Figure 3. ATP acyl phosphates enable gel-based activity-based profiling of DGKα.

(A) In vitro IC₅₀ values for DGKα inhibition by ritanserin as measured by DAG phosphorylation substrate assay described in Figure S2. Data shown are mean +/− SEM. for triplicate measurements. Results are representative of two independent biological replicates. 95% confidence intervals for IC₅₀ values: 20–30 μM. (B) Gel-based ATP acyl phosphate assay was used to determine in vitro IC₅₀ values for DGKα inhibition by ritanserin (95% confidence intervals for IC₅₀ values: 17–192 μM). Details of the assay and representative gels used to calculate potency values can be found in Figure S3. (C) DGKα-HEK293T soluble proteomes were pretreated with ritanserin (100 μM), ketanserin (100 μM), or ATP (1 mM) for 30 min prior to addition of ATP acyl phosphate probe (10 μM, 30 min) and gel-based analysis as described in Figure S3. Pretreatment with ritanserin but not ketanserin blocked probe labeling of ~80 kDa recombinant DGKα. Western blot analysis (anti-FLAG, 0.8 μg/mL) confirmed equivalent recombinant protein expression across treatment conditions.