Atp γ s

Cytotoxic activity of NK cells was determined using a Lactate Dehydrogenase (LDH) Detection Kit (Dojindo, Japan). Briefly, the target tumor cells (2.5 × 10⁴/well) were co-cultured with NK-92MI cells in 96-well microplates at different effector-to-target (E: T) ratios for 6 h. In experiments exploring the effect of ATP and Ado analogs on the cytotoxicity of NK cells, NK-92MI cells were pre-treated with ATP-gamma-S (ATP-γ-S, Abcam, USA) or 2-Chloroadenosine (CADO, Abcam) for 3 h before co-culture. In P2Rs and P1Rs blocking experiments, NK-92MI cells were pre-inoculated with 10 μM P2Y11R antagonist NF340 (Santa Cruz Biotechnology, USA) and/or 1 μM A_2AR antagonist SCH58261 (Sigma Aldrich) for 30 min before ATP-γ-S or CADO treatment. Anti-DNAM-1 antibody (Ab) mediated blocking assays were performed by incubating NK-92MI cells with anti-human DNAM-1 Ab (clone 102511, R&D Systems, USA) at a final concentration of 10 μg/mL for 1 h before cytotoxicity assay. After 6 h of co-culture, the LDH released in the supernatant was detected and calculated as the manufacturer’s instructions.

For cryo-EM, purified full-length P5CS was diluted to approximately 2 μM and dissolved in buffer containing 25 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgSO₄, and incubated with 20 mM l-glutamate for the P5CS^Glu filament preparation. The P5CS^Glu/ATPγS filament was added with an additional 0.5 mM ATPγS (Abcam) compared to the P5CS^Glu filament. For the P5CS^Mix filament, P5CS proteins (2 μM) were incubated with 100 mM KCl, 10 mM MgSO₄, 20 mM l-glutamate, 2 mM ATP, and 0.5 mM NADPH. All the samples were incubated for 1 hr on ice before vitrification. The P5CS filament samples were placed on H₂/O₂ glow-discharged holey carbon grids (Quantifoil Cu 300 mesh, R1.2/1.3) or amorphous alloy film (CryoMatrix M024-Au300-R12/13). Then, the grids were immediately blotted for 3.0 s and plunge-frozen in liquid ethane cooled by liquid nitrogen using Vitrobot (Thermo Fisher) at 4°C with 100% humidity. Images were collected on Titan Krios G3 (FEI) equipped with a K3 Summit direct electron detector (Gatan), operating in counting super-resolution mode at 300 kV with a total dose of 72 e⁻/Ų, subdivided into 50 frames in 4 s exposure using SerialEM (Mastronarde, 2005 (link)). The images were recorded at a nominal magnification of 22,500 × and a calibrated pixel size of 1.06 Å, with defocus ranging from 0.8 to 2.5 μm.

HEK293T cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a. At 48 h after transfection, cells were lysed in 1.0 mL IP lysis buffer supplemented with protease inhibitor cocktail. Cell lysates were centrifuged at 17,700 × g for 10 min at 4°C. The supernatant was incubated with 50 μL anti-HA magnetic beads for 2 h at 4°C. The beads were washed five times with IP lysis buffer and once with kinase buffer (40 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, and 50 μM dithiothreitol [DTT]). The immunocomplex was incubated with GST-IκBα (catalog no. ab59981; Abcam) as the substrates in kinase buffer with 1 mM ATP-γ-S (catalog no. ab138911; Abcam) at 37°C for 30 min. Reactions were followed by addition of 2.5 mM p-nitrobenzyl mesylate (catalog no. ab138910; Abcam) to start alkylation for 2 h at room temperature. The reaction was stopped by boiling in 4× sample buffer and subjected to Western blotting. Membranes were probed with anti-thiophosphate ester antibody and horseradish peroxidase-conjugated secondary antibody. Labeled proteins were visualized using a LuminoGraph I.

TAP-purified MAP4K4 eluted in standard lysis buffer with protease and phosphatase inhibitors were added to kinase assay buffer (25 mM Tris-HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate and 10 mM MgCl₂) containing 20 μM ATPγS (Abcam) and 1 μg of myelin basic protein (MBP) (Sigma). Where specified, ATPγS was left out of the reaction as a negative control. Kinase reactions were carried out as previously described (Allen et al., 2007 (link)). Reactions were carried out at 30°C for 30 min. P-nitrobenzyl mesylate (PNBM) (Abcam) was then added (2.5 mM final) and the reaction was incubated at room temperature for 2 hr, followed by addition of 6x SDS loading buffer, boiling of samples, SDS-PAGE and subsequent immunoblotting for phosphorylated MBP. Relative activity was calculated as the ratio of the band intensities (measured with ImageJ) between the thiophosphate ester signal (phospho-MBP) and HA signal (NTAP-MAP4K4).

General biochemicals, unless otherwise stated, were purchased from Sigma–Aldrich. Primers for molecular cloning and site directed mutagenesis were produced by integrated DNA technologies (IDT). Reagents for metabolic labelling of acylated PSKH2 were purchased from Click Chemistry Tools. ATP-γ-S, Thiophosphate ester antibody and para-nitrobenzyl mesylate were purchased from Abcam. All commercial antibodies were purchased from Cell Signalling Technology. A PSKH2 polyclonal antibody was raised towards a unique peptide in the C-lobe of the human PSKH2 pseudokinase domain (KGKYNYTGEPWPSISC) and affinity-purified prior to testing (Abgent). This antibody did not detect endogenous PSKH2, but specifically recognises exogenous PSKH2 in human cell extracts.