Fig 1: The transcription factor c-Myb can negatively regulate DTX4 to induce the K48-linked polyubiquitination profile of TBK1: (a) the interaction between TBK1 and DTX4 in KCs was verified by immunoprecipitation (IP); (b) the protein expression of NLRP4, c-Myb, and DTX4 in KCs was detected by Western blotting. Densitometric analysis of the Western blotting data; (c) position weight matrix of transcription factor c-Myb binding site (right panel). The c-Myb binding sites and the first three binding sequences of the DTX4 promoter regions were obtained from the JASPAR website (left panel); (d) the ChIP assay was performed in HEK293T cells to verify the three binding sequences of c-Myb in the DTX4 promoter region; (e) luciferase reporter assay was performed in HEK293T cells to verify that transcription factor c-Myb could negatively regulate DTX4; (f) the regulation of c-Myb on DTX4 in RAW264.7 cells was detected by Western blotting; (g) K48-linked polyubiquitination of TBK1 in RAW264.7 cells was detected. Data are represented as the mean ± SD of at least three independent experiments; one-way ANOVA with Dunnett's multiple comparisons was used. **p < 0.01, ****p < 0.0001.
Fig 2: TBK1 induces endotoxin tolerance by negatively regulating p100 processing: (a) Western blotting analysis of p-TBK1, TBK1, NIK, P-P100, and NF-?B/p100 protein expression. Densitometric analysis of the Western blotting data; (b) the expression of the TNF-a and IL-10 in the culture supernatant of RAW264.7 cells were detected by ELISA. Data are represented as the mean ± SD of at least three independent experiments; one-way ANOVA with Dunnett's multiple comparisons was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3: The OST complex is indispensable for antiviral IFN production.(A) THP-1 cells were infected with HSV-1 (MOI = 2) for the indicated time, and endogenous MITA was then immunoprecipitated with the indicated antibodies before immunoblotting analysis. ACTB: ß-actin. (B) Consensus transcript levels of the indicated genes in human tissues were adopted from The Human Protein Atlas version 21.0 (www.proteinatlas.org) and are shown as the relative value to the transcript level of each gene in the brain. The values in the brackets represent the relative value to the transcript level of DDOST in the brain. (C) Different mouse organs were obtained and analyzed for the expression of Ddost by immunoblots. We discriminated proteins from humans or other species by a certain naming rule, for example, DDOST, as all letters capitalized to describe human proteins, and Ddost, as the first letter capitalized to describe mouse proteins. (D) HEK293T cells in 24-well plates were transfected with DDOST-RNAi NO.1 (Di#1), NO.2 (Di#2), RPN1-RNAi, RPN2-RNAi, STT3B-RNAi, STT3A-RNAi or empty vector control (Coni) by calcium phosphate transfection for 24 hours and then retransfected with pRK-HA-cGAS and pRK-HA-MITA together with the IFNß firefly luciferase reporter gene (50 ng/well) and TK Renilla luciferase reporter gene (5 ng/well) by Lipofectamine 2000. A dual luciferase reporter assay was performed after 24 hours. Data displayed are the mean ± SD of each technical repeat (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test). (E-I) Stable DDOST-, RPN1-, RPN2-, STT3A- or STT3B-knockdown THP-1 cells were constructed and infected with HSV-1 (MOI = 2) for the indicated time before immunoblotting analysis. The relative ratio (Rel. ratio) of phosphorylated TBK1 (pTBK1) to TBK1 or phosphorylated IRF3 (pIRF3) to IRF3 was calculated by measuring the grayscale values of the bands and then normalized by the value at the time of the activation peak in the control group. One representative result from at least three independent experiments is shown. (J) DDOST knockdown THP-1 cells and control cells were transfected with ISD45 (2 µg/mL) by Lipofectamine 2000 for the indicated time before immunoblotting analysis. The relative ratio (Rel. ratio) of pTBK1 to TBK1 or pIRF3 to IRF3 was calculated by measuring the grayscale values of the bands and then normalized by the value at the time of the activation peak in the control group. One representative result from at least three independent experiments is shown.
Fig 4: Serine/threonine-protein kinase TBK1 antibody screening by immunofluorescence.U2OS WT and TBK1 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated TBK1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with yellow and magenta dashed line, respectively. Antibodies were tested at 1.0 µg/ml. When the concentration was not indicated by the supplier, we tested the antibodies at 1/500 or 1/1000. At this concentration, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: NB100-56705 at 1/1000; 28397-1-AP at 1/500; 67211-1-Ig at 1/1000; PA5-17478 at 1/1000; 703154 at 1/500; ab12116 at 1/1000; ab40676 at 1/1500; ab109735 at 1/500; GTX113057 at 1/700, 38066 at 1/500, 3504 at 1/500. Bars = 10 µm.
Fig 5: Serine/thronine-protein kinase TBK1 screening by immunoprecipitation.U2OS lysates were prepared and IP was performed using 1.0 µg of the indicated TBK1 antibodies pre-coupled to either protein G or protein A Sepharose beads. Samples were washed and processed for immunoblot with the indicated TBK1 antibody. For immunoblot, ab40676, ab12116 and 67211-1-Ig were used. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitate.
Supplier Page from Abcam for Anti-NAK/TBK1 antibody [EPR2867(2)-19]