Dataset Information

Overexpress S-protein tagged mouse TBK1 protein and identify the TBK1-interacting proteins by mass spectrometry

ABSTRACT: HEK293T cells were transfected with SFB-tagged mTBK1 or an empty vector for 24 h, the cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM ethylenediaminetetraacetic acid) containing a complete protease inhibitor cocktail, followed by centrifugation at 16000xg for 10 min at 4 °C. The supernatants were subjected to immunoprecipitation using S-protein agarose (Millipore, Cat# 69704) for 3 h at 4 °C. Immunoprecipitates were washed with lysis buffer containing 150 mM NaCl three times and incubated with cell lysates from RAW 264.7 cells overnight at 4 °C. The immunoprecipitated complexes were washed three times again and separated by SDS-PAGE, and the gel was stained with Coomassie brilliant blue. The entire lane was cut into 2-mm gel slices, digested with Trypsin, and subjected to LC-MS/MS assays using an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). The mass spectrometry data were analyzed using Thermo Proteome Discovery (Version2.3), and tandem mass spectra were searched against the UniProt- the UniProt Mus musculus database 2022.11.16.

ORGANISM(S): Homo Sapiens Mus Musculus

SUBMITTER: Qinmiao Sun

PROVIDER: PXD045961 | iProX | Fri Oct 27 00:00:00 BST 2023

REPOSITORIES: iProX

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Publications

PTK2B promotes TBK1 and STING oligomerization and enhances the STING-TBK1 signaling.

Lin Yongfang Y Yang Jing J Yang Qili Q Zeng Sha S Zhang Jiayu J Zhu Yuanxiang Y Tong Yuxin Y Li Lin L Tan Weiqi W Chen Dahua D Sun Qinmiao Q

Nature communications 20231121 1

TANK-binding kinase 1 (TBK1) is a key kinase in regulating antiviral innate immune responses. While the oligomerization of TBK1 is critical for its full activation, the molecular mechanism of how TBK1 forms oligomers remains unclear. Here, we show that protein tyrosine kinase 2 beta (PTK2B) acts as a TBK1-interacting protein and regulates TBK1 oligomerization. Functional assays reveal that PTK2B depletion reduces antiviral signaling in mouse embryonic fibroblasts, macrophages and dendritic cells ...[more]

PMID: 37989995

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Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

Dataset Information

Overexpress S-protein tagged mouse TBK1 protein and identify the TBK1-interacting proteins by mass spectrometry

Publications

PTK2B promotes TBK1 and STING oligomerization and enhances the STING-TBK1 signaling.

Similar Datasets

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