Bx795

In vitro kinase assays were performed by incubating 0.5 μg active TBK1 (SignalChem, T02-10G-10) with 1 μg tau (441 residues) purified recombinant proteins (SignalChem, T08-55BN-20) in kinase assay buffer (25 mM MOPS, pH 7.2, 12.5 mM β-glycerol-phosphate, 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA and 0.25 mM dithiothreitol (DTT)) plus 100 μM ATP for 30 min at 37 °C (SignalChem, K01-09). TBK1 was expressed form Baculovirus expression vector in Sf9 insect cells and purified in an active form. The specific activity of TBK1 has been determined to be 290 nmol/min/mg. TBK1 kinase assay was quenched by adding 2 M urea for MS analysis. For both time course in vitro kinase assay and TBK1 inhibitor dose–response reactions, 400 ng of purified tau and 200 ng of active TBK1 were used and reactions were quenched by boiling at 98 °C in Laemmli sample buffer (BioRad, 161-0737) for 5 min prior to western blot analysis. For TBK1 inhibition, increasing concentrations (5, 10, 20, and 40 μM) of BX795 (Enzo Life Sciences, ENZ-CHM189-0005) (53 (link), 78 (link)) were added to in vitro reactions. After boiling, a fraction (100 ng) of protein was used for western blot analysis.

Human embryonic kidney (HEK)-293 cells (ATCC, CRL-3216) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, 11965-092) supplemented with 10% (v/v) fetal bovine serum (Gibco, 97068-091) and penicillin/streptomycin (Gibco, 15140-122) and maintained at 37 °C under a humidified atmosphere of 5% (v/v) CO₂. For transient transfection, cells grown to 80–90% confluency were transfected with expression plasmids using JetPrime reagent (Polyplus, 114-07) according to the manufacturer’s instructions. A TBK1 inhibitor BX795 (Enzo Life Sciences, ENZ-CHM189-0005) was added at the reported concentrations 24 h post transfection and further incubated for 6 h. Subsequently, cells were rinsed with cold 1× phosphate buffered saline (PBS) and harvested in NP-40 lysis buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, plus HALT protease inhibitor cocktail, (ThermoFisher, 87786)).