Figure 2.

(A) DARTS with methotrexate (Mtx) shows interaction with its known target dihydrofolate reductase (DHFR) but not eukaryotic elongation factor 1 alpha (eEF1A), which serves as a control protein. Jurkat lysates were incubated with varying concentrations of methotrexate or vehicle (in equal volume, with final 1% DMSO), followed by digestion with 1:900 Pronase:protein ratios for 15 min. The dissociation constant for purified recombinant DHFR is ~10 nM. Its IC₅₀ for cell lines varies greatly, and some cells have nM IC₅₀ values corresponding to its binding affinity. We found that with ~30 nM of Mtx, there is the same level of protection of DHFR against proteolysis as with ~100 μM of Mtx. (B) DARTS with olaparib (O) (IC₅₀ ~1 nM) confirms its interaction with its known target poly(ADP-ribose) polymerase (PARP), but not DHFR, which is instead the target of Mtx. Performed as in A using varying concentrations of olaparib or vehicle (in equal volume, with final 1% DMSO). (C) DARTS with CGP 3466B confirms its interaction with GAPDH while eEF1A serves as a control protein. HEK293 cell lysates were incubated with 100 μM CGP 3466B or 1% DMSO, followed by digestion with 1:1600, 1:800, 1:400, and 1:200 Pronase:protein ratios for 15 min (see Note 23). Although CGP was reported to show strong neuroprotective effects at 1 nM (21), it is not clear that this is mediated by GAPDH.