Journal: bioRxiv
Article Title: Complex Intracellular Mechanisms of TBK1 Kinase Activation Revealed by a Specific Small Molecule Inhibitor
doi: 10.1101/2022.10.11.511671
Figure Lengend Snippet: A . GSK8612 is a more specific TBK1 inhibitor compared to BX795 and MRT67307. HT1080 cells were either untreated, or pretreated with GSK8612, MRT67307 or BX795 (10µM) for 1hr, then treated by lipofectamine 2000 mediated transfection of dsDNA (poly dG:dC, 1.6µg/ml) or dsRNA (poly I:C, 1.6µg/ml) for 2hrs. Total cell lysates were prepared, and the phosphorylation and expression of various proteins determined by Western blotting with specific antibodies. A nonspecific band from A20 blot is labeled with an asterisk. The target bands of A20 and OPTN are marked by arrows, and a cleavage product of OPTN is labeled by an arrowhead. B . Kinetic study of HT1080 cells infected with Sendai virus in the absence or presence of GSK8612. Control cells or cells pretreated with GSK8612 (10µM) for 1hr were infected with Sendai virus (300 HAU/ml) for increasing time ( , and 6hrs). Total cell lysates were prepared at specified time points post infection, and the phosphorylation and expression of TBK1, IRF3, p62, Sendai virus C protein and LC3B were determined by Western blotting with specific antibodies. C . Kinetic study of HT1080 cells stimulated with TNFꭤ in the absence or presence of GSK8612. Control cells or cells pretreated with GSK8612 (10µM) for 1hr were treated with TNFꭤ (10ng/ml) for increasing time (20 to 120 minutes). Total cell lysates were prepared at specified time points post stimulation, and the profile of phosphorylation and expression of various proteins were determined by Western blotting. D . Kinetic study of HT1080 cells stimulated with dsDNA in the absence or presence of GSK8612. Control cells or cells pretreated with GSK8612 (10µM) for 1hr were challenged with dsDNA (poly dG:dC) for increasing time (30 minutes to 6hrs). Total cell lysates were prepared at specified time post stimulation. The phosphorylation profile and expression of various proteins were determined by Western blotting with specific antibodies. E . Kinetic study of HT1080 cells stimulated with dsRNA in the absence or presence of GSK8612. Control cells or cells pretreated with GSK8612 (10µM) for 1hr were challenged with dsRNA (poly I:C) for increasing time (30 min to 4hrs). Total cell lysates were prepared at specified time points post stimulation. The expression and phosphorylation of various proteins were determined by Western blotting with specific antibodies.
Article Snippet: Concentrated Sendai virus stock was purchased from Charles River Lab (Cantell strain) and used at a concentration of 300 HAU/ml for indicated infection time.
Techniques: Transfection, Phospho-proteomics, Expressing, Western Blot, Labeling, Infection, Virus, Control
