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. 2016 Apr 22;7:11391. doi: 10.1038/ncomms11391

Figure 4. FINDY affects the folding of DYRK1A, targeting the ATP-binding pocket.

Figure 4

(a) Schematic diagram of the biotin-transfer experiment. Expression of FLAG-DYRK1A is induced by doxycycline (Dox) with epoxomicin (Epo) and kinase inhibitors (10 μM). The resulting FLAG-DYRK1A proteins are immunopurified and reacted with the biotinylated acyl phosphate of ATP (20 μM). If the inhibitor occupies the ATP-binding pocket, then biotin transfer is prevented. (b) FINDY competes with the biotin-ATP analogue for the DYRK1A intermediate. FINDY, INDY and RD0392 were used as described in a. As a positive control for the competition experiment, the FLAG-DYRK1A protein was pre-treated with ATP (100 μM) before the reaction with the biotin-ATP analogue. The labelled proteins were blotted with streptavidin and anti-FLAG antibody. Representative data from the triplicate experiments are shown. IP, immunoprecipitation.