras Genes
Neoplasms
RELA protein, human
RNA Interference
Short Hairpin RNA
TBK1 protein, human
Large-scale, arrayed format RNAi screens to identify genes essential for proliferation/viability were performed as described3 (link),14 (link). The effect of introducing each of the 5002 shRNAs (targeting 957 genes) was determined in 19 cell lines, and normalized using the B-score metric4 (link). Feature selection of shRNA B-score data was performed using the Comparative Marker Application Suite in GenePattern5 (link) and was independently analyzed using RIGER analysis6 (link) to compute NES for each gene. Secondary screen viability data was normalized using a percent of control statistic, given the biased nature of the candidate shRNA plate. Expression profiling was used to generate a signature that correlates with KRAS activation and implicated NF-κB signaling in cell lines and tumors dependent on KRAS. Regulation of NF-κB by TBK1 was shown using biochemical and cell biological approaches. Details of the analytical methods are provided in the Full Methods.
Biopharmaceuticals
Cell Lines
Cells
Genes
K-ras Genes
Neoplasms
RELA protein, human
RNA Interference
Short Hairpin RNA
TBK1 protein, human
pMXs-puro-GFP-WIPI1 and pMXs-puro-GFP-DFCP1 were a kind gift from Dr. N. Mizushima (University of Toyko, Japan) and pMXs-IP-GFP-ULK1 was purchased from Addgene (#38193). To generate pBMN-mEGFP-C1, mEGFP-C1 (Addgene #36412) was PCR amplified (together with the multiple cloning site) and cloned into pBMN-Z at BamHI/SalI sites using the Gibson Cloning kit (New England BioLabs) according to manufacturer's instructions. The BamHI and SalI sites used to insert mEGFP-C1 were not regenerated. The following GFP-tagged plasmids were generated by PCR amplification of open reading frames followed by ligation into pBMN-mEGFP-C1: OPTN, NDP52, p62, TAX1BP1, NBR1, LC3A, LC3B, LC3C, GABARAP, GABARAPL1, GABARAPL2. The Gateway Cloning (Invitrogen) system was used to generate GFP-, mCherry-, myc- and FLAG/HA-constructs. Briefly, TBK1, TBK1-K38M, NDP52, OPTN, p62, DFCP1, WIPI1 and ULK1 were cloned into pDONR2333. Mutations in cDNA sequences were introduced using PCR site directed mutagenesis in the pDONR2333 vector, (sequences of mutagenesis primers used are available upon request) then recombined into pHAGE-N-FLAG/HA, pHAGE-N-GFP, pHAGE-N-mCherry and/or pDEST-N-myc using LR Clonase (Invitrogen) as per the manufacturer's protocol. All constructs generated in this study were verified by sequencing.
To generate stably transfected cell lines, retroviruses (for pBMN-mEGFP-C1 constructs, pBMN-mCherry-Parkin, pBMN-puro-P2A-FRB-Fis1, pCHAC-mt-mKeima-IRES-MCS2) and lentiviruses (for pHAGE- and pDEST- constructs) were packaged in HEK293T cells. HeLa cells were transduced with virus for 24 h with 8 μg/ml polybrene (Sigma) then optimized for protein expression via selection (puromycin or blasticidin) or fluorescence sorting.
To generate stably transfected cell lines, retroviruses (for pBMN-mEGFP-C1 constructs, pBMN-mCherry-Parkin, pBMN-puro-P2A-FRB-Fis1, pCHAC-mt-mKeima-IRES-MCS2) and lentiviruses (for pHAGE- and pDEST- constructs) were packaged in HEK293T cells. HeLa cells were transduced with virus for 24 h with 8 μg/ml polybrene (Sigma) then optimized for protein expression via selection (puromycin or blasticidin) or fluorescence sorting.
Bacteriophages
Cell Lines
Cells
Cloning Vectors
DNA, Complementary
Fluorescence
HeLa Cells
Internal Ribosome Entry Sites
Lentivirus
Ligation
Mutagenesis
Mutagenesis, Site-Directed
Mutation
Oligonucleotide Primers
Open Reading Frames
PARK2 protein, human
Plasmids
Polybrene
Proteins
Puromycin
Retroviridae
TBK1 protein, human
ULK1 protein, human
Virus
Actins
Amino Acids
Antibodies
Avalanches
Biological Factors
Cells
Chickens
Culture Media
cytochrome C oxidase subunit II
Eagle
Effectene
Fetal Bovine Serum
GAPDH protein, human
HeLa Cells
HEPES
Homo sapiens
Lipofectamine
Monoclonal Antibodies
Mus
Mycoplasma
Novus
OPTN protein, human
PARK2 protein, human
Progens
PTGS2 protein, human
Pyruvate
Rabbits
Sodium
TBK1 protein, human
Tissues
Transfection
Ubiquitin
ULK1 protein, human
C57BL/6 wild-type mice were commercially obtained (Jackson Laboratories, Bar Harbor, ME). IFN-αβ receptor (IFN-αβR)−/− congenic C57BL/6 mice were the kind gift of J. Sprent (La Jolla, CA). The congenic backcrossed IRF-3−/−[37] (link), IRF-5−/−[41] (link), and IRF-7−/−[14] (link) mice were the kind gift of T. Taniguchi (Tokyo, Japan) and provided generously by colleagues in the United States (I. Rifkin, Boston, MA and K. Fitzgerald, Worcester, MA). The IRF-1−/− mice were obtained commercially (Jackson Laboratories). The IRF-8−/− mDC were obtained from bone marrow of IRF-8−/− mice [36] (link) and were a generous gift of P. Tailor and K. Ozato (Bethesda, MD). The TRAF3−/− and TRAF6−/− MEF were kindly provided by G. Cheng (UCLA, Los Angeles, CA) and T. Mak (University of Toronto, Canada), respectively. The TBK1 MEF were obtained from B. TenOever (Mount Sinai Hospital, NY). IRF-3−/−× IRF-7−/− DKO mice were generated after large-scale crossing and recombination in the F1 generation because of the 1 cM linkage of the two loci. DKO mice were genotyped and bred in the animal facilities of the Washington University School of Medicine, and experiments were performed with approval of the Washington University Animal Studies Committee. Eight to twelve week-old age-matched mice were used for all in vivo studies. 102 PFU of WNV was diluted in Hanks balanced salt solution (HBSS) supplemented with 1% heat-inactivated fetal bovine serum (FBS) and inoculated by footpad injection in a volume of 50 µl.
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Animals
Bone Marrow
Fetal Bovine Serum
Hanks Balanced Salt Solution
IRF1 protein, human
IRF3 protein, human
IRF5 protein, human
IRF7 protein, human
IRF8 protein, human
Mice, Congenic
Mice, Inbred C57BL
Mus
Recombination, Genetic
TBK1 protein, human
TNF Receptor-Associated Factor 3
TNF Receptor Associated Factor 6
Most recents protocols related to «TBK1 protein, human»
PARP1, PARP7, STING, and TBK1 single‐gene knockout cells were generated by lentiviral transfection followed by puromycin selection. The pLenti CRISPRv2 vectors inserted with specific sgRNA oligos were transfected together with psPAX2 and pMD2G to product lentiviruses. The sgRNA sequences designed for human PARP1 knockout were 5′‐CGATGCCTATTACTGCACTG‐3′, 5′‐AGCTAGGCATGATTGACCGC‐3′, and 5′‐CCGGCACCCTGACGTTGAGG‐3′; sgRNA sequence designed for human PARP7 knockout was 5′‐CACTGAAGCTCCAGAACGAG‐3′; sgRNA sequence designed for human STING knockout was 5′‐GGCTGTCACTCACAGGTACC‐3′; sgRNA sequence designed for human TBK1 knockout was 5′‐AGAGCACTTCTAATCATCTG‐3′; sgRNA sequence designed for mouse Parp1 knockout was 5′‐ CGAGTGGAGTACGCGAAGAG‐3′; sgRNA sequence designed for mouse Parp7 knockout was 5′‐AAGGATGCGCTTCTGGTAAT‐3′.
HT‐29 PARP1−/− PARP7−/− double genes knockout cells were conducted by transfecting HT29 PARP7 KO clone cells with lentiviral containing specifically edited pLX‐sgRNA followed by blasticidin selection. The sgRNA sequences designed for PARP1 knockout was 5′‐ CGATGCCTATTACTGCACTG‐3′.
HT‐29 PARP1−/− PARP7−/− double genes knockout cells were conducted by transfecting HT29 PARP7 KO clone cells with lentiviral containing specifically edited pLX‐sgRNA followed by blasticidin selection. The sgRNA sequences designed for PARP1 knockout was 5′‐ CGATGCCTATTACTGCACTG‐3′.
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2',5'-oligoadenylate
Cells
Clone Cells
Cloning Vectors
Gene Knockout Techniques
Genes
Homo sapiens
HT29 Cells
Lentivirus
Mice, Knockout
PARP1 protein, human
Parp1 protein, mouse
Puromycin
TBK1 protein, human
Transfection
The standard western blotting protocol was conducted to detect the levels of indicated proteins as described previously (Yuan et al, 2017 (link); Li et al, 2021 (link)). Antibody against GAPDH (AF0006) was from Beyotime (Shanghai, China). Antibodies against γ‐H2AX (#2577), CDT1 (#8064), Chk1 (#2360), p‐Chk1(S317, #2344), p‐Chk2 (Thr68, #2661), caspase‐3 (#9662), caspase‐7 (#12827), caspase 9 (#9502), PARP (#9542), Bak (#12105), BID (#2002), Puma (#4976), Noxa (#14766), Bcl‐XL (#2764), XIAP (#14334), MRE11 (#4895), c‐IAP1 (#7943), c‐IAP2 (#3130), STAT1 (#14994), p‐STAT1 (Tyr701, #9167), p‐STAT1 (Ser727, #8826), STAT3 (#9139), p‐STAT3 (Ser727, #9145), STING (#13647), p‐STING (#19781), TBK1 (#3504), p‐TBK1 (#5483), IRF3 (#11904), p‐IRF3 (Ser386, #37829), p38 (#9212), p‐p38 (#9211), ERK1/2 (#9102), p‐ERK1/2 (#4370), MEK1/2 (#4694), p‐MEK1/2 (#9154), JNK (#9252), Axin2 (#2151), KU70 (#4588) and KU80 (#2753) were from Cell Signaling Technology. Antibodies against RPA32 (sc‐271578), Bax (sc‐493), MCL1 (sc‐819), PTIP (sc‐367459), Chk2 (sc‐9604), PARP2 (sc‐30622), XRCC1 (sc‐11429), XRCC3 (sc‐271714), MLH1 (sc‐581), MSH2 (sc‐494), TNKS1/2 (sc‐365897), p‐JNK (sc‐6254), PARP1 (sc‐7150) and PAR [pADPr (10H) (sc‐56198)] were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against p‐RPA32 (PLA0071) was from Sigma (Shanghai, China). Antibody against RAD51 (ab63801), CTIP (ab70163), MCRS1/MSP58 (ab247013) and PALB2 (ab202970) were from Abcam. Antibody against MAD2L2/REV7 (BD‐612266) was from BD Biosciences. Antibody against BRCA1 (OP92) and BRCA2 (OP95) were from Millipore. Goat anti‐mouse IgG horseradish peroxidase antibody was provided by Merk/Calbiochem (Darmstadt, Germany). All of the primary antibodies, except for GAPDH, were used after 1:1,000 dilution, and GAPDH primary antibody was used after 1:5,000 dilution. Second antibodies were used following 1:2,000 dilution.
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Antibodies
Antibodies, Anti-Idiotypic
AXIN2 protein, human
BIRC2 protein, human
BIRC3 protein, human
BRCA1 protein, human
Caspase-7
Caspase 3
Caspase 9
GAPDH protein, human
Gene, BRCA2
Goat
IGG-horseradish peroxidase
Immunoglobulins
IRF3 protein, human
MAP2K1 protein, human
MCL1 protein, human
Mitogen-Activated Protein Kinase 3
MLH1 protein, human
Mus
PALB2 protein, human
PARP1 protein, human
PARP2 protein, human
Proteins
Puma
RBBP8 protein, human
STAT1 protein, human
STAT3 protein, human
TBK1 protein, human
Technique, Dilution
XRCC1 protein, human
XRCC3 protein, human
Xrcc6 protein, human
Protein was extracted from the cells using RIPA buffer, resolved by SDS–polyacrylamide gels and then transferred to PVDF membranes. Primary antibodies against STING (1:1000; CST, 13647), Phospho-TBK1 (1:1000; CST, 5483), TBK1 (1:1000; CST, 38066), Phospho-IRF-3 (1:1000; CST, 29047), IRF-3 (1:1000; CST, 4302), Phospho-p65 (1:1000; CST, 3033), p65 (1:1000; CST, 8242), GAPDH (1:1000; CST, 5174) and β-actin (1:1000; CST, 4970) were used. Peroxidase-conjugated secondary antibody (CST, 7077/7076) was used, and the antigen–antibody reaction was visualized by enhanced chemiluminescence assay (ECL, ThermoFisher, 34580).
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Actins
Antibodies
Antigen-Antibody Reactions
Buffers
Chemiluminescent Assays
GAPDH protein, human
Immunoglobulins
IRF3 protein, human
Peroxidase
polyacrylamide gels
polyvinylidene fluoride
Proteins
Radioimmunoprecipitation Assay
TBK1 protein, human
Tissue, Membrane
The expression plasmids of cGAS, STING, RIG-I N, TBK1, IRF3, IRF3-5D, IκB-sr, HA-Ub-WT and HA-Ub-K0, and luciferase reporter plasmids IFNB-Luc, IRF3-Luc, TK-Luc have been described previously [44 (link),46 (link),81 (link)]. The EBV protein expression library was kindly provided by Lori Frappier from University of Toronto, and the p2089 bacmid was provided by Wolfgang Hammerschmidt from German Center for Infection Research (Munich, Germany). The expression constructs of BPLF1 mutant C61A, HA-Ub-K63, HA-Ub-K48, HA-Ub-K27, HA-Ub-K63R, HA-Ub-K48R were constructed by Q5 Site-Directed Mutagenesis Kit (New England Biolabs). Lenti-MPHv2 and lenti-SAMv2 plasmids [45 (link)] were gifts from Feng Zhang from Broad Institute.
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cDNA Library
Chromogranin A
DDX58 protein, human
Genus Loris
Gifts
Infection
Interferon, beta
IRF3 protein, human
Luciferases
Mutagenesis, Site-Directed
Plasmids
Proteins
TBK1 protein, human
Mouse anti-Myc (9E10), rabbit anti-HA (F-7), mouse anti-α-tubulin (B-7) and mouse anti-β-tubulin (3F3-G2) antibodies were purchased from Santa Cruz. Mouse anti-Flag (F3165), rabbit anti-Flag (F7425) and mouse anti-ubiquitin (P4D1) antibodies were purchased from Sigma-Aldrich. Rabbit anti-cGAS (D1D3G), rabbit anti-STING (D2P2F) and rabbit anti-TBK1 (D1B4) antibodies were from Cell Signaling Technology. Mouse anti-V5 (E10/V4RR) antibodies were from Invitrogen.
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alpha-Tubulin
Antibodies
Chromogranin A
Mus
Rabbits
TBK1 protein, human
Tubulin
Ubiquitin
Top products related to «TBK1 protein, human»
Sourced in United States
Anti-TBK1 is a research-use-only antibody product from Cell Signaling Technology. It is designed to detect the expression of TBK1 (TANK-binding kinase 1), a serine/threonine protein kinase that plays a key role in innate immune signaling pathways.
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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
Sourced in United States, China, United Kingdom
P-TBK1 is a phospho-specific antibody that recognizes TBK1 (TANK-binding kinase 1) when phosphorylated at Ser172. TBK1 is a serine/threonine protein kinase that plays a role in innate immune response and inflammatory signaling pathways.
Sourced in United States, United Kingdom
P-IRF3 is a primary antibody that specifically recognizes the phosphorylated form of the transcription factor Interferon Regulatory Factor 3 (IRF3). IRF3 plays a key role in the activation of type I interferon genes in response to viral infection or other stimuli.
Sourced in United States, United Kingdom, China
Anti-IRF3 is a primary antibody that targets the Interferon Regulatory Factor 3 (IRF3) protein. IRF3 is a transcription factor that plays a key role in the activation of type I interferon genes in response to viral infection or other cellular stressors. This antibody can be used to detect and quantify IRF3 expression or activation in various experimental systems.
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β-actin is a cytoskeletal protein that is ubiquitously expressed in eukaryotic cells. It is an important component of the microfilament system and is involved in various cellular processes such as cell motility, structure, and integrity.
Sourced in United States, China
Anti-phospho-TBK1 is a laboratory reagent that detects the phosphorylated form of TBK1 (TANK-Binding Kinase 1), a serine/threonine protein kinase involved in cellular signaling pathways. This product is intended for research use only.
Sourced in United States, China, Germany, Japan, United Kingdom, France, Canada, Italy, Australia, Switzerland, Denmark, Spain, Singapore, Belgium, Lithuania, Israel, Sweden, Austria, Moldova, Republic of, Greece, Azerbaijan, Finland
Lipofectamine 3000 is a transfection reagent used for the efficient delivery of nucleic acids, such as plasmid DNA, siRNA, and mRNA, into a variety of mammalian cell types. It facilitates the entry of these molecules into the cells, enabling their expression or silencing.
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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GAPDH is a protein that functions as an enzyme involved in the glycolysis process, catalyzing the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. It is a common reference or housekeeping protein used in various assays and analyses.